[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Microbiol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Microbiol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Microbiol.</journal-id><journal-title-group><journal-title>Frontiers in Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-302X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849322</article-id><article-id pub-id-type=\"pmc\">PMC7431629</article-id><article-id pub-id-type=\"doi\">10.3389/fmicb.2020.01532</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Microbiology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Antimicrobial Peptide Cec4 Eradicates the Bacteria of Clinical Carbapenem-Resistant <italic>Acinetobacter baumannii</italic> Biofilm</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Liu</surname><given-names>Weiwei</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/913841/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wu</surname><given-names>Zhaoying</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/951282/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Mao</surname><given-names>Chengju</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Guo</surname><given-names>Guo</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zeng</surname><given-names>Zhu</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Fei</surname><given-names>Ying</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/948233/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wan</surname><given-names>Shan</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Peng</surname><given-names>Jian</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/886981/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wu</surname><given-names>Jianwei</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Immune Cells and Antibody Engineering Research Center of Guizhou Province, Guizhou Medical University</institution>, <addr-line>Guiyang</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University</institution>, <addr-line>Guiyang</addr-line>, <country>China</country></aff><aff id=\"aff3\"><sup>3</sup><institution>The Key and Characteristic Laboratory of Modern Pathogen Biology, Basic Medical College, Guizhou Medical University</institution>, <addr-line>Guiyang</addr-line>, <country>China</country></aff><aff id=\"aff4\"><sup>4</sup><institution>The Center for Clinical Laboratories, The Affiliated Hospital of Guizhou Medical University</institution>, <addr-line>Guiyang</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Luis Cláudio Nascimento da Silva, Universidade Ceuma, Brazil</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Cesar de la Fuente-Nunez, University of Pennsylvania, United States; Maria Bagattini, University of Naples Federico II, Italy; Lucas Pinheiro Dias, Federal University of Ceará, Brazil</p></fn><corresp id=\"c001\">*Correspondence: Jian Peng, <email>jianpeng@gmc.edu.cn</email></corresp><corresp id=\"c002\">Jianwei Wu, <email>wjw@gmc.edu.cn</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>These authors have contributed equally to this work</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1532</elocation-id><history><date date-type=\"received\"><day>16</day><month>2</month><year>2020</year></date><date date-type=\"accepted\"><day>12</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Liu, Wu, Mao, Guo, Zeng, Fei, Wan, Peng and Wu.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Liu, Wu, Mao, Guo, Zeng, Fei, Wan, Peng and Wu</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>The drug resistance rate of <italic>Acinetobacter baumannii</italic> increases year on year, and the drugs available for the treatment of carbapenem-resistant <italic>A. baumannii</italic> (CRAB) infection are extremely limited. <italic>A. baumannii</italic>, which forms biofilms, protects itself by secreting substrates such as exopolysaccharides, allowing it to survive under adverse conditions and increasing drug resistance. Antimicrobial peptides are small molecular peptides with broad-spectrum antibacterial activity and immunomodulatory function. Previous studies have shown that the antimicrobial peptide Cec4 has a strong effect on <italic>A. baumannii</italic>, but the antibacterial and biofilm inhibition of this antimicrobial peptide on clinical carbapenem resistance <italic>A. baumannii</italic> is not thoroughly understood. In this study, it was indicated that most of the 200 strains of CRAB were susceptible to Cec4 with a MIC of 4 μg/ml. Cec4 has a strong inhibitory and eradication effect on the CRAB biofilm; the minimum biofilm inhibition concentration (MBIC) was 64–128 μg/ml, and the minimum biofilm eradication concentration (MBEC) was 256–512 μg/ml. It was observed that Cec4 disrupted the structure of the biofilm using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). A comparative transcriptome analysis of the effects of the antimicrobial peptide Cec4 on CRAB biofilm, identified 185 differentially expressed genes, including membrane proteins, bacterial resistance genes, and pilus-related genes. The results show that multiple metabolic pathways, two-component regulation systems, quorum sensing, and antibiotic synthesis-related pathways in <italic>A. baumannii</italic> biofilms were affected after Cec4 treatment. In conclusion, Cec4 may represent a new choice for the prevention and treatment of clinical infections, and may also provide a theoretical basis for the development of antimicrobial peptide drugs.</p></abstract><kwd-group><kwd>antimicrobial peptide</kwd><kwd>Cec4</kwd><kwd>CRAB</kwd><kwd>biofilm</kwd><kwd>transcriptome</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Natural Science Foundation of China<named-content content-type=\"fundref-id\">10.13039/501100001809</named-content></funding-source></award-group><award-group><funding-source id=\"cn002\">Natural Science Foundation of Guizhou Province<named-content content-type=\"fundref-id\">10.13039/501100005329</named-content></funding-source></award-group><award-group><funding-source id=\"cn003\">Guizhou Science and Technology Department<named-content content-type=\"fundref-id\">10.13039/501100004001</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"6\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"56\"/><page-count count=\"13\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p><italic>Acinetobacter baumannii</italic> is a non-fermenting Gram-negative bacterium, and infections often occur in patients with poor immunity, especially in patients in intensive care units or in patients undergoing invasive surgery (<xref rid=\"B21\" ref-type=\"bibr\">Handal et al., 2017</xref>). It can cause hospital-acquired pneumonia, especially ventilator-associated pneumonia, bacteremia, urinary tract infection, secondary meningitis, etc., with a high mortality rate (<xref rid=\"B10\" ref-type=\"bibr\">Bentancor et al., 2012</xref>). At the same time, <italic>A. baumannii</italic> is prone to drug resistance and is even resistant to carbapenem antibiotics, including imipenem and meropenem (<xref rid=\"B34\" ref-type=\"bibr\">Maragakis and Perl, 2008</xref>). At present, most antibiotics, except tigecycline and polymyxin, do not affect it (<xref rid=\"B14\" ref-type=\"bibr\">Dafopoulou et al., 2019</xref>). Although tigecycline has a broad antibacterial spectrum and good antibacterial activity, the blood concentration is low, only 0.7–0.8 mg/L (<xref rid=\"B13\" ref-type=\"bibr\">Cai and Wang, 2011</xref>). Therefore, it remains controversial to use tigecycline to treat blood-related infections (<xref rid=\"B3\" ref-type=\"bibr\">Akalay et al., 2020</xref>); moreover, polymyxin has greater renal and neurotoxicity, limiting its use (<xref rid=\"B48\" ref-type=\"bibr\">Spapen et al., 2011</xref>). By organism, the highest overall rates of multidrug resistance reported in a study were among <italic>A. baumannii</italic> isolates, for which 44% of isolates collected globally were multidrug-resistant bacteria. Additionally, the treatment options for infections caused by such organisms are limited, which deserves attention (<xref rid=\"B20\" ref-type=\"bibr\">Giammanco et al., 2017</xref>). Recently, the World Health Organization (WHO) classified carbapenem-resistant <italic>A. baumannii</italic> (CRAB) as the first in the list of key pathogens for the development of new antibiotics (<xref rid=\"B38\" ref-type=\"bibr\">O’Shea, 2012</xref>).</p><p>When <italic>A. baumannii</italic> forms biofilms, its resistance increases rapidly (<xref rid=\"B17\" ref-type=\"bibr\">Eze et al., 2018</xref>). Biofilms are composed of bacteria that irreversibly adhere to the surface of living or non-living organisms and are surrounded by a secreted matrix of extracellular polysaccharide, protein, and DNA. Once the special structure forms, the bacteria express completely different genes from planktonic bacteria, with significant differences in morphology, physical and chemical properties, and antibiotic susceptibility (<xref rid=\"B26\" ref-type=\"bibr\">Jamal et al., 2018</xref>). The ability to form biofilms on abiotic surfaces under adverse conditions makes the biofilm phenotype an important virulence factor for <italic>A. baumannii</italic> infection (<xref rid=\"B17\" ref-type=\"bibr\">Eze et al., 2018</xref>). At the same time, it is beneficial for bacteria to survive on nutritionally limited abiotic surfaces and stressful environmental conditions (<xref rid=\"B5\" ref-type=\"bibr\">Alvarez-Fraga et al., 2016</xref>). Bacterial biofilms cause at least 65% of human infections, particularly implantable device-related infections and chronic disease infections (<xref rid=\"B53\" ref-type=\"bibr\">Williams and Costerton, 2012</xref>). Therefore, there is an urgent need for drugs that effectively treat biofilm-associated infections. At present, it has been reported that some natural product extracts and compounds can treat bacterial biofilms. Water extract of <italic>Galla chinensis</italic> suppressed biofilm and extracellular matrix formation of <italic>Staphylococcus aureus</italic> (<xref rid=\"B54\" ref-type=\"bibr\">Wu et al., 2019</xref>). <xref rid=\"B42\" ref-type=\"bibr\">Qin et al. (2014)</xref> reported the effects of two natural compounds on methicillin-resistant <italic>Staphylococcus aureus</italic> (MRSA). Ursolic acid can inhibit the formation of biofilms, while resveratrol combined with vancomycin can inhibit pre-formed mature biofilms. Silver nanoparticles do not affect the growth of planktonic <italic>Staphylococcus aureus</italic> but can reduce the production of biofilms at a concentration of 50 μg/ml (<xref rid=\"B47\" ref-type=\"bibr\">Singh et al., 2019</xref>). However, in many cases, these anti-biofilm active ingredients are not sufficient enough to completely inhibit or eliminate bacterial biofilms and lack broad-spectrum anti-biofilm efficacy.</p><p>Antimicrobial peptides (AMPs) have a broad antibacterial spectrum and a wide range of sources, and have unique antibacterial mechanisms, making them less prone to drug resistance. It is generally believed that AMPs exert their microbicidal activity mainly through targeting the cell membrane by penetration and cell lysis activities (<xref rid=\"B2\" ref-type=\"bibr\">Aisenbrey et al., 2019</xref>). The MIC of human-derived cationic peptide LL-37 and its truncated fragments against drug-resistant <italic>A. baumannii</italic> is 16–32 μg/ml, and it inhibits the formation of biofilms (<xref rid=\"B18\" ref-type=\"bibr\">Feng et al., 2013</xref>). The peptide IDR-1018 exhibits broad-spectrum anti-biofilm activity against a variety of hospital pathogens, including <italic>Pseudomonas aeruginosa</italic> and <italic>Klebsiella pneumonia</italic> (<xref rid=\"B16\" ref-type=\"bibr\">de la Fuente-Nunez et al., 2014</xref>). Therefore, antimicrobial peptides are expected to become new drugs for the treatment of bacterial biofilm and associated infections (<xref rid=\"B28\" ref-type=\"bibr\">Kim et al., 2020</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Neshani et al., 2020</xref>). Our previous study found that the peptide Cec4 had a minimum inhibitory concentration (MIC) of 4 μg/ml against an <italic>A. baumannii</italic> reference strain (ATCC19606), which is superior to the reported similar cecropin antimicrobial peptides Cec1, cecropin A and fusion peptide CA. (1–8) M (1–18) (<xref rid=\"B46\" ref-type=\"bibr\">Saugar et al., 2002</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Batoni et al., 2011</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Long et al., 2017</xref>). It was confirmed in the previous report that the semi-inhibitory concentration of Cec4 on the formation of standard <italic>A. baumannii</italic> biofilms (to inhibit the formation of biofilm by 50%) is about 4 μg/ml; this peptide is non-hemolytic on human red blood cells at high concentrations (100 × MIC) (<xref rid=\"B40\" ref-type=\"bibr\">Peng et al., 2019</xref>). However, whether the antimicrobial peptide Cec4 can inhibit CRAB and its biofilm to the same extent as with the <italic>A. baumannii</italic> (ATCC19606), remains elusive. Therefore, 200 strains of clinical CRAB were collected, and their ability to form biofilms was tested. Furthermore, the susceptibility and biofilm formation of <italic>A. baumannii</italic> isolates to the antimicrobial peptide Cec4 were evaluated, and the molecular mechanism of Cec4 on bacterial biofilm was analyzed by transcriptome analysis. In conclusion, this study is expected to provide new ideas for the treatment of clinical infections and presents a theoretical basis for research and development into new antibiotics.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Synthesis and Preparation of Peptides</title><p>Antimicrobial peptide Cec4 (GWLKKIGKKIERVGQNTRD ATIQAIGVAQQAANVAATLKGK) was synthesized by Gil Biochemical Co., Ltd., Shanghai. Using solid-phase chemical synthesis, the purity (HPLC) > 97% and the mass of the peptide determined by spectrometry. It was dissolved to 10 mg/ml with distilled deionized H<sub>2</sub>O (ddH<sub>2</sub>O) and stored at −80°C for further analysis.</p></sec><sec id=\"S2.SS2\"><title>Bacterial Isolates and Growth Conditions</title><p>From October 2017 to December 2018, 200 isolates of CRAB from clinical samples of patients from the affiliated hospital of Guizhou Medical University were collected and duplicate samples from the same patient were excluded. All of them come from sputum, blood, urine and so on. The collection and use of clinically isolated strains were approved by the Institutional review board (IRB) of Guizhou Medical University, China. The studies involving human participants were reviewed and approved by Guizhou Medical University and the affiliation of the ethics committee. The patients provided written informed consent to participate in this study. Minimum inhibitory concentrations (MICs) of these strains to nine antibiotics including amikacin, ertapenem, imipenem, meropenem, ceftazidime, ciprofloxacin, ceftriaxone, levofloxacin, and cefepime were assessed on MicroScan WalkAway 40-SI Analyzer (SIEMENS, Germany), according to the manufacturer’s instructions. Resistance to cefotaxime was assessed using the standard disc diffusion method (Oxoid, Hampshire, United Kingdom). Interpretive breakpoints for susceptible, intermediate, and resistant were consistent with Clinical and Laboratory Standards Institute guidelines (<xref rid=\"B25\" ref-type=\"bibr\">Humphries et al., 2018</xref>). In order to ensure the accuracy of bacterial identification, the 16<italic>SrRNA</italic> and <italic>rpoB</italic> genes of all strains were amplified, and PCR products were sequenced and analyzed. <italic>A. baumannii</italic> (ATCC19606) is a biofilm-forming positive strain. <italic>Escherichia coli</italic> ATCC25922 and <italic>Pseudomonas aeruginosa</italic> ATCC27853 were used as quality control bacteria. The above strains are stored in the Pathogen Biology Laboratory of Guizhou Medical University. Strains were grown on Mueller-Hinton Broth (MHB), Typic Soy Broth (TSB) or Luria-Bertani (LB) agar plates and incubated at 37°C.</p></sec><sec id=\"S2.SS3\"><title>Detecting the Minimum Inhibitory Concentration (MIC) Value</title><p>According to a previous study (<xref rid=\"B52\" ref-type=\"bibr\">Wiegand et al., 2008</xref>), the MIC of Cec4 peptide against 200 strains of CRAB was assessed using the broth microdilution assay in MHB. After adding different concentrations of antimicrobial peptide Cec4, the 96-well plate was placed in a constant temperature incubator at 37°C. After incubation for 24 h, it was observed. The MIC was defined as the lowest drug concentration that can inhibit bacterial growth by visual evaluation.</p></sec><sec id=\"S2.SS4\"><title>Quantitative Biofilm Formation Assay</title><p>According to the crystal violet staining method of a previous study (<xref rid=\"B39\" ref-type=\"bibr\">O’Toole, 2011</xref>), the 96-well tissue culture plate method was utilized for a quantitative evaluation of biofilm formation by 200 strains of CRAB. <italic>A. baumannii</italic> ATCC19606 was used as the positive control, and TSB medium without bacteria was used as the negative control. Absorbance at 570 nm (OD570) was measured for each well to obtain quantitative data on biofilm formation as described previously (<xref rid=\"B7\" ref-type=\"bibr\">Badmasti et al., 2015</xref>). The mean ± standard deviation of the OD value of the negative control was defined as ODc. Based on the OD value, the strains were divided into the following four groups: the OD value of the test strain was compared with ODc, and OD<sub>570</sub> ≤ ODc was negative for biofilm formation (−); ODc < OD<sub>570</sub> ≤ 2 × ODc indicates weak biofilm formation (+); 2 × ODc < OD570 ≤ 4 × ODc indicates moderate biofilm formation (++); 4 × ODc < OD570 indicates strong biofilm formation (+++).</p></sec><sec id=\"S2.SS5\"><title>Detection of the Minimum Biofilm Inhibition Concentration (MBIC) and Minimum Biofilm Eradication Concentration (MBEC)</title><p>In order to detect the inhibitory effect of Cec4 peptide on the growth of biofilms, a method described in the literature was used with modifications (<xref rid=\"B1\" ref-type=\"bibr\">Abouelhassan et al., 2017</xref>). Briefly, 200 μl of bacterial cells (1 × 10<sup>6</sup> CFU/ml) of 10 strains (CRAB 3, 4, 53, 55, 78, 117, 120, 128, 130, 136) with the strongest biofilm formation ability was inoculated in a polyethylene 96-well plate; TSB culture medium containing no bacteria was used as the blank control, and the plate was incubated at 37°C for 24 h. The culture medium was subsequently removed, and wells were carefully washed with PBS three times to remove planktonic bacteria. And then, 200 μl of TSB culture medium containing Cec4 in serial doubling dilutions was added to each well. TSB medium without an antimicrobial peptide was used as a negative control, and plates were incubated at 37°C for 24 h. If OD600 < 0.1, there was no bacterial growth, and the lowest concentration without bacterial growth at this time point was recorded; this is the MBIC. Then, cells and peptide in the 96-well cell culture plate were washed with PBS, and 200 μl of TSB culture medium was added to each well. The plate was incubated at 37°C for 24 h to re-grow the surviving biofilm bacteria. An OD600 < 0.1 indicated that there was no bacterial growth. The lowest concentration at which no bacterial growth was recorded is the MBEC. In order to evaluate the eradication efficiency of Cec4 on the biofilm, the culture in the wells was removed and washed with PBS to remove non-adherent cells. The biofilm was quantified by the aforementioned method, and calculated using the equation <inline-formula><mml:math id=\"INEQ7\"><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mn>1</mml:mn><mml:mo>-</mml:mo><mml:mfrac><mml:mrow><mml:mrow><mml:mtext>OD</mml:mtext></mml:mrow><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mn>570</mml:mn></mml:mpadded><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>of</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>the</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>test</mml:mi></mml:mrow><mml:mrow><mml:mrow><mml:mrow><mml:mtext>OD</mml:mtext></mml:mrow><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mn>570</mml:mn></mml:mpadded><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>of</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>non</mml:mi></mml:mrow><mml:mo rspace=\"5.3pt\">-</mml:mo><mml:mrow><mml:mpadded width=\"+2.8pt\"><mml:mi>treated</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>control</mml:mi></mml:mrow></mml:mrow></mml:mfrac></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo rspace=\"5.8pt\">×</mml:mo><mml:mn>100</mml:mn></mml:mrow></mml:math></inline-formula>.</p></sec><sec id=\"S2.SS6\"><title>Scanning Electron Microscopy (SEM)</title><p>According to a previous report (<xref rid=\"B43\" ref-type=\"bibr\">Ramalingam and Lee, 2018</xref>), the biofilm of CRAB 55 was cultured <italic>in vitro</italic> in a 6-well plate, and a sterile polylysine-treated cover glass and a sterile medical catheter cut at a length of 1 cm were added in advance as a biofilm growth carrier. Samples were processed by gradient dehydration with 20, 50, 70, 90, and 100% ethanol/tert-butanol mixture. The samples were dried in a critical point dryer and placed in a high vacuum evaporator, then sprayed gold with an ion sprayer and observed using a scanning electron microscope (Hitachi S-3400).</p></sec><sec id=\"S2.SS7\"><title>Confocal Laser Scanning Microscope Analysis</title><p>Confocal laser scanning microscopy analysis was carried out according to a previous study (<xref rid=\"B12\" ref-type=\"bibr\">Bortolin et al., 2016</xref>). A sterile polylysine-treated cover glass was used as the carrier, and the biofilm of CRAB 55 was cultivated according to the method of the previous step. Then, 1 mM SYTO9 and 10 mM propidium iodide (PI) were added and incubated for 15 min in the dark. An Olympus Fluoview FV1000 confocal microscope (Olympus, Markham, ON, Canada) was used to obtain a fluorescence image. The bottom of the biofilm to the surface was scanned layer by layer along the <italic>Z</italic>-axis to record the earliest and last disappearance of fluorescence, and the corresponding biofilm thickness was calculated accordingly; each layer was 1 μm. The resulting stacks of images were quantified using an image processing package (ImageJ, United States) and subsequently rendered into three-dimensional mode using image analysis software (Imaris 7.2.3, Bitplane, Switzerland).</p></sec><sec id=\"S2.SS8\"><title>Motility Assays</title><p>As described by a previous study (<xref rid=\"B35\" ref-type=\"bibr\">McQueary and Actis, 2011</xref>), twitching plates were made with 10 g/l tryptone, 5 g/l yeast extract and 5 g/l NaCl and 1% Eiken agar, with different concentrations of Cec4 (or none). A single colony of CRAB 55 was picked from a normal LB agar (1.5%, wt/vol) plate and inoculated vertically to the bottom of the plate, so that it could grow at the intersection of the bottom of the agar layer and the culture dish. The plate was incubated at 37°C for 24 h, and motility was evaluated by observing the formation of a transparent halo around the growing colonies and measuring the diameter. Twitching motility assays were conducted on at least three separate occasions. As described in a previous study (<xref rid=\"B22\" ref-type=\"bibr\">Harding et al., 2013</xref>), surface motility plates are comprised of 5 g/l tryptone, 2.5 g/l NaCl and 0.35% Eiken agar, with different concentrations of Cec4 or not. The bacteria were cultured to the logarithmic phase, then a 2 μl of aliquot of an overnight culture was stabbed into the surface of the center of the plate, and motility was measured after incubating the plate at 37°C for 24 h. Surface-associated motility assays were conducted on at least three separate occasions.</p></sec><sec id=\"S2.SS9\"><title>Quantitative RT-PCR</title><p>According to a previous study (<xref rid=\"B24\" ref-type=\"bibr\">He et al., 2015</xref>), biofilm formation of CRAB 55 was conducted as described above, and after the biofilm had been treated with Cec4 or not, it was scraped from the plate with a cell spatula. Total RNA was extracted using Trizol. RNA was quantified and quality was assessed using a NanoDrop spectrophotometer (ND-2000, Thermo Scientific, Loughborough, United Kingdom), and the final RNA concentration was adjusted to 1 ng/μl. cDNA was synthesized in a 20 μl reaction mixture using a PrimeScript RT reagent Kit with gDNA Eraser. According to the SYBR Premix Ex Taq TM Kit (Takara, Dalian, China) protocol, the reactions were run on an ABI7300 real-time PCR system using a 20 μl reaction volume. Gene expression levels were normalized to the abundance of <italic>A. baumannii</italic> 16<italic>S rRNA</italic>. Target genes included <italic>CsuE</italic>, <italic>BfmR</italic>, <italic>BfmS</italic>, <italic>AbaI</italic>, and <italic>Bap</italic>. The primers were designed with reference to the GenBank sequences. The primer sequences are shown in the <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table S1</xref>.</p></sec><sec id=\"S2.SS10\"><title>RNA-Seq</title><sec id=\"S2.SS10.SSS1\"><title>RNA Isolation, Library Construction and Sequencing</title><p>The four strains CRAB 4, 55, 78, and 117 with the strong biofilm-forming ability and similar characteristics were selected as four biological replicates. The logarithmic growth culture was washed twice in sterile PBS and diluted to 10<sup>6</sup> cells/ml in TSB. Then, 4 ml of the culture was added to a flat-bottomed 6-well plate (Corning, United States) and incubated at 37°C for 24 h to form a biofilm. Samples were collected as a control group (Control 1, Control 2, Control 3 and Control 4). For the Cec4 treatment group (ABF1, ABF2, ABF3, and ABF4), after forming mature biofilms, non-adherent cells were removed, and TSB culture solution containing 4 × MIC (16 μg/ml) of Cec4 was added. Biofilm samples were collected 24 h later.</p><p>The total RNA of these samples was extracted using Trizol reagent (Sigma-Aldrich, United States) according to the previously described method. The quality and quantity were determined by a NanoPhotometer spectrophotometer (IMPLEN, CA, United States) and an Agilent 2100 bioanalyzer (Agilent Technologies, CA, United States). RNA-seq library construction and RNA sequencing were performed by the Novogene Corporation (Beijing, China). Sequence reads were deposited at the National Center for Biotechnology Information under BioProject <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"PRJNA607078\">PRJNA607078</ext-link> as <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN14120700\">SAMN14120700</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN14120701\">SAMN14120701</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN14120702\">SAMN14120702</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN14120703\">SAMN14120703</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN14120704\">SAMN14120704</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN14120705\">SAMN14120705</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN14120706\">SAMN14120706</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN14120707\">SAMN14120707</ext-link><sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup>.</p></sec><sec id=\"S2.SS10.SSS2\"><title>Identification of Differentially Expressed Genes and Annotation</title><p>Raw reads were generated from the image data and stored as FASTQ format. Raw data were filtered to remove adaptor contaminated and low-quality sequences and to obtain clean reads. Genomic mapping of the filtered sequence was performed using Bowtie2-2.2.3 (<xref rid=\"B29\" ref-type=\"bibr\">Langmead and Salzberg, 2012</xref>). The reference genome and gene model annotation file of <italic>A. baumannii</italic> AB030 was downloaded from GenBank (NZ_CP009257.1). HTSeq v0.6.1 was used to count the read numbers mapped to each gene. The expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) of each gene was calculated based on the length of the gene and reads count mapped to this gene (<xref rid=\"B50\" ref-type=\"bibr\">Trapnell et al., 2012</xref>). Differential expression analysis of two groups (four biological replicates per group) was performed using the DEGSeq R package (??) (<xref rid=\"B6\" ref-type=\"bibr\">Anders and Huber, 2012</xref>). The resulting <italic>p</italic>-values were adjusted using the Benjamini and Hochberg approach for controlling the false discovery rate. Genes with a corrected <italic>p</italic>-value < 0.05 and log<sub>2</sub>(Fold change)>1 found by DEGSeq were assigned as differentially expressed. Gene ontology (GO) enrichment analysis of differentially expressed genes was implemented by the GOseq R package. The GO enrichment analysis of differentially expressed genes was achieved by GOseq software, and KOBAS software was used to test the statistical enrichment of differentially expressed genes in KEGG pathways (<xref rid=\"B27\" ref-type=\"bibr\">Kanehisa et al., 2008</xref>; <xref rid=\"B56\" ref-type=\"bibr\">Young et al., 2010</xref>). Significantly enriched KEGG pathways and GO terms were identified by a <italic>p</italic>-value < 0.05 using Fisher’s exact test and a <italic>p</italic>-value < 0.01 in the hypergeometric distribution, respectively, and adjusted by false discovery rates (FDR) (<xref rid=\"B45\" ref-type=\"bibr\">Rivals et al., 2007</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Boca and Leek, 2018</xref>).</p></sec><sec id=\"S2.SS10.SSS3\"><title>Statistical Analysis</title><p>Statistical analysis was performed using GraphPad Prism software version 6.0 (Graph Pad Software, San Diego, CA, United States) and Student’s <italic>t</italic>-test. Data are expressed as mean ± standard deviation (SD). All experiments were performed in triplicate. A <italic>p</italic>-value < 0.05 was considered statistically significant.</p></sec></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Cec4 Inhibits Clinical Resistant Bacteria</title><p>It was reported that large differences have been shown between <italic>A. baumannii</italic> strains. In previous studies, Cec4 showed outstanding antibacterial activity against <italic>A. baumannii</italic>. Its MIC against standard <italic>A. baumannii</italic>(ATCC19606)was 4 μg/ml. In order to study its antibacterial effect on clinical CRAB, the susceptibility of CRAB to Cec4 was tested. As shown in <xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>, 98.5% of the 200 isolates of CRAB were susceptive to the peptide Cec4 with a MIC ≤ 4 μg/ml. Only 1.5% of the strains had MIC > 4 μg/ml, which were 8 and 16 μg/ml, respectively (<xref ref-type=\"supplementary-material\" rid=\"TS2\">Supplementary Table S2</xref>). Therefore, the peptide Cec4 has great antibacterial effects on the majority of clinical carbapenem resistant bacteria.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Cec4 inhibits clinical resistant bacteria and biofilms. <bold>(A)</bold> Distribution of MIC value of Cec4 against 200 CRAB clinical isolates (μg/mL). <bold>(B)</bold> Quantification of biofilm formation in 200 CRAB clinical isolates. <italic>A. baumannii</italic> ATCC19606 was used as a positive control. Experiments were performed in triplicate and each bar represents the mean ± standard deviation. <bold>(C)</bold> The effects of Cec4 on mature biofilms of CRAB. The adherent biofilm was stained by crystal violet, and then the dye was extracted with ethanol, measured at a 570-nm absorbance, and presented as percentage of biofilm remains compared to untreated wells (0 μg/mL). All experiments were done in triplicate for statistical significance. *<italic>p</italic> < 0.05; **<italic>p</italic> < 0.01.</p></caption><graphic xlink:href=\"fmicb-11-01532-g001\"/></fig></sec><sec id=\"S3.SS2\"><title>The Biofilm Formation Ability of CRAB Strains</title><p>Bacteria in the form of biofilms have increased resistance to antibacterial drugs, external environmental pressures, and the host’s immune system, which has brought great challenges to clinical treatment. Based on crystal violet staining, the ability of 200 isolates of CRAB to form biofilms was studied. As shown in <xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>, the OD570 value of the positive control strain ATCC19606 in the experimental plate was 1.191 ± 0.012, and the negative control was 0.111 ± 0.005. The OD570 value of the 200 resistant bacteria was between 0.1 and 1.9 (<xref ref-type=\"supplementary-material\" rid=\"TS2\">Supplementary Table S2</xref>). All 200 strains of CRAB were able to form biofilms. Among them, 53 strains (26.5%) had strong biofilm formation ability, 120 strains (60%) were moderate and 27 strains (13.5%) were weak.</p></sec><sec id=\"S3.SS3\"><title>Cec4 Inhibits and Eradicates Biofilm of CRAB Strains</title><p>After bacteria formed the biofilm, their resistance to drugs was greatly enhanced. Our previous studies have demonstrated that antimicrobial peptide Cec4 at 0.5 μg / mL can inhibit the formation of <italic>A. baumannii</italic> biofilm (<xref rid=\"B40\" ref-type=\"bibr\">Peng et al., 2019</xref>). Regarding biofilm formation ability, the 10 strongest strains were selected to evaluate the ability of Cec4 to inhibit and eradicate their biofilm. As shown in <xref ref-type=\"supplementary-material\" rid=\"TS2\">Supplementary Table S2</xref>, the antimicrobial peptide Cec4 can inhibit the biofilm of the strains with the strongest CRAB biofilm formation ability at 64–128 μg/ml, and can eradicate the biofilm at 256–512 μg/ml. In order to determine the effect of Cec4 on the removal of biofilms, after the formation of mature biofilms, different concentrations of Cec4 were added to the culture for 24 h. The crystal violet staining method was used to measure the absorbance at 570 nm to calculate the clearance rate of different concentrations of the peptide on mature biofilms. The results showed that 1 × MIC (4 μg/ml) could clear more than 20% of mature biofilms, with MBEC<sub>50</sub> of 16 μg/ml and MBEC<sub>80</sub> of 128 μg/ml (<xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref>).</p></sec><sec id=\"S3.SS4\"><title>Cec4 Destroys the Structure of Biofilms</title><p><italic>Acinetobacter baumannii</italic> colonizes the surface of medical equipment and indwelling medical devices (including urinary catheters) to form biofilms, leading to long-term and recurrent infections in patients (<xref rid=\"B31\" ref-type=\"bibr\">Lin et al., 2019</xref>). Coverslips and urinary catheters were used as carriers to evaluate the ability of Cec4 to remove the biofilms formed on them. Clinical CRAB forms biofilm on coverslips and catheters, and the effects of Cec4 on the biofilm are shown in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>. After 24 h in culture, for the untreated group, the <italic>A. baumannii</italic> that adhered to the coverslip mostly was bacilliform; the biofilm was dense and the cell membrane was intact, forming a typical “mushroom cloud” three-dimensional structure. After treatment with Cec4 at a concentration of 1 × MIC (4 μg/ml), the biofilm structure was destroyed, and the cells were loosely distributed, and some cell surfaces appeared to be attacked. <italic>A. baumannii</italic> adhered to the catheter was mostly spherical and formed a thick biofilm, covered with extracellular substrates. After treatment with Cec4 (4 μg/ml), the biofilm structure was destroyed. Moreover, small vesicles appeared on the surface of the cell membrane, and even the collapse and disintegration of cells were observed.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Ultrastructural images of CRAB biofilm on sterile coverslip and catheters after treated with Cec4. <bold>(A)</bold> Coverslip without treatment, <bold>(B)</bold> Coverslip after Cec4 treatment, <bold>(C)</bold> catheter without treatment, and <bold>(D)</bold> catheter after Cec4 treatment. The selected images are the best representation of biofilms on coverslips and catheters.</p></caption><graphic xlink:href=\"fmicb-11-01532-g002\"/></fig><p>To better understand the destructive effect of Cec4 on the biofilm, the fluorescent dyes SYTO<sup>®</sup>9 and propidium iodide were used to characterize bacteria in different states. Under the laser confocal scanning microscope, a large number of biofilm bacteria were aggregated into the control group, mainly living bacteria with green fluorescence (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). The total biomass was about 9 × 10<sup>6</sup> μm<sup>3</sup> and the average biofilm thickness was > 16 μm. The number of dead bacteria increased gradually after treated with Cec4 (4 μg/ml) for 2 h. It was found that many bacteria in the visual field emitted red fluorescence. The total biomass was about 4 × 10<sup>6</sup> μm<sup>3</sup>, and the thickness of the biofilm was reduced to 7 μm. Therefore, the CLSM results are consistent with the SEM observations, showing that Cec4 has a strong damaging effect on CRAB strain biofilm.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>The CRAB biofilm was removed effectively with Cec4. Confocal microscopy of CRAB biofilm without treatment <bold>(A)</bold> and after treatment with Cec4 <bold>(B)</bold>. The three-dimensional reconstruction of CRAB biofilm without treatment <bold>(C)</bold> and after treatment with Cec4 <bold>(D)</bold>. The total biomass volume <bold>(E)</bold> and thickness <bold>(F)</bold> of biofilm were calculated based on the fluorescence intensity. The results were averaged from three randomly selected positions of each sample. Data are expressed as mean ± standard deviation (SD). *<italic>p</italic> < 0.05; **<italic>p</italic> < 0.01.</p></caption><graphic xlink:href=\"fmicb-11-01532-g003\"/></fig></sec><sec id=\"S3.SS5\"><title>Cec4 Decreases Twitching Motility and Surface-Associated Motility in CRAB</title><p><italic>Acinetobacter baumannii</italic> adheres to the surface of the object through pili, which is the initial stage of biofilm formation. Twitching motility is a unique form of movement mediated by type IV pili. <italic>A. baumannii</italic> relies on the contraction and extension of type IV pili on the surface of the Petri dish, with the inoculation point as the center, and spreads around and forms interstitial colony expansion halo. We observed that, after incubation at 37°C overnight, two types of the colony grew on the Petri plate. There were colonies on the surface of the agar around the inoculation point (top colony) and a visible halo of bacteria that had twitched across the plate between the bottom of the agar and the plate (interstitial colony) (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1</xref>). Adding different concentrations of Cec4 decreased twitching motility. As the concentration of Cec4 increased, the twitching motility of bacteria decreased (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>). <italic>A. baumannii</italic> can also move on the surface of a semi-solid medium, forming round colonies at the inoculation site of the moving plate (<xref ref-type=\"supplementary-material\" rid=\"FS2\">Supplementary Figure S2</xref>). The average colony diameter of the group with Cec4 was lower than that of the control group, which was consistent with the experimental results on twitching motility (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>).</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>The motility of CRAB and biofilm involved genes were affected after treated with Cec4. Twitching motility <bold>(A)</bold> and surface-associated motility <bold>(B)</bold> of CRAB were quantitated from three separate experiments at 37°C. Error bars represent standard deviation of the mean. *<italic>p</italic> < 0.05; **<italic>p</italic> < 0.01; ***<italic>p</italic> < 0.001; ****<italic>p</italic> < 0.0001. <bold>(C)</bold> Effect of Cec4 on the expression levels of biofilm involved genes in CRAB strains. Error bars represent the standard deviations. **<italic>p</italic> < 0.01.</p></caption><graphic xlink:href=\"fmicb-11-01532-g004\"/></fig></sec><sec id=\"S3.SS6\"><title>Cec4 Alters the Expression of Biofilm-Related Genes</title><p>Many studies have shown that several genes of <italic>A. baumannii</italic> are involved in the formation of biofilms and adhesion to abiotic surfaces. In order to understand the effect of Cec4 on these genes, the RNA of biofilm bacteria under different conditions was extracted. Next, the effect of Cec4 on the expression of <italic>CsuE</italic>, <italic>BfmR</italic>, <italic>BfmS</italic>, <italic>AbaI</italic>, and <italic>Bap</italic> related to biofilm formation was evaluated using qRT-PCR (<xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>). The results show that, compared with the control group, the pilus-related gene <italic>CsuE</italic> was down-regulated 1.8-fold; the two-component regulatory system genes <italic>BfmR</italic> and <italic>BfmS</italic> were down-regulated 3.6-fold and 3.9-fold, respectively; the quorum-sensing regulatory gene <italic>AbaI</italic> was down-regulated 6.3-fold, and the biofilm-related gene <italic>Bap</italic> was down-regulated 2.6-fold. These results suggest that Cec4 inhibited the expression of these genes involved in biofilm formation.</p></sec><sec id=\"S3.SS7\"><title>Transcriptional Stress Response of CRAB Biofilm to Cec4</title><p>The RNA sequencing results revealed the differences in gene expression between ABF and control. There were 3403 genes co-expressed in these two samples; 40 genes were specificity expressed in ABF, and 6 genes were specificity expressed in control (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>). The volcano plots of the differentially expressed genes (DEGs) show that 185 genes were differentially expressed after Cec4 treatment, including 132 genes that were up-regulated (red) and 53 down-regulated genes (green) (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>).</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p><bold>(A)</bold> Venn diagram analysis of gene expression. The number in each circle represents the total number of genes that are expressed in each sample, and the overlapping part of circles indicates that the gene is co-expressed in both samples. <bold>(B)</bold> The volcano plots of the differentially expressed genes. Significantly differentially expressed genes were treated with red dots (up-regulated) or green dots (down-regulated). The abscissa represents fold change, and the ordinate represents statistical significance.</p></caption><graphic xlink:href=\"fmicb-11-01532-g005\"/></fig></sec><sec id=\"S3.SS8\"><title>Analysis of Differential Gene Expression</title><p>Transcriptome analysis revealed that the differential genes mainly included membrane protein-related genes, drug-resistant genes and pilus-related genes (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). The expression of the amino acid ABC transporter permease, ATP-binding proteins (IX87_RS02655, IX87_RS02660, and artP) in the ABC transport system was down-regulated 1.68-fold, 1.61-fold, and 1.66-fold; the MFS transporter (IX87_RS20020) was down-regulated 2.96-fold, and the EamA family transporter (IX87_RS12100) expression was down-regulated 2.29-fold (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). The expression of MBL fold metallo-hydrolase (IX87_RS19070) was down-regulated about 3-fold, ADC family cephalosporin-hydrolyzing class C beta-lactamase (IX87_RS08365) was down-regulated 1.27-fold; adeC/adeK/oprM family multidrug efflux complex outer membrane factor (IX87_RS16845) expression was up-regulated 1.37-fold (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). The protein CsuA (IX87_RS06910) was down-regulated 1.51-fold, while the expression of pili assembly chaperone (IX87_RS02530) and type 4 fimbria biogenesis proteins FimT (IX87_RS10830) was up-regulated 1.8 and 1.42-fold (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>The genes up-regulated or down-regulated response to Cec4.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Gene name</bold></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Description</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>log <sup>2</sup> Fold_change</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Corrected <italic>p</italic>-value</bold></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Membrane protein</bold></td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IX87_RS20020</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MFS transporter</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–2.96</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.87 × 10<sup>–4</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IX87_RS02655</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">amino acid ABC transporter permease</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.68</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.86 × 10<sup>–3</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">artP</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">amino acid ABC transporter ATP-binding protein</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.66</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.30 × 10<sup>–2</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IX87_RS02660</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">amino acid ABC transporter permease</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.61</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.77 × 10<sup>–3</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IX87_RS12100</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EamA family transporter</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–2.29</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.85 × 10<sup>–4</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Bacterial resistance</bold></td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IX87_RS19070</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MBL fold metallo-hydrolase</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–2.97</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.11 × 10<sup>–8</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IX87_RS08365</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ADC family cephalosporin-hydrolyzing class C beta-lactamase</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.86 × 10<sup>–2</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IX87_RS16845</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">adeC/adeK/oprM family multidrug efflux complex outer membrane factor</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.90 × 10<sup>–2</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Pili</bold></td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IX87_RS06910</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">protein CsuA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.51</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.64 × 10<sup>–3</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IX87_RS02530</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pili assembly chaperone</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.23 × 10<sup>–2</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IX87_RS10830</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">type 4 fimbrial biogenesis protein FimT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.63 × 10<sup>–2</sup></td></tr></tbody></table></table-wrap></sec><sec id=\"S3.SS9\"><title>Enrichment Analysis of GO and KEGG Pathways</title><p>To further understand the function of the DEGs underlying the effect of low concentrations of Cec4 on CRAB biofilms, GO enrichment analysis was performed with the DEGs (<xref ref-type=\"fig\" rid=\"F6\">Figure 6A</xref>). Based on sequence homology, DEGs were assigned to one or more GO terms and categorized into three main categories of GO function (biological process, molecular function, and cellular component). It is noteworthy that in the category of biological process, protein folding, extracellular polysaccharide biosynthetic process, cellular polysaccharide metabolic process, secondary metabolite biosynthetic process, protein catabolic process, dsRNA transport and regulation of DNA-templated transcription are significantly enriched. Outer membrane-bounded periplasmic space and pilus are significantly enriched in the cellular component category. Enzyme activity and nucleic acid transmembrane transporter activity are enriched in the molecular function category.</p><fig id=\"F6\" position=\"float\"><label>FIGURE 6</label><caption><p><bold>(A)</bold> Gene Ontology (GO) analysis of differentially expressed genes. <bold>(B)</bold> Statistical enrichment of differentially expressed genes in KEGG pathways.</p></caption><graphic xlink:href=\"fmicb-11-01532-g006\"/></fig><p>By enrichment analysis, the main KEGG metabolic pathways involved in DEGs are carbon metabolism, oxidative phosphorylation, the two-component system, ABC transporters, nucleotide excision repair, biosynthesis of antibiotics, quorum sensing, microbial metabolism in diverse environments, and β-lactam resistance (<xref ref-type=\"fig\" rid=\"F6\">Figure 6B</xref>). It is indicated that Cec4 affected the energy metabolism of biofilm bacteria and the bacteria two-component system. By interfering with the expression of type IV pili assembly protein in the two-component system, bacterial motility function was affected. In addition, the β-lactam antibiotics are the most widely used group of antibiotics, which exert their effect by interfering with the structural cross-linking of peptidoglycans in bacterial cell walls. Bacterial resistance to β-lactam antibiotics can be achieved by producing inactivating enzymes called β-lactamases, altering the β-lactam targets of penicillin-binding proteins (PBPs), and so on. After Cec4 treatment, genes in the β-lactam resistance pathway were down-regulated. More importantly, quorum sensing (QS) allows bacteria to share information about cell density and adjust gene expression accordingly, and controls including virulence, motility, sporulation and biofilm formation. Therefore, the transcriptome results indicate that Cec4 mainly affected the expression of energy metabolism and quorum sensing signaling pathway genes.</p></sec></sec><sec id=\"S4\"><title>Discussion</title><p>It has been reported that after bacteria form a biofilm, the MIC value for antibiotics can be increased by 1000 times or more (<xref rid=\"B4\" ref-type=\"bibr\">Alhede et al., 2011</xref>). The antimicrobial peptide can down-regulate quorum sensing, prevent the initial adhesion of bacteria to the surface, target bacteria before they form a biofilm, kill bacteria embedded in biofilms, or destroy mature biofilms to achieve the effect of inhibiting or eradicating biofilms (<xref rid=\"B9\" ref-type=\"bibr\">Batoni et al., 2016</xref>). Previous experiments have shown that Cec4 has the best antibacterial effect on <italic>A. baumannii</italic>, and it has very low hemolysis to human red blood cells even in high concentrations (600 μg/mL) (<xref rid=\"B40\" ref-type=\"bibr\">Peng et al., 2019</xref>). Our experiment results also indicated that Cec4 had little cytotoxic effect on the two human cell lines HepG2 and Hela (data not shown). In this study, 200 isolates of CRAB from different clinical patients were collected to detect their biofilm formation ability and to evaluate the inhibitory effect of Cec4 on biofilms. Screening at a concentration of 4 μg/ml revealed that 98.5% of the strains were susceptive to Cec4. Thus, Cec4 has a strong inhibitory activity against standard <italic>A. baumannii</italic> and can also inhibit clinical CRAB. The crystal violet staining method showed that the collected clinical CRAB could form biofilms, and 26.5% of the strains could strongly form a biofilm. Cec4 eradicated more than 20% of mature biofilms at a concentration of 4 μg/ml, and the MBEC<sub>50</sub> was 16 μg/ml. When treated with Cec4 with a concentration of 256–512 μg/ml, the biofilm was completely eradicated. The SEM results indicated that the biofilm formed on the medical silicone catheter was thick, with extracellular polysaccharides and other substrates that completely covered the cells. After Cec4 was treated for 2 h, the biofilm was easily detached, and the cells were damaged or collapsed. The CLSM results further quantified the thickness and volume of the biofilms. The results show that, after 24 h, the biofilm that formed on the coverslips was thick and dense, and mainly composed of live bacteria with a thickness of 10–20 μm. After treatment with Cec4, the number of dead bacteria gradually increased, and the biofilm became significantly thinner. Therefore, these results indicate that Cec4 has a good eradication ability on biofilm forming of clinical CRAB.</p><p>It has been reported that <italic>A. baumannii</italic> adheres to the surface of living or non-living organisms by pili participating in the initial stage of biofilm formation, and thereby forming a biofilm. The results show that Cec4 decreased pili-mediated motility in a concentration dependent manner. Under the action of Cec4 with a sub-inhibitory concentration (2 μg/ml), motility was also suppressed, and no transparent halo was formed. It has been reported that peptide 1037 inhibited the swimming of <italic>P. aeruginosa</italic> PA14, but stimulated twitching motility (<xref rid=\"B15\" ref-type=\"bibr\">de la Fuente-Nunez et al., 2012</xref>). The expression of the CsuA/BABCDE chaperone complex is necessary for the assembly and production of pili associated with abiotic surface adhesion, while the Csu operon is controlled by a two-component system consisting of a BfmS-encoded sensor kinase and a BfmR-encoded response regulator. Insertion inactivation of BfmR results in the loss of expression of the CsuA/BABCDE operon, and impaired pilus production and biofilm formation (<xref rid=\"B49\" ref-type=\"bibr\">Tomaras et al., 2008</xref>). The qRT-PCR results show that Cec4 reduced the expression of <italic>CsuE</italic>, <italic>BfmR</italic> and <italic>BfmS</italic> genes. Clinical isolates of <italic>A. baumannii</italic> M2 can produce N-acyl-homoserine lactone (3-OH-C12-HSL), a product of the <italic>abaI</italic> autoinducer synthetase gene, which is very important for the formation of complete biofilms on abiotic surfaces (<xref rid=\"B37\" ref-type=\"bibr\">Niu et al., 2008</xref>). Our research shows that Cec4 significantly reduced the expression of <italic>AbaI</italic>. Therefore, we speculate that Cec4 may affect quorum sensing in bacteria. In addition, <italic>A. baumannii</italic> encodes for a protein associated with the biofilm, Bap. <italic>A. baumannii</italic> strain 307–0294, mutations in large outer membrane proteins that are highly similar to <italic>Staphylococcus</italic> biofilm-associated protein (Bap) were lost, resulting in a reduction in the volume and thickness of the biofilm formed by the strain (<xref rid=\"B32\" ref-type=\"bibr\">Loehfelm et al., 2008</xref>). Cec4 reduced the expression of the <italic>Bap</italic> gene, which was consistent with its effect on the biofilm. In conclusion, Cec4 affects the expression of genes involved in the formation of <italic>A. baumannii</italic> biofilms, such as quorum-sensing and motility genes.</p><p>Through the neutralization or decomposition of LPS and the interference of gene expression, abnormal regulation of the genes for biofilm survival, thus inhibiting the formation of biofilm (<xref rid=\"B30\" ref-type=\"bibr\">Li et al., 2004</xref>). It has been reported that peptide 1018 can inhibit bacterial stress response via the (p)ppGpp signaling molecule (<xref rid=\"B44\" ref-type=\"bibr\">Reffuveille et al., 2014</xref>); however, there are few reports on the mechanism of inhibition and eradication of biofilm by antimicrobial peptides. To further investigate the mechanism of action of Cec4, we used RNA-Seq to study the transcriptomic profile of CRAB biofilms treated with Cec4. The RNA-Seq results show that gene expression of CRAB biofilms was extensively altered after Cec4 treatment. Compared with the control group, membrane proteins, bacterial resistance and pilus-related genes were differentially expressed. Iron ion as an important signal regulator mediates the expression of adhesion, and then affects the formation of biofilms (<xref rid=\"B19\" ref-type=\"bibr\">Gentile et al., 2014</xref>). Our results show that bacterial ferritin was down-regulated, iron ion uptake and storage-related gene expression were increased. Besides, drug efflux-related genes and ABC transporter-related genes expressions in iron-containing cells were up-regulated. GO terms were abundant in biological process categories, indicating that bacterial biological processes were significantly affected, including the synthesis and metabolism of polysaccharides and proteins. In addition, KEGG analysis showed that multiple metabolic pathways, two-component regulation systems, quorum sensing and antibiotic synthesis-related pathways in <italic>A. baumannii</italic> biofilms were affected after Cec4 treatment. Most importantly, the two-component signal transduction system regulates ABC transporters and type IV pili assembly proteins and participates in the growth and formation of biofilm bacteria (<xref rid=\"B55\" ref-type=\"bibr\">Xue et al., 2020</xref>). <xref rid=\"B51\" ref-type=\"bibr\">Whiteley et al. (2017)</xref> mentioned that quorum sensing is involved in the formation of biofilms. Cec4 may inhibit the growth of biofilms by affecting the quorum sensing signaling pathway. Therefore, the results show that Cec4 regulates the CRAB biofilm through multiple targets, and further experiments such as gene knockout verification are required to determine the key anti-biofilm mechanisms.</p><p>In summary, this study showed that Cec4 has good antibacterial activity against planktonic clinical CRAB and its biofilm. However, the biofilm formation of clinical strains has great differences. It is reported that resistant strains achieve high levels of biofilm-specific resistance despite producing weak biofilms (<xref rid=\"B41\" ref-type=\"bibr\">Qi et al., 2016</xref>). So, the epidemiological analysis of the 200 clinic CRAB strains is very important in understanding their genetic relationship. For example, it was shown that, of the cases of <italic>A. baumannii</italic> acquisition, at least 17% were cases of patient-to-patient transmission in the Intensive Care Unit (<xref rid=\"B23\" ref-type=\"bibr\">Harris et al., 2019</xref>). Furthermore, deeper explorations of epidemiologic studies, such as bacterial molecular typing, drug resistance, and virulence factors detecting clinical strains, would improve our understanding of their relationship. In conclusion, these results provide a new strategy for the treatment of clinical biofilm-related infections, and also lay the foundation for the development of antimicrobial peptides as new antibacterial drugs.</p></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>The sequencing data in the article have been deposited at the National Center for Biotechnology Information under BioProject <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"PRJNA607078\">PRJNA607078</ext-link> as <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN14120700\">SAMN14120700</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN14120701\">SAMN14120701</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN14120702\">SAMN14120702</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN14120703\">SAMN14120703</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN14120704\">SAMN14120704</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN1412\">SAMN1412</ext-link> 0705, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN14120706\">SAMN14120706</ext-link>, and <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SAMN14120707\">SAMN14120707</ext-link> (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/sra/PRJNA607078\">https://www.ncbi.nlm.nih.gov/sra/PRJNA607078</ext-link>).</p></sec><sec id=\"S6\"><title>Author Contributions</title><p>ZW and WL conceived and designed the experiments. ZW, WL, and CM performed the experiments. WL, ZW, ZZ, and GG analyzed the data. YF, SW, and ZZ contributed materials and analysis tools. JP and JW wrote the manuscript. All authors analyzed the data and contributed to the manuscript, gave final approval of the version to be published, and agreed to be accountable for all aspects of the work.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This research was funded by the National Natural Science Foundation of China (No. 81660347), the Guizhou Provincial Science and Technology Plan Project ([2017]1154), and the Key Technologies R&D Program for Science and Technology Department of Guizhou Province ([2019]2823) is co-financed. Funders had no role in study design, data collection or analysis, preparation of the manuscript or the decision to publish it. We also thank proof-reading-service for proofreading the manuscript (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.proof-reading-service.com\">www.proof-reading-service.com</ext-link>).</p></fn></fn-group><fn-group><fn id=\"footnote1\"><label>1</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/sra/PRJNA607078\">https://www.ncbi.nlm.nih.gov/sra/PRJNA607078</ext-link></p></fn></fn-group><sec id=\"S8\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fmicb.2020.01532/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fmicb.2020.01532/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"FS1\"><label>FIGURE S1</label><caption><p>Images of twitching motility after treated with Cec4. From left to right <bold>(A–E)</bold>, the concentration of Cec4 is 0, 2, 4, 8, 16 μg/ml.</p></caption><media xlink:href=\"Image_1.tif\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS2\"><label>FIGURE S2</label><caption><p>Images of surface-associated motility after treated with Cec4. From left to right <bold>(A–E)</bold>, the concentration of Cec4 is 0, 2, 4, 8, 16 μg/ml.</p></caption><media xlink:href=\"Image_2.tif\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS1\"><label>TABLE S1</label><caption><p>Oligonucleotide primers used in this study.</p></caption><media xlink:href=\"Table_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS2\"><label>TABLE S2</label><caption><p>The MICs, MBIC, MBEC, sample source and drug susceptibility of 200 CRAB strains.</p></caption><media xlink:href=\"Table_2.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abouelhassan</surname><given-names>Y.</given-names></name><name><surname>Yang</surname><given-names>Q.</given-names></name><name><surname>Yousaf</surname><given-names>H.</given-names></name><name><surname>Nguyen</surname><given-names>M. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Bioeng Biotechnol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Bioeng Biotechnol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Bioeng. Biotechnol.</journal-id><journal-title-group><journal-title>Frontiers in Bioengineering and Biotechnology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-4185</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850767</article-id><article-id pub-id-type=\"pmc\">PMC7431630</article-id><article-id pub-id-type=\"doi\">10.3389/fbioe.2020.00949</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Bioengineering and Biotechnology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Estimation of Trunk Muscle Forces Using a Bio-Inspired Control Strategy Implemented in a Neuro-Osteo-Ligamentous Finite Element Model of the Lumbar Spine</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Sharifzadeh-Kermani</surname><given-names>Alireza</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/992330/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Arjmand</surname><given-names>Navid</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/992023/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Vossoughi</surname><given-names>Gholamreza</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Shirazi-Adl</surname><given-names>Aboulfazl</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1048157/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Patwardhan</surname><given-names>Avinash G.</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Parnianpour</surname><given-names>Mohamad</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/816432/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Khalaf</surname><given-names>Kinda</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/198416/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Mechanical Engineering, Sharif University of Technology</institution>, <addr-line>Tehran</addr-line>, <country>Iran</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Division of Applied Mechanics, Department of Mechanical Engineering, Polytechnique Montréal</institution>, <addr-line>Montreal, QC</addr-line>, <country>Canada</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Musculoskeletal Biomechanics Laboratory, Edward Hines, Jr. VA Hospital</institution>, <addr-line>Hines, IL</addr-line>, <country>United States</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Biomedical Engineering, Khalifa University of Science and Technology</institution>, <addr-line>Abu Dhabi</addr-line>, <country>United Arab Emirates</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Jerome Noailly, Pompeu Fabra University, Spain</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Rizwan Arshad, Royal Military College of Canada, Canada; Fabio Galbusera, Galeazzi Orthopedic Institute (IRCCS), Italy</p></fn><corresp id=\"c001\">*Correspondence: Navid Arjmand, <email>arjmand@sharif.edu</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Biomechanics, a section of the journal Frontiers in Bioengineering and Biotechnology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>949</elocation-id><history><date date-type=\"received\"><day>29</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>23</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Sharifzadeh-Kermani, Arjmand, Vossoughi, Shirazi-Adl, Patwardhan, Parnianpour and Khalaf.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Sharifzadeh-Kermani, Arjmand, Vossoughi, Shirazi-Adl, Patwardhan, Parnianpour and Khalaf</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Low back pain (LBP), the leading cause of disability worldwide, remains one of the most common and challenging problems in occupational musculoskeletal disorders. The effective assessment of LBP injury risk, and the design of appropriate treatment modalities and rehabilitation protocols, require accurate estimation of the mechanical spinal loads during different activities. This study aimed to: (1) develop a novel 2D beam-column finite element control-based model of the lumbar spine and compare its predictions for muscle forces and spinal loads to those resulting from a geometrically matched equilibrium-based model; (2) test, using the foregoing control-based finite element model, the validity of the follower load (FL) concept suggested in the geometrically matched model; and (3) investigate the effect of change in the magnitude of the external load on trunk muscle activation patterns. A simple 2D continuous beam-column model of the human lumbar spine, incorporating five pairs of Hill’s muscle models, was developed in the frontal plane. Bio-inspired fuzzy neuro-controllers were used to maintain a laterally bent posture under five different external loading conditions. Muscle forces were assigned based on minimizing the kinematic error between target and actual postures, while imposing a penalty on muscular activation levels. As compared to the geometrically matched model, our control-based model predicted similar patterns for muscle forces, but at considerably lower values. Moreover, irrespective of the external loading conditions, a near (<3°) optimal FL on the spine was generated by the control-based predicted muscle forces. The variation of the muscle forces with the magnitude of the external load within the simulated range at the L1 level was found linear. This work presents a novel methodology, based on a bio-inspired control strategy, that can be used to estimate trunk muscle forces for various clinical and occupational applications toward shedding light on the ever-elusive LBP etiology.</p></abstract><kwd-group><kwd>spine</kwd><kwd>model</kwd><kwd>controller</kwd><kwd>muscle force</kwd><kwd>follower load</kwd><kwd>stability</kwd></kwd-group><counts><fig-count count=\"7\"/><table-count count=\"3\"/><equation-count count=\"10\"/><ref-count count=\"51\"/><page-count count=\"12\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Low back pain (LBP) as the leading cause for work loss and years lived with disability emerges also as the most common and costliest problem in occupational musculoskeletal disorders (<xref rid=\"B7\" ref-type=\"bibr\">Clark and Horton, 2018</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Hartvigsen et al., 2018</xref>). In the United States alone, the annual cost of LBP was estimated at ∼$200 billion in 2006 (<xref rid=\"B27\" ref-type=\"bibr\">Katz, 2006</xref>). This asserts the important role of biomechanical investigations to mitigate and manage the associated risk of injury through quantitative assessment of the mechanical loads on the spine during various daily and occupational activities. In the absence of adequate non-invasive <italic>in vivo</italic> measurement techniques, a number of musculoskeletal spine models, with different degrees of complexities, have been developed to estimate the internal loads in the active-passive structures of the trunk (<xref rid=\"B10\" ref-type=\"bibr\">Dreischarf et al., 2016</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Ghezelbash et al., 2020</xref>). Due to the large number of trunk muscles spanning the intervertebral joints, the available equations are insufficient to solve this mechanically indeterminate system toward a unique solution, i.e., joint kinetics redundancy. The kinematic redundancies in the multi-joint spinal column, while providing flexibility in performing a specific task, add further complexity to the motor control strategies (<xref rid=\"B38\" ref-type=\"bibr\">Parnianpour, 2013</xref>). They can be viewed as the abundance to manage the conflicting objectives due to alterations in the environmental conditions and/or changes in task demand priorities (<xref rid=\"B28\" ref-type=\"bibr\">Latash et al., 2010</xref>).</p><p>Two distinct approaches are generally used to resolve the redundancies in such musculoskeletal models: inverse (e.g., equilibrium- and equilibrium-stability-based) and forward (e.g., control-based) dynamic. Equilibrium-based models leverage the available kinematics and governing equilibrium equations at various levels/joints/directions, and employ an optimization algorithm [often combined with limited recording of surface muscle electromyography (EMG)], to compute muscle forces and internal loads (<xref rid=\"B6\" ref-type=\"bibr\">Cholewicki and McGill, 1996</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Parnianpour et al., 1997</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Sparto and Parnianpour, 1998</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Gagnon et al., 2011</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Mohammadi et al., 2015</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Dreischarf et al., 2016</xref>). In these models, the system, maintains equilibrium (static and/or dynamic) with no attention to crucial stability requirements. Imposing stability, in addition to the equilibrium, has led to the development of multi-criteria equilibrium-stability-based models, in which the kinetics redundancy can once again be resolved either by using an optimization/control theory-based algorithm (<xref rid=\"B23\" ref-type=\"bibr\">Hemami and Katbab, 1982</xref>; <xref rid=\"B16\" ref-type=\"bibr\">Granata and Orishimo, 2001</xref>; <xref rid=\"B51\" ref-type=\"bibr\">Zeinali-Davarani et al., 2008</xref>; <xref rid=\"B49\" ref-type=\"bibr\">Vakilzadeh et al., 2011</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Hajihosseinali et al., 2014</xref>), or an EMG-driven algorithm (<xref rid=\"B41\" ref-type=\"bibr\">Samadi and Arjmand, 2018</xref>). The stability criterion in these models is typically investigated through the positive definiteness of the Hessian matrix of the system’s potential energy (<xref rid=\"B8\" ref-type=\"bibr\">Crisco and Panjabi, 1991</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Cholewicki and McGill, 1996</xref>; <xref rid=\"B46\" ref-type=\"bibr\">Shirazi-Adl et al., 2005</xref>), or equivalently by the eigenvalues of the dynamic system (<xref rid=\"B23\" ref-type=\"bibr\">Hemami and Katbab, 1982</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Bazrgari et al., 2008</xref>, <xref rid=\"B3\" ref-type=\"bibr\">2009</xref>; <xref rid=\"B51\" ref-type=\"bibr\">Zeinali-Davarani et al., 2008</xref>; <xref rid=\"B45\" ref-type=\"bibr\">Shahvarpour et al., 2015</xref>). In general, considering stability requirements when calculating trunk muscle forces yields stronger correlation between predicted muscle activation and experimentally measured EMG data (<xref rid=\"B16\" ref-type=\"bibr\">Granata and Orishimo, 2001</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Hajihosseinali et al., 2014</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Samadi and Arjmand, 2018</xref>).</p><p>Unlike the inverse dynamics approaches, forward control-based dynamic models assign forces to muscles, either individually or synergistically grouped, in alignment with the central nervous system’s (CNS) neural control strategies applied in trunk movements. The controller used in these models commonly adjusts muscle forces in search of target postural trajectories, while maintaining dynamic equilibrium and stability requirements (<xref rid=\"B9\" ref-type=\"bibr\">Dariush et al., 1998</xref>). Predictions of control-based models have been successfully validated against EMG data (<xref rid=\"B43\" ref-type=\"bibr\">Sedighi et al., 2011</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Nasseroleslami et al., 2014</xref>). Due to the challenging geometrical complexity and intricate multi-joint structures in the human trunk, previous control-based models have mainly simplified the upper trunk as an inverted pendulum with a single ball-and-socket (spherical) joint fixed at its base [i.e., the lumbosacral (L5/S1) junction]. This approach neglects relative deformations at the upper levels, translational degrees of freedom (DoFs), and changes in the centers of rotation (CoRs) under varying motions/loading conditions (<xref rid=\"B33\" ref-type=\"bibr\">Nasseroleslami et al., 2014</xref>). Recent investigations have demonstrated the variable effects of both the joint positioning (<xref rid=\"B14\" ref-type=\"bibr\">Ghezelbash et al., 2018</xref>) and joint translational DoFs (<xref rid=\"B5\" ref-type=\"bibr\">Cashaback et al., 2013</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Ghezelbash et al., 2015</xref>) on the kinematics, as well as, muscle forces and spinal loads. While a control-based model of the whole body is used to provide more geometrical details, it is based on multi-body simulations of the spine thus neglecting the intervertebral joint complexities (<xref rid=\"B40\" ref-type=\"bibr\">Rupp et al., 2015</xref>). Up to date, however, only one control-based FE model of the entire body included translational DoFs (with movement restricted to the sagittal plane), while using a simplistic proportional-integral-derivative (PID) controller, to determine muscle activations (<xref rid=\"B36\" ref-type=\"bibr\">Östh, 2010</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Andersson, 2013</xref>). To provide more geometrical details, deformable elements, based on fitting a curve on the forces and moments previously obtained by a finite element model of the intervertebral disc, were added to the multi-body model of the lumbar spine; again neglecting the intervertebral joint complexities (<xref rid=\"B26\" ref-type=\"bibr\">Karajan et al., 2013</xref>, <xref rid=\"B25\" ref-type=\"bibr\">2014</xref>; <xref rid=\"B40\" ref-type=\"bibr\">Rupp et al., 2015</xref>). Moreover, a previous study included active muscle models in a reduced musculoskeletal finite element model of the lumbar spine to explore possible functional relationships between muscle function and intervertebral disc condition (<xref rid=\"B48\" ref-type=\"bibr\">Toumanidou and Noailly, 2015</xref>).</p><p>The objectives of the present study are as follows:</p><p>(1)To develop a novel 2D beam-column control-based model of the lumbar spine and compare its muscle force predictions with an existing geometrically matched equilibrium-based model (<xref rid=\"B39\" ref-type=\"bibr\">Patwardhan et al., 2001</xref>). The model incorporate (1) the DoFs at all levels of the spine [via implementing the controller in a finite element model of plant (passive spine)] thus also approximating changes in the joint CoRs, (2) force-length and force-velocity relationships in muscles using a Hill-based muscle model (<xref rid=\"B50\" ref-type=\"bibr\">Zajac, 1989</xref>), and (3) a bio-inspired control strategy to estimate muscle forces using fuzzy neuro-controllers with an emotional learning algorithm that adequately mimics the adaptive mechanism of the CNS. The controller minimizes kinematic deviations between actual and target postures, while calculating muscle activations by penalizing the controller unit for muscle activation level (<xref rid=\"B33\" ref-type=\"bibr\">Nasseroleslami et al., 2014</xref>).</p><p>(2)To investigate, using the foregoing control-based finite element model, the follower load (FL) concept as suggested in the geometrically matched equilibrium-based model (<xref rid=\"B39\" ref-type=\"bibr\">Patwardhan et al., 2001</xref>). In that model, the muscle forces were estimated based on the premise that the resultant compressive load on the spine behaves as a follower load (FL) (i.e., a load that follows the curvature of the lumbar spine, at all lumbar levels and postures), thus providing inherent spinal stability, as observed in <italic>in vitro</italic> studies. This strategy implicitly leverages the stability requirement by minimizing horizontal translations/rotations along the spine. We hypothesize that our control strategy (selected to mimic the role of the CNS in resolving the kinetic redundancy) automatically leads to trunk muscle forces consistent with a FL on the spine, thereby maximizing the mechanical stability of the spine. This suggests that the controller used in our model would learn to activate muscles in a manner that not only minimizes the kinematic deviations, but also the destabilizing shear forces and moments.</p><p>(3)To investigate the effect of external load magnitude on the trunk muscle activation patterns. It is hypothesized that the predicted pattern of muscle activation is scaled with the external load magnitude, thus providing evidence for a synergistic activation.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Geometry and Musculature of the Lumbar Spine Model</title><p>For the sake of comparison and hypothesis testing, the geometry of our deformable beam-column model of the lumbar spine and musculature were selected to be identical to those introduced in a previous work (<xref rid=\"B39\" ref-type=\"bibr\">Patwardhan et al., 2001</xref>). A simple 2D model of the lumbar spine, as a continuous elastic beam-column in the frontal plane, was constructed in LS-DYNA<sup>®</sup> (Livermore Software Technology Corporation, Livermore, CA, United States) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Five distinct pairs of muscles were attached to a fixed base (representing the pelvis/sacrum) of the deformable beam at various L1–L5 lumbar levels. The simulation at the steady-state condition was quasi-static; upper body masses and inertias were hence neglected. The gravitational effect of masses was, however, accounted for by either a concentrated force at the L1 or distributed forces at various nodes (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). The exact beam geometry, flexural rigidity (EI = 1.9 Nm<sup>2</sup>), and coordinates of upper/lower muscle insertions were all adopted from earlier work (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Patwardhan et al., 2001</xref>). The cross sectional area of the column was assumed constant at 1225 mm<sup>2</sup>. The model was fixed at the sacrum (lower node) and restricted elsewhere to solely move in the frontal plane. The lumbar spine model consisted of five Hughes-Liu beam elements. The Hughes-Liu beam is a degenerated 8-node solid element (linear displacement and rotation field) with high computational efficiency and robustness (<xref rid=\"B19\" ref-type=\"bibr\">Hallquist, 2006</xref>). Sensitivity of the model predictions to the number of beam elements in the model (i.e., mesh refinement) was verified.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Geometry and musculature of the lumbar spine model in a laterally flexed posture in the frontal plane (<xref rid=\"B39\" ref-type=\"bibr\">Patwardhan et al., 2001</xref>).</p></caption><graphic xlink:href=\"fbioe-08-00949-g001\"/></fig><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Simulation cases.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Loading case</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>P</italic><sub><italic>1</italic></sub> (N)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>P</italic><sub><italic>2</italic></sub> (N)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>P</italic><sub><italic>3</italic></sub> (N)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>P</italic><sub><italic>4</italic></sub> (N)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>P</italic><sub><italic>5</italic></sub> (N)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>D (mm)</bold></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">635</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">350</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">75</td></tr></tbody></table><table-wrap-foot><attrib><italic><italic><italic>P</italic><sub><italic>i</italic></sub> represents the gravitational force of the upper trunk acting on the lumbar vertebra <italic>L</italic><sub><italic>i</italic></sub>. Gravitational loads increase from zero to <italic>P</italic><sub><italic>i</italic></sub> during 0.2 s. D represent the lateral distance of muscle origins.</italic></italic></attrib></table-wrap-foot></table-wrap><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Nodal coordinates of the deformed lumbar spine model (see <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>L1</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>L2</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>L3</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>L4</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>L5</bold></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">X (mm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">190</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">150</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">105</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">38</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Z (mm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.4</td></tr></tbody></table></table-wrap></sec><sec id=\"S2.SS2\"><title>Hill’s Muscle Model</title><p>A Hill muscle model (<xref rid=\"B50\" ref-type=\"bibr\">Zajac, 1989</xref>) is used as follows:</p><disp-formula id=\"S2.E1\"><label>(1)</label><mml:math id=\"M1\"><mml:mrow><mml:mi>F</mml:mi><mml:mo>=</mml:mo><mml:msub><mml:mi>f</mml:mi><mml:mi mathvariant=\"italic\">max</mml:mi></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mi mathvariant=\"normal\">α</mml:mi><mml:mo>.</mml:mo><mml:msub><mml:mi>f</mml:mi><mml:mi>l</mml:mi></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mi>l</mml:mi><mml:mo>)</mml:mo></mml:mrow><mml:mo>.</mml:mo><mml:msub><mml:mi>f</mml:mi><mml:mi>v</mml:mi></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mover accent=\"true\"><mml:mi>l</mml:mi><mml:mo>.</mml:mo></mml:mover><mml:mo>)</mml:mo></mml:mrow><mml:mo>+</mml:mo><mml:msub><mml:mi>f</mml:mi><mml:mi>p</mml:mi></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mi>l</mml:mi><mml:mo>)</mml:mo></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:math></disp-formula><disp-formula id=\"S2.E2\"><label>(2)</label><mml:math id=\"M2\"><mml:mrow><mml:mrow><mml:msub><mml:mpadded lspace=\"2.8pt\" width=\"+2.8pt\"><mml:mi>f</mml:mi></mml:mpadded><mml:mi>l</mml:mi></mml:msub><mml:mo>⁢</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mi>l</mml:mi><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mo>=</mml:mo><mml:mrow><mml:mrow><mml:mrow><mml:mrow><mml:mn>5.1</mml:mn><mml:mo>-</mml:mo><mml:mrow><mml:mn>29</mml:mn><mml:mo>⁢</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mfrac><mml:mi>l</mml:mi><mml:msub><mml:mi>l</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mfrac><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:mn>56</mml:mn><mml:mo>⁢</mml:mo><mml:msup><mml:mrow><mml:mo>(</mml:mo><mml:mfrac><mml:mi>l</mml:mi><mml:msub><mml:mi>l</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mfrac><mml:mo>)</mml:mo></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:mrow><mml:mo>-</mml:mo><mml:mrow><mml:mn>41</mml:mn><mml:mo>⁢</mml:mo><mml:msup><mml:mrow><mml:mo>(</mml:mo><mml:mfrac><mml:mi>l</mml:mi><mml:msub><mml:mi>l</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mfrac><mml:mo>)</mml:mo></mml:mrow><mml:mn>3</mml:mn></mml:msup></mml:mrow></mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:mn>10</mml:mn><mml:mo>⁢</mml:mo><mml:msup><mml:mrow><mml:mo>(</mml:mo><mml:mfrac><mml:mi>l</mml:mi><mml:msub><mml:mi>l</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mfrac><mml:mo>)</mml:mo></mml:mrow><mml:mn>4</mml:mn></mml:msup></mml:mrow></mml:mrow></mml:mrow></mml:math></disp-formula><disp-formula id=\"S2.E3\"><label>(3)</label><mml:math id=\"M3\"><mml:mrow><mml:mrow><mml:msub><mml:mi>f</mml:mi><mml:mi>v</mml:mi></mml:msub><mml:mo>⁢</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mover accent=\"true\"><mml:mi>l</mml:mi><mml:mo>.</mml:mo></mml:mover><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow><mml:mo>=</mml:mo><mml:mfrac><mml:mn>0.1433</mml:mn><mml:mrow><mml:mn>0.1074</mml:mn><mml:mo>+</mml:mo><mml:mrow><mml:mi>exp</mml:mi><mml:mo>⁡</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mo>-</mml:mo><mml:mrow><mml:mn>1.409</mml:mn><mml:mo>⁢</mml:mo><mml:mrow><mml:mi>sinh</mml:mi><mml:mo>⁡</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mrow><mml:mn>3.2</mml:mn><mml:mo>⁢</mml:mo><mml:mfrac><mml:mover accent=\"true\"><mml:mi>l</mml:mi><mml:mo>_</mml:mo></mml:mover><mml:msub><mml:mover accent=\"true\"><mml:mi>l</mml:mi><mml:mo>_</mml:mo></mml:mover><mml:mi mathvariant=\"italic\">max</mml:mi></mml:msub></mml:mfrac></mml:mrow><mml:mo>+</mml:mo><mml:mn>1.6</mml:mn></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:mrow></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula><disp-formula id=\"S2.E4\"><label>(4)</label><mml:math id=\"M4\"><mml:mrow><mml:mrow><mml:msub><mml:mi>f</mml:mi><mml:mi>p</mml:mi></mml:msub><mml:mo>⁢</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mi>l</mml:mi><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mo>=</mml:mo><mml:mrow><mml:mi>exp</mml:mi><mml:mo>⁡</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mrow><mml:mo>-</mml:mo><mml:mn>10.671</mml:mn></mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:mn>7.675</mml:mn><mml:mo>⁢</mml:mo><mml:mfrac><mml:mi>l</mml:mi><mml:msub><mml:mi>l</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:mfrac></mml:mrow></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:mrow></mml:math></disp-formula><p>In the above equations, <italic>F</italic>, <italic>f <sub>l</sub></italic> (<italic>l</italic>) (<xref rid=\"B34\" ref-type=\"bibr\">Nussbaum and Chaffin, 1998</xref>), f<sub>v</sub>⁢(l) (<xref rid=\"B22\" ref-type=\"bibr\">Hatze, 1977</xref>), and <italic>f</italic><sub><italic>p</italic></sub>(<italic>l</italic>) (<xref rid=\"B30\" ref-type=\"bibr\">McGill and Norman, 1986</xref>) are muscle force, as well as force-length, force-velocity and passive force-length relationships, respectively. Moreover, <italic>f<sub>max</sub></italic>, α, <italic>l</italic>, <inline-formula><mml:math id=\"INEQ14\"><mml:mover accent=\"true\"><mml:mi>l</mml:mi><mml:mo>.</mml:mo></mml:mover></mml:math></inline-formula>, <italic>l</italic><sub>0</sub>, <inline-formula><mml:math id=\"INEQ16\"><mml:msub><mml:mover accent=\"true\"><mml:mi>l</mml:mi><mml:mo>.</mml:mo></mml:mover><mml:mi mathvariant=\"italic\">max</mml:mi></mml:msub></mml:math></inline-formula> represent muscle maximum force, muscle activation level, muscle length, muscle velocity, muscle resting length and maximum muscle velocity, respectively. The value of <inline-formula><mml:math id=\"INEQ17\"><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>l</mml:mi><mml:mo>.</mml:mo></mml:mover><mml:mi mathvariant=\"italic\">max</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:msub><mml:mi>l</mml:mi><mml:mn>0</mml:mn></mml:msub><mml:mrow><mml:mn>0.1</mml:mn><mml:mo>⁢</mml:mo><mml:mi>s</mml:mi></mml:mrow></mml:mfrac></mml:mrow></mml:math></inline-formula> is assumed in this study (<xref rid=\"B50\" ref-type=\"bibr\">Zajac, 1989</xref>). <italic>f<sub>max</sub></italic> is assumed to be 800 N in all muscles. In muscles, the damping is represented intrinsically by the force-velocity relationship (Eq. 3), while the stiffness alters with the current length according to the force-length relationships (Eqs. 2 and 4). Inspection of the Hill type muscle response used (Eq. 1) reveals that the muscle activation affects the system response by modulating the muscle force and stiffness (<xref rid=\"B24\" ref-type=\"bibr\">Hogan, 1990</xref>). The spine structural stiffness matrix consists of contributions from both active and passive systems.</p></sec><sec id=\"S2.SS3\"><title>Controller</title><p>One single-input and single-output (SISO) controller for each muscle was used to control the continuous beam model. The main idea behind the control structure assumes that each pair of bilateral muscles attached to a particular point increases the active stiffness at that point. The SISO controller used in this study is a fuzzy neuro-controller whose weights are tuned according to two critic signals (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Lucas et al., 2004</xref>). The purpose of the controller is to minimize the general error function displayed below (Eq. 5) with the steepest descent algorithm:</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Feedback control loop with SISO fuzzy neuro-controller unit. C1 and C2 are the critics of the system that generate <inline-formula><mml:math id=\"INEQ2\"><mml:mrow><mml:msub><mml:mi>r</mml:mi><mml:mi>e</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mrow><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi></mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mo>⁢</mml:mo><mml:mover accent=\"true\"><mml:mi>e</mml:mi><mml:mo>.</mml:mo></mml:mover></mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mn>3</mml:mn></mml:msub><mml:mo>⁢</mml:mo><mml:mrow><mml:mo mathsize=\"90%\" stretchy=\"false\">∫</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>e</mml:mi></mml:mpadded></mml:mrow></mml:mrow></mml:mrow></mml:mrow></mml:math></inline-formula> and <italic>r</italic><sub>α</sub> = abs(α), respectively. α<sub><italic>j</italic></sub> is the level of activation of muscle (j) attached to <italic>L</italic><sub><italic>i</italic></sub> and <italic>e</italic> is the difference between desired (<italic>Z<sub>id</sub></italic>) and actual (<italic>Z</italic><sub><italic>i</italic></sub>) Z-coordinate of the node <italic>L</italic><sub><italic>i</italic></sub> (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p></caption><graphic xlink:href=\"fbioe-08-00949-g002\"/></fig><disp-formula id=\"S2.E5\"><label>(5)</label><mml:math id=\"M5\"><mml:mrow><mml:mi>E</mml:mi><mml:mo>=</mml:mo><mml:mrow><mml:msub><mml:mi>E</mml:mi><mml:mi>e</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>E</mml:mi><mml:mi mathvariant=\"normal\">α</mml:mi></mml:msub></mml:mrow><mml:mo>=</mml:mo><mml:mrow><mml:mrow><mml:mfrac><mml:mn>1</mml:mn><mml:mn>2</mml:mn></mml:mfrac><mml:mo>⁢</mml:mo><mml:msub><mml:mi>k</mml:mi><mml:mi>e</mml:mi></mml:msub><mml:mo>⁢</mml:mo><mml:msup><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi></mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mo>⁢</mml:mo><mml:mover accent=\"true\"><mml:mi>e</mml:mi><mml:mo>.</mml:mo></mml:mover></mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mn>3</mml:mn></mml:msub><mml:mo>⁢</mml:mo><mml:mrow><mml:mo mathsize=\"90%\" movablelimits=\"false\" stretchy=\"false\">∫</mml:mo><mml:mi>e</mml:mi></mml:mrow></mml:mrow></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:mfrac><mml:mn>1</mml:mn><mml:mn>2</mml:mn></mml:mfrac><mml:mo>⁢</mml:mo><mml:msub><mml:mi>k</mml:mi><mml:mi mathvariant=\"normal\">α</mml:mi></mml:msub><mml:mo>⁢</mml:mo><mml:msup><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi mathvariant=\"italic\">abs</mml:mi><mml:mo>⁢</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mi mathvariant=\"normal\">α</mml:mi><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:mrow></mml:mrow></mml:math></disp-formula><p>In the above equation, <italic>e</italic>, <inline-formula><mml:math id=\"INEQ20\"><mml:mover accent=\"true\"><mml:mi>e</mml:mi><mml:mo>.</mml:mo></mml:mover></mml:math></inline-formula>, and ∫<italic>e</italic>represent the error (difference between the actual and target kinematics), error rate, and the integral of error, where <italic>h</italic><sub>1</sub>, <italic>h</italic><sub>2</sub>, and <italic>h</italic><sub>3</sub> represent error, error rate and error integral coefficients. Moreover, <italic>k</italic><sub><italic>e</italic></sub> and <italic>k</italic><sub>α</sub> represent the weighting functions for the priority of the error signal components. α is the level of muscle activation (between 0 and 1). As can be seen in Eq. 5, the error function consists of two parts <italic>E<sub>e</sub></italic>and <italic>E</italic><sub>α</sub>,where<italic>E<sub>e</sub></italic>represents the kinematics error, while <italic>E</italic><sub>α</sub>penalizes the controller for the control activation signal and plays an essential role in resolving the system redundancy in terms of muscle forces (<xref rid=\"B33\" ref-type=\"bibr\">Nasseroleslami et al., 2014</xref>). The above cost function is defined for each muscle, where the error terms are based on the Z-coordinates of the nodes to which muscles are attached. Eq. (6), formulated below, is defined as the Jacobian of the SISO controller. In MIMO applications, it is necessary to calculate the exact value of the Jacobian. However, in SISO systems, only the sign of the Jacobian is sufficient for control (<xref rid=\"B33\" ref-type=\"bibr\">Nasseroleslami et al., 2014</xref>). The overall weight tuning rule can be calculated from Eq. 7.</p><disp-formula id=\"S2.E6\"><label>(6)</label><mml:math id=\"M6\"><mml:mrow><mml:mi>j</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mi>Z</mml:mi></mml:mrow><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi mathvariant=\"normal\">α</mml:mi></mml:mpadded></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula><disp-formula id=\"S2.E7\"><label>(7)</label><mml:math id=\"M7\"><mml:mrow><mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:mo>⁢</mml:mo><mml:msub><mml:mi>w</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mrow><mml:mo>=</mml:mo><mml:mrow><mml:mo>-</mml:mo><mml:mi>η</mml:mi></mml:mrow></mml:mrow><mml:mo>.</mml:mo><mml:mfrac><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mi>E</mml:mi></mml:mrow><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:msub><mml:mi>w</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mpadded></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula><p>Where <italic>w</italic><sub><italic>i</italic></sub> is the <italic>i</italic>th neuron weight of the neural controller and η (learning rate) represents the rate of change in weights. Finally, by using the chain derivative rule and combining the relevant equations, Eq. (7) is rewritten as Eq. (10):</p><disp-formula id=\"S2.E8\"><label>(8)</label><mml:math id=\"M8\"><mml:mrow><mml:mrow><mml:mfrac><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:msub><mml:mi>E</mml:mi><mml:mi>e</mml:mi></mml:msub></mml:mrow><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:msub><mml:mi>w</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mpadded></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:msub><mml:mi>E</mml:mi><mml:mi>e</mml:mi></mml:msub></mml:mrow><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:msub><mml:mi>r</mml:mi><mml:mi>e</mml:mi></mml:msub></mml:mpadded></mml:mrow></mml:mfrac></mml:mrow><mml:mo>.</mml:mo><mml:mfrac><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:msub><mml:mi>r</mml:mi><mml:mi>e</mml:mi></mml:msub></mml:mpadded></mml:mrow><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>Z</mml:mi></mml:mpadded></mml:mrow></mml:mfrac><mml:mo>.</mml:mo><mml:mfrac><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>Z</mml:mi></mml:mpadded></mml:mrow><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi mathvariant=\"normal\">α</mml:mi></mml:mpadded></mml:mrow></mml:mfrac><mml:mo>.</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi mathvariant=\"normal\">α</mml:mi></mml:mpadded></mml:mrow><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:msub><mml:mi>w</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mpadded></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:mrow><mml:mo>-</mml:mo><mml:msub><mml:mi>k</mml:mi><mml:mi>e</mml:mi></mml:msub></mml:mrow></mml:mrow><mml:mo>.</mml:mo><mml:msub><mml:mi>h</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mo>.</mml:mo><mml:msub><mml:mi>r</mml:mi><mml:mi>e</mml:mi></mml:msub><mml:mo>.</mml:mo><mml:mi>j</mml:mi><mml:mo>.</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mfrac><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi mathvariant=\"normal\">α</mml:mi></mml:mpadded></mml:mrow><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:msub><mml:mi>w</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mpadded></mml:mrow></mml:mfrac></mml:mpadded></mml:mrow></mml:math></disp-formula><disp-formula id=\"S2.E9\"><label>(9)</label><mml:math id=\"M9\"><mml:mrow><mml:mrow><mml:mfrac><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:msub><mml:mi>E</mml:mi><mml:mi mathvariant=\"normal\">α</mml:mi></mml:msub></mml:mrow><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:msub><mml:mi>w</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mpadded></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:msub><mml:mi>E</mml:mi><mml:mi mathvariant=\"normal\">α</mml:mi></mml:msub></mml:mrow><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:msub><mml:mi>r</mml:mi><mml:mi mathvariant=\"normal\">α</mml:mi></mml:msub></mml:mpadded></mml:mrow></mml:mfrac></mml:mrow><mml:mo>.</mml:mo><mml:mfrac><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:msub><mml:mi>r</mml:mi><mml:mi mathvariant=\"normal\">α</mml:mi></mml:msub></mml:mpadded></mml:mrow><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi mathvariant=\"normal\">α</mml:mi></mml:mpadded></mml:mrow></mml:mfrac><mml:mo>.</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi mathvariant=\"normal\">α</mml:mi></mml:mpadded></mml:mrow><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:msub><mml:mi>w</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mpadded></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:msub><mml:mi>k</mml:mi><mml:mi mathvariant=\"normal\">α</mml:mi></mml:msub></mml:mrow><mml:mo>.</mml:mo><mml:mrow><mml:mi mathvariant=\"italic\">sgn</mml:mi><mml:mo>⁢</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi mathvariant=\"normal\">α</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow><mml:mo>.</mml:mo><mml:msub><mml:mi>r</mml:mi><mml:mi mathvariant=\"normal\">α</mml:mi></mml:msub><mml:mo>.</mml:mo><mml:mfrac><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi mathvariant=\"normal\">α</mml:mi></mml:mpadded></mml:mrow><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:msub><mml:mi>w</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mpadded></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula><disp-formula id=\"S2.E10\"><label>(10)</label><mml:math id=\"M10\"><mml:mrow><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:msub><mml:mi>w</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mi>η</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:msub><mml:mi>k</mml:mi><mml:mi>e</mml:mi></mml:msub><mml:mo>.</mml:mo><mml:msub><mml:mi>h</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mo>.</mml:mo><mml:msub><mml:mi>r</mml:mi><mml:mi>e</mml:mi></mml:msub><mml:mo>.</mml:mo><mml:mi>j</mml:mi><mml:mo>-</mml:mo><mml:msub><mml:mi>k</mml:mi><mml:mi mathvariant=\"normal\">α</mml:mi></mml:msub><mml:mo>.</mml:mo><mml:msub><mml:mi>r</mml:mi><mml:mi mathvariant=\"normal\">α</mml:mi></mml:msub><mml:mo>)</mml:mo></mml:mrow><mml:mo rspace=\"5.3pt\">.</mml:mo><mml:mfrac><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi mathvariant=\"normal\">α</mml:mi></mml:mpadded></mml:mrow><mml:mrow><mml:mo>∂</mml:mo><mml:mo>⁡</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:msub><mml:mi>w</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mpadded></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula><p>In these equations, <inline-formula><mml:math id=\"INEQ31\"><mml:mrow><mml:msub><mml:mi>r</mml:mi><mml:mi>e</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mrow><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi></mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mo>⁢</mml:mo><mml:mover accent=\"true\"><mml:mi>e</mml:mi><mml:mo>.</mml:mo></mml:mover></mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mn>3</mml:mn></mml:msub><mml:mo>⁢</mml:mo><mml:mrow><mml:mo mathsize=\"90%\" stretchy=\"false\">∫</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>e</mml:mi></mml:mpadded></mml:mrow></mml:mrow></mml:mrow></mml:mrow></mml:math></inline-formula>and <italic>r</italic><sub>α</sub> = <italic>abs</italic>(α). <italic>h</italic><sub>1</sub>, <italic>h</italic><sub>2</sub>, <italic>h</italic><sub>3</sub>, <italic>k</italic><sub>α</sub>, are assumed as 2, 2, 2, and 0.2, respectively.<italic>k<sub>e</sub></italic> = 15, 7, 2.5, 1, and 0.1 for levels L1 through L5, respectively. Each muscle is considered as a SISO controller, thus the Jacobian sign would be adequate for control. Each controller-muscle unit minimizes the kinematic error of the node to which it is attached while minimizing its muscle activation. Initial muscle activations were neglected as the muscle forces were adjusted through a feedback strategy.</p></sec><sec id=\"S2.SS4\"><title>Simulations</title><p>A total of five simulations (loading cases 1–5), based on the external load distributions and lateral distances of the muscle origins, were considered in this study (<xref rid=\"B39\" ref-type=\"bibr\">Patwardhan et al., 2001</xref>; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Gravitational loads attained their values in 0.2 s. All simulation cases were modeled with and without the muscle/controller. Identical boundary conditions were considered for all simulated cases. The purpose of the controller in this study (i.e., target posture), was to maintain the primary Z-coordinates (minimize lateral deviations between the target and actual positions to remain bounded within 2 mm during the learning process) of the beam, as specified in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>. It is noteworthy that the foregoing restrictions on the lateral translations automatically limit any changes in the nodal lateral rotations. The vertical (X direction) displacement of the beam as well as the orientation of the vertebrae were left free to change under the external loads and muscle forces. In addition, in order to investigate the effect of the external load magnitude on the pattern of trunk muscle activations in loading case 2 (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>), the muscle forces were recalculated for different external vertical loads (150-750 N) applied at the L1 level.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Comparison With the Matched Equilibrium-Based Model</title><p>In the absence of any controller (i.e., without any muscle activation), the simulated system, expectedly, exhibited large deformations and became unstable. In all five loading cases (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>), the controllers in the model successfully learned, over time, to maintain the model close to the target kinematics at equilibrium under the estimated muscle exertions and applied external loads (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). The actual and target nodal Z-coordinates were different by <0.2 mm during the steady-state condition after 20 s. Initially in the transient period, when the controller was not fully trained, the model deviated slightly (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>) from its target kinematics, and the muscle forces substantially increased. By training the controller, the muscle forces subsequently considerably decreased, and the model reached its steady state. In all five loading conditions, the controllers unilaterally activated only one muscle at each level (i.e., no coactivation). As compared to the matched equilibrium-based model mentioned earlier (<xref rid=\"B39\" ref-type=\"bibr\">Patwardhan et al., 2001</xref>), our model predicted similar patterns for muscle forces (i.e., the muscles were activated unilaterally, and their forces decreased going downwards from the upper levels, although generally at lower values (RMSE = ∼41, 16, 12, 44, and 9 N for loading cases 1 through 5, with an overall normalized (to mean) RMSE of 121% for all loading cases) (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). Consequently, our control-based model predicted smaller compressive loads as compared to its matched equilibrium-based model (<xref rid=\"B39\" ref-type=\"bibr\">Patwardhan et al., 2001</xref>; <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). However, the equilibrium-based model predicted near zero shear loads in keeping with its own strategy to use the joint reaction forces as an FL (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Error, error rate, and muscle forces at different lumbar levels vs. time (up to 0.5 s) for the loading case 1 (with controllers and muscles). Gravitational loads increase from zero to <italic>P</italic><sub><italic>i</italic></sub> (see <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>) during 0.2 s. The controller tries to maintain the primary Z-coordinates of beam (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>) with a penalty on muscle activation level. For the sake of a clarified visualization, the horizontal axis is cut at 0.5 s while the convergence occurs at ∼20 s.</p></caption><graphic xlink:href=\"fbioe-08-00949-g003\"/></fig><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Predicted muscle forces in the current study (middle) as compared to those predicted by a matched equilibrium-based model (<xref rid=\"B39\" ref-type=\"bibr\">Patwardhan et al., 2001</xref>) <bold>(right)</bold> for different loading cases <bold>(left)</bold> (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p></caption><graphic xlink:href=\"fbioe-08-00949-g004\"/></fig><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>Predicted spinal loads (compression and shear) in the current control-based model as compared to those predicted by a matched equilibrium-based model (<xref rid=\"B39\" ref-type=\"bibr\">Patwardhan et al., 2001</xref>) for different loading cases (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" colspan=\"2\" rowspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\"><bold>Loading case 2</bold><hr/></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\"><bold>Loading case 3</bold><hr/></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\"><bold>Loading case 4</bold><hr/></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\"><bold>Loading case 5</bold><hr/></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Load (N)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Levels</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Control</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Equilibrium</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Control</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Equilibrium</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Control</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Equilibrium</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Control</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Equilibrium</bold></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Shear</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">L1–L2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−5.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−7.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−6.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−7.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.1</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">L2–L3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−5.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−5.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.8</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">L3–L4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.5</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">L4–L5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.2</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">L5–S1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−13.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−8.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−10.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−7.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Compression</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">L1–L2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">522</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">564</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">191</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">175</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">292</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">423</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">161</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">146</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">L2–L3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">619</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">638</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">325</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">300</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">433</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">541</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">285</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">267</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">L3–L4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">690</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">707</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">435</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">410</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">575</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">667</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">396</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">378</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">L4–L5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">752</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">774</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">547</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">526</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">701</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">802</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">507</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">491</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">L5–S1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">804</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">837</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">658</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">644</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">811</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">938</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">618</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">606</td></tr></tbody></table><table-wrap-foot><attrib><italic><italic>Negative shear forces are toward left (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Results for Loading case 1 are not reported in the equilibrium-based model; hence no comparison was made.</italic></italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS2\"><title>Follower Load (FL) Hypothesis</title><p>By minimizing the errors between the actual and target nodal Z-coordinates, the control-based model, predicted muscle forces that also generated a near FL on the spine (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>). Regardless of the loading case, the angle between the resultant force on the lumbar spine in our model and an optimal hypothetical FL remained <3°.</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>The absolute value of angle between the resultant force on the spine and a hypothetical follower load (FL).</p></caption><graphic xlink:href=\"fbioe-08-00949-g005\"/></fig></sec><sec id=\"S3.SS3\"><title>Effect of External Load on Muscle Activations</title><p>The controllers activated the trunk muscles unilaterally (no bilateral co-activation) regardless of the magnitude of the external loads (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>). Both models produced similar, although not identical activation patterns in the five loading cases. Variation of the muscle forces with the magnitude of external loading within the range simulated at the L1 was found linear.</p><fig id=\"F6\" position=\"float\"><label>FIGURE 6</label><caption><p>Predicted muscle forces for different external compressive loads acting on the L1 and the distributed loads of 50 N at the L2 to L5 similar to loading Case 2 (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). External force increases from zero to its final value in 0.2 s.</p></caption><graphic xlink:href=\"fbioe-08-00949-g006\"/></fig></sec></sec><sec id=\"S4\"><title>Discussion</title><p>This study developed a novel geometrically simple control-based model of the lumbar spine and compared its predictions for a slightly bent posture in the frontal plane with those of a geometrically matched equilibrium-based model (<xref rid=\"B39\" ref-type=\"bibr\">Patwardhan et al., 2001</xref>). Moreover, the FL concept suggested as an input constraint in the matched equilibrium-based model, as well as, the effect of changes in the externally applied loads on muscle forces, were investigated. The present learning algorithm is classified as a reinforcement-based one, where the controller tends to decrease the defined cost function based on the critic’s signals (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). The findings indicated that, similar to the equilibrium-based model, the fuzzy neuro-controllers balanced the spine at a given deformed posture using a unilateral muscle force pattern, albeit with generally smaller muscle forces (the sums of muscle forces in our model were smaller by ∼ 159, 40, −11, 132, and −8 N for loading cases 1 through 5, and hence, the L5-S1 compression forces were smaller by ∼ 145, 33, −14, 127, and −12 N for loading cases 1 through 5, respectively). This unilateral muscle activation pattern did not change with the variation of the magnitude of external loads (i.e., the spine was balanced by the controllers without bilateral coactivations). Moreover, for the loading conditions at a slightly laterally bent posture (i.e., quiet standing posture) considered in this study, and consistent with the objective function, the controllers activated the muscles such that the net load on the lumbar spine approached an ideal FL condition. In the future, our control-based approach will be applied to our 3D musculoskeletal model of the spine (<xref rid=\"B2\" ref-type=\"bibr\">Arjmand and Shirazi-Adl, 2006</xref>) while simulating various physiological tasks. This model incorporates a realistic geometry of the spine, including ∼80 thoracolumbar muscles, and 6 degrees-of-freedom intervertebral joint with non-linear passive properties. The controllers will aim to determine optimal muscle forces accounting for all the degrees-of-freedom in all anatomical planes. In particular, it would also be interesting to simulate, amongst others, some passive-active injuries and pathological conditions (e.g., altered passive stiffness-muscle coordination/muscle areas).</p><sec id=\"S4.SS1\"><title>Interpretations</title><p>Application of the external loads in 0.2 s resulted in an increase in the initial position and velocity beyond those in the target condition (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). In response, and to maintain equilibrium and stability, the controllers bilaterally and significantly activated the muscles at all levels. Following a transient period with large fluctuations, the controllers succeeded in reducing the errors, such that at the final steady state conditions, the velocity errors completely disappeared, while the position errors diminished to less than 1 mm (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). At this final static configuration, and in agreement with the matched equilibrium-based model, the controllers activated the muscles unilaterally with no coactivation to balance the spine (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). The only difference between the two models was observed in loading cases 3 and 5, during which the unilaterally opposite muscles were activated at the L2 level (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). The control-based model generally balanced the external loads at smaller muscle forces (differences reached ∼159, 40, −11, 132, and −8 N for loading cases 1 through 5). This in in alignment with objective functions minimizing the sum of linear, squared, or cubed muscle forces/stresses, commonly considered in optimization-driven models. While at some levels in the loading cases 3 and 5, our model predicted larger muscular forces, as compared to its matched equilibrium-based model, the sums of muscle forces in these loading conditions, were only moderately larger (11 and 12% increase for loading case 3 and 5, respectively) (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref> and <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). This suggests that the cost function used by the CNS to assign forces to muscles may additionally depend on loading conditions and posture. It is to be noted that even smaller total (resultant) spinal loads were estimated in our model when compared to the equilibrium-based model. This highlights the crucial role of our controller (Eq. 5).</p><p>Interestingly, without imposing any constraints on the magnitude or direction of muscle or reaction forces in the lumbar spine, a near FL condition was found in various cases (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>). This was in agreement with the matched equilibrium-based model, which constrained activation in muscles to generate an FL on the spine at all levels. It appears, therefore, that the controllers (i.e., the CNS) learned to balance and stabilize the spine by generating conditions approaching that under an FL. This is also in agreement with findings from another detailed musculoskeletal equilibrium-based model of the spine, in which the muscle forces were predicted to create compressive FLs on the spine during a quiet standing posture (<xref rid=\"B20\" ref-type=\"bibr\">Han et al., 2011</xref>). The outcome in internal loading is also consistent with the minimization of changes in horizontal translations. Moreover, unlike the equilibrium-based model which predicted no shear loads on the spine, small spinal shear loads were predicted in our model (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). The structure and nature of the constitutive components of the objective function in our model (Eq. 5) allow for diverse simulation possibilities to explore the competing goals of the system toward emulating the sophisticated physiological system and its intricate strategies. The addition of more state variables can be another intriguing motivation for future investigation.</p><p>The recruitment of trunk muscles has been shown to be strongly direction dependent (<xref rid=\"B35\" ref-type=\"bibr\">Nussbaum et al., 1995</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Hadizadeh et al., 2014</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Sedaghat-Nejad et al., 2015</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Eskandari et al., 2016</xref>). In quasi-static conditions the emergent synergies responsible for a direction of external load will be linearly scaled. The invariance in set of activated muscles under varying magnitude of external load (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>) is in line with the theories of using muscle synergy in multiple muscle systems across the cost functions (<xref rid=\"B31\" ref-type=\"bibr\">Moghadam et al., 2013</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Eskandari et al., 2016</xref>). Future studies must test this in more physiological models with realistic posture/loading and non-linear properties. Future studies can also benefit by incorporating more physiologically based detailed architectural/geometrical muscle models and structure/function data obtained from neuroimaging studies.</p></sec><sec id=\"S4.SS2\"><title>Limitations</title><p>This model was idealized in terms of the geometry of the active-passive tissues, material properties, and loading conditions in the frontal plane, as we primarily aimed to (1) implement a novel bio-inspired control strategy that mimics the adaptive mechanism of the CNS and (2) compare its predictions with an existing matched equilibrium-based model. As the current model was idealized based on simplifying assumptions in terms of the geometry of the spine, loading, boundary conditions, and musculature, caution should be exercised when extrapolating results to clinical applications. The maximal force in all muscles was considered to be 800 N, in order to accommodate large fluctuations in muscle forces during the transient period (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). In the final steady-state, however, much smaller muscular forces were estimated (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). Non-zero muscle pre-activation values (initial values) could subdue the fluctuations observed in the transient state. While the stability was not formally examined in our model, different perturbations (e.g., the addition of a moment at the L1 and the reduction of Young’s modulus of the beams; <xref rid=\"B33\" ref-type=\"bibr\">Nasseroleslami et al., 2014</xref>) did not cause instability, as the controllers prevented large deformations and maintained the final steady-state position. For example, <xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref> depicts the model response under a perturbation, where the addition and removal of a 100 N load to impose external compression of 635 N at L1 for a duration of 0.5 s, caused the muscle force to appropriately rise and fall, respectively, to maintain the required objective posture (<italic>Z</italic> coordinates). The error terms, which approached nil at the end of the 20 s simulation, are not shown in <xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref> for clarity. The closed loop response could include multiple loops with varying gains and time delays (<xref rid=\"B51\" ref-type=\"bibr\">Zeinali-Davarani et al., 2008</xref>). We have neither considered the spindle nor the reflexive responses in the feedback loop, and we have not used an internal model to assist with the initial exploration of activation selection (<xref rid=\"B9\" ref-type=\"bibr\">Dariush et al., 1998</xref>; <xref rid=\"B44\" ref-type=\"bibr\">Shadmehr and Mussa-Ivaldi, 2012</xref>), all warranting future investigation. The objective function should be designed considering stability in the Lyapunov sense, while setting the performance criterion to maintain the system within the safe normal physiological limits of the passive and active spinal structures. This provides an envelope with margins of safety to avoid pain, discomfort, muscle fatigue, instability and ultimately failure/injury.</p><fig id=\"F7\" position=\"float\"><label>FIGURE 7</label><caption><p>External compressive force, error, error rate and muscle forces at L1 vs. time (up to 2.2 s) for the loading case 1 under application of −100 N and 100 N vertical force at L1 during [0.5 s, 1 s] and [1.5 s, 2 s] time intervals, respectively, in order to model perturbations (with controllers and muscles). The controller tries to maintain the primary Z-coordinates of beam (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>) with a penalty on muscle activation level.</p></caption><graphic xlink:href=\"fbioe-08-00949-g007\"/></fig></sec></sec><sec id=\"S5\"><title>Conclusion</title><p>This work presents a new method to estimate muscle forces using a control-based FE model of the lumbar spine. The model incorporates a control strategy that mimics the adaptive mechanism of the CNS to adjust muscle forces. Steady state muscle forces have similar patterns to a geometrically matched equilibrium-based model and spine reaction forces resemble a FL on the spine. Additionally, controllers linearly scale muscle forces in a specific loading condition with varying magnitude of external load. The phenomenon of FL is the predicted behavior of this adaptive neuro-fuzzy control system and not the explicit objective of the mathematical theory or conjecture. That creates a fertile paradigm to consider clinical ideas (i.e., spinal injuries and/or fusion) to be investigated in future studies with a more detailed architecture for muscles under more general loading conditions during daily activities at work, leisure and sport.</p></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>All datasets generated for this study are included in the article/supplementary material.</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>All authors listed above have made substantial contributions to the conception and design of the study, analysis and interpretation of data, preparing the manuscript, and have read the final approval of the version to be submitted and listed on the title page have read the manuscript and also attest to the validity and legitimacy of the data and its interpretation, and agreed to its submission.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by grants from the Sharif University of Technology (Tehran, Iran).</p></fn></fn-group><ack><p>Assistance of Mr. Mohammad Shahiri in model simulation is greatly appreciated.</p></ack><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Andersson</surname><given-names>S.</given-names></name></person-group> (<year>2013</year>). <source><italic>Active Muscle Control in Human Body Model Simulations: Implementation of a Feedback Control Algorithm with Standard Keywords in LS-DYNA.</italic></source>\n<comment>M. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Neurosci</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Neurosci</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Neurosci.</journal-id><journal-title-group><journal-title>Frontiers in Neuroscience</journal-title></journal-title-group><issn pub-type=\"ppub\">1662-4548</issn><issn pub-type=\"epub\">1662-453X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32848535</article-id><article-id pub-id-type=\"pmc\">PMC7431631</article-id><article-id pub-id-type=\"doi\">10.3389/fnins.2020.00681</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Neuroscience</subject><subj-group><subject>Perspective</subject></subj-group></subj-group></article-categories><title-group><article-title>Toward Long-Term Communication With the Brain in the Blind by Intracortical Stimulation: Challenges and Future Prospects</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Fernández</surname><given-names>Eduardo</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/8392/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Alfaro</surname><given-names>Arantxa</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/168406/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>González-López</surname><given-names>Pablo</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/876435/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Institute of Bioengineering, Universidad Miguel Hernández</institution>, <addr-line>Elche</addr-line>, <country>Spain</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Center for Biomedical Research in the Network in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN)</institution>, <addr-line>Madrid</addr-line>, <country>Spain</country></aff><aff id=\"aff3\"><sup>3</sup><institution>John A. Moran Eye Center, University of Utah</institution>, <addr-line>Salt Lake City, UT</addr-line>, <country>United States</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Hospital Vega Baja</institution>, <addr-line>Orihuela</addr-line>, <country>Spain</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Hospital General Universitario de Alicante</institution>, <addr-line>Alicante</addr-line>, <country>Spain</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Alejandro Barriga-Rivera, The University of Sydney, Australia</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Frank Rattay, Vienna University of Technology, Austria; Mohit Naresh Shivdasani, University of New South Wales, Australia</p></fn><corresp id=\"c001\">*Correspondence: Eduardo Fernández, <email>e.fernandez@umh.es</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Neural Technology, a section of the journal Frontiers in Neuroscience</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>14</volume><elocation-id>681</elocation-id><history><date date-type=\"received\"><day>24</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>03</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Fernández, Alfaro and González-López.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Fernández, Alfaro and González-López</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>The restoration of a useful visual sense in a profoundly blind person by direct electrical stimulation of the visual cortex has been a subject of study for many years. However, the field of cortically based sight restoration has made few advances in the last few decades, and many problems remain. In this context, the scientific and technological problems associated with safe and effective communication with the brain are very complex, and there are still many unresolved issues delaying its development. In this work, we review some of the biological and technical issues that still remain to be solved, including long-term biotolerability, the number of electrodes required to provide useful vision, and the delivery of information to the implants. Furthermore, we emphasize the possible role of the neuroplastic changes that follow vision loss in the success of this approach. We propose that increased collaborations among clinicians, basic researchers, and neural engineers will enhance our ability to send meaningful information to the brain and restore a limited but useful sense of vision to many blind individuals.</p></abstract><kwd-group><kwd>visual prostheses</kwd><kwd>blindness</kwd><kwd>biocompatibility</kwd><kwd>biotolerability</kwd><kwd>neuroplasticity</kwd><kwd>visual cortex</kwd></kwd-group><counts><fig-count count=\"2\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"89\"/><page-count count=\"8\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Visual impairment affects personal independence, reduces quality of life, and has a significant impact on the lives of those who suffer it (<xref rid=\"B10\" ref-type=\"bibr\">Bourne et al., 2017</xref>). Although some visual pathologies can be effectively treated, and there are some novel approaches to slow down the progression of several eye diseases, including gene and stem cell therapies (<xref rid=\"B38\" ref-type=\"bibr\">Higuchi et al., 2017</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Artero Castro et al., 2018</xref>; <xref rid=\"B49\" ref-type=\"bibr\">Llonch et al., 2018</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Benati et al., 2019</xref>; <xref rid=\"B87\" ref-type=\"bibr\">West et al., 2019</xref>), unfortunately, there are not treatments for all causes of blindness (<xref rid=\"B26\" ref-type=\"bibr\">Fernandez, 2018</xref>). Therefore, many scientists have long dreamed of the possibility of restoring vision by using neural prosthetic devices that bypass the damaged visual pathways.</p><p>The concept of artificially producing a visual sense in the blind is based on our current understanding of the structure of the mammalian visual system and the relationship between electrical stimulation of any part of the visual pathways and the resulting visual perceptions (<xref rid=\"B28\" ref-type=\"bibr\">Fernandez and Normann, 1995</xref>; <xref rid=\"B53\" ref-type=\"bibr\">Maynard, 2001</xref>). Thus, several research groups are focusing their efforts on the development of new approaches for artificial vision based on electric stimulation of the retina (<xref rid=\"B19\" ref-type=\"bibr\">Da Cruz et al., 2016</xref>; <xref rid=\"B50\" ref-type=\"bibr\">Lorach et al., 2016</xref>; <xref rid=\"B79\" ref-type=\"bibr\">Stingl et al., 2017</xref>), optic nerve (<xref rid=\"B24\" ref-type=\"bibr\">Duret et al., 2006</xref>; <xref rid=\"B51\" ref-type=\"bibr\">Lu et al., 2013</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Gaillet et al., 2020</xref>), lateral geniculate nucleus (<xref rid=\"B84\" ref-type=\"bibr\">Vurro et al., 2014</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Killian et al., 2016</xref>), or visual cortex (<xref rid=\"B29\" ref-type=\"bibr\">Fernandez et al., 2005</xref>; <xref rid=\"B60\" ref-type=\"bibr\">Normann et al., 2009</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Kane et al., 2013</xref>; <xref rid=\"B59\" ref-type=\"bibr\">Normann and Fernandez, 2016</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Fernandez, 2018</xref>; <xref rid=\"B57\" ref-type=\"bibr\">Niketeghad et al., 2019</xref>). All of these prosthetic devices work by exchanging information between the electronic devices and different types of neurons, and although most of them are still in development, they show promise of restoring vision in many forms of blindness.</p><p>At present, retinal prostheses are the most successful approach in this field, and several retinal devices have already been approved for patients with retinal dystrophies (<xref rid=\"B19\" ref-type=\"bibr\">Da Cruz et al., 2016</xref>; <xref rid=\"B79\" ref-type=\"bibr\">Stingl et al., 2017</xref>). However, the inner layers of the retina can degenerate in many retinal diseases. Consequently, a retinal prosthesis may not be useful, for example, in patients with advanced retinal degenerations, glaucoma, or optic atrophy. Therefore, there are compelling reasons for the development of other approaches able to restore a functional sense of vision bypassing the retina.</p><p>In this framework, since the neurons in the higher visual regions of the brain are usually spared from the damage to the retina and optic nerve, several researchers are trying to develop visual prostheses designed to directly stimulate the brain. Even if only a crude representation of the surrounding physical world can be evoked, a blind individual could use this artificially encoded neural information for tasks such as orientation and mobility. This functional performance has already been attained in the field of auditory prostheses. These devices have already allowed many deaf patients to hear sounds and acquire language capabilities (<xref rid=\"B56\" ref-type=\"bibr\">Merkus et al., 2014</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Glennon et al., 2019</xref>), and the same hope exists in the field of neuroprosthetic devices designed for electrical stimulation of the visual cortex.</p><p>However, in spite of all the progress in materials and neuroelectronic interfaces, the scientific and technological problems associated with the long-term biocompatibility and biotolerability of cortical electrodes, together with the difficulties associated with the encoding of visual information, are very complex. Moreover, it is still unclear how to identify the ideal candidates for a cortical prosthesis (<xref rid=\"B55\" ref-type=\"bibr\">Merabet et al., 2007</xref>). Therefore, there are still many unresolved issues delaying its development. We summarize herein some of the main biological and technical issues that still remain to be fully solved, related mainly to the field of intracortical devices, and discuss some of the challenges in this highly multidisciplinary field.</p></sec><sec id=\"S2\"><title>Electrodes That Interact With the Brain in the Blind: General Remarks</title><p>Otfried Foerster was the first neurosurgeon who exposed the occipital area of one cerebral hemisphere in an awake patient (under local anesthesia) and electrically stimulated it (<xref rid=\"B31\" ref-type=\"bibr\">Foerster, 1929</xref>). He found that electrical stimulation of this region of the brain induced the perception of small spots of light directly in front of the subject. These early findings, together with the studies of Wilder Penfield and co-workers in epileptic patients (<xref rid=\"B65\" ref-type=\"bibr\">Penfield and Rasmussen, 1950</xref>; <xref rid=\"B64\" ref-type=\"bibr\">Penfield and Jaspers, 1974</xref>), established the anatomical and physiological basis for the development of a cortical visual prosthesis for the blind. Later on, Giles Brindley in England (<xref rid=\"B11\" ref-type=\"bibr\">Brindley and Lewin, 1968a</xref>, <xref rid=\"B12\" ref-type=\"bibr\">b</xref>; <xref rid=\"B71\" ref-type=\"bibr\">Rushton and Brindley, 1978</xref>) and William Dobelle in the United States (<xref rid=\"B22\" ref-type=\"bibr\">Dobelle and Mladejovsky, 1974</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Dobelle et al., 1976</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Dobelle, 2000</xref>) showed that simultaneous stimulation of several electrodes placed on the surface of the brain allowed blind volunteers to see some predictable simple patterns, including Braille characters and letters (<xref rid=\"B2\" ref-type=\"bibr\">Bak et al., 1990</xref>; <xref rid=\"B74\" ref-type=\"bibr\">Schmidt et al., 1996</xref>). However, there were also some problems, such as the induction of epileptic seizures and the appearance of pain due to meningeal or scalp stimulation. These issues were associated with the large active surface of the electrodes, which required high electrical currents of the order of milliamps to evoke phosphenes. In addition, these large electrodes interacted with relatively large volumes of cortex (∼1 cm<sup>3</sup>), resulting in very low spatial resolution of the perceived phosphenes (<xref rid=\"B16\" ref-type=\"bibr\">Christie et al., 2016</xref>; <xref rid=\"B57\" ref-type=\"bibr\">Niketeghad et al., 2019</xref>). These later findings have recently been confirmed by <xref rid=\"B5\" ref-type=\"bibr\">Beauchamp et al. (2020)</xref>, who implanted two different types of electrodes on the surface of the visual cortex of two blind individuals and found that when multiple electrodes were stimulated simultaneously, phosphenes fused into larger formless perceptions, making shape recognition impossible.</p><p>Cortical artificial vision did not seem feasible until we could find a way to provide a much more focal stimulation of neurons in the visual cortex (<xref rid=\"B61\" ref-type=\"bibr\">Normann et al., 1996</xref>). This led a number of investigators to develop new approaches such as smaller intracortical electrodes designed to be similar in size to the cell bodies of the neurons they are trying to stimulate and able to penetrate through the surface of the cortex (<xref rid=\"B62\" ref-type=\"bibr\">Normann et al., 1999</xref>; <xref rid=\"B81\" ref-type=\"bibr\">Troyk et al., 2003</xref>; <xref rid=\"B88\" ref-type=\"bibr\">Wise, 2005</xref>). These new microelectrodes can be located very close to the neurons they intend to stimulate, which are situated generally at 1–1.5 mm from the cortical surface, avoiding the relatively high electrical currents required by surface electrodes. Thus, we recently implanted an array of 100 penetrating electrodes (a Utah Electrode Array) in the occipital cortex of a 57-year-old person during a six-month period, and we found that stimulation thresholds to excite neurons were in the 1-100 microamp range (<xref rid=\"B30\" ref-type=\"bibr\">Fernandez et al., 2019</xref>). This is clearly two to three orders of magnitude smaller than the currents required to evoke phosphenes using surface electrodes.</p><p>Some examples of these new penetrating neural interfaces are the arrays built with metal microelectrodes, the Utah Electrode Array, the implantable microcoils for intracortical magnetic stimulation (<xref rid=\"B46\" ref-type=\"bibr\">Lee et al., 2016</xref>), and other penetrating devices made of a variety of other materials (<xref rid=\"B27\" ref-type=\"bibr\">Fernandez and Botella, 2017</xref>). However, although these penetrating microelectrodes have been used successfully in both the central (CNS) and peripheral (PNS) nervous systems, the brain imposes some specific conditions such as the absence of regeneration and the presence of different types of glial cells. Moreover, the requirements for electrical stimulation and recording in the brain are clearly different from those in the peripheral nervous system. Thus, the brain hosts different types of neurons arranged in several superficial layers and in deep nuclei and various types of glial cells that interact in very intricate ways. Furthermore, the brain is protected by the meninges, a multi-layered structure formed by connective tissue, bone, and skin. This means that it is impossible to reach the desired cortical neurons without affecting neighboring parts of the nervous system. Likewise, the brain tissue includes a complex network of blood vessels that are likely to be injured by the introduction of any external device (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Human cerebral vascular architecture. <bold>(A)</bold> Section of human primary visual cortex visualized with an intravascular injection of India ink and gelatin (courtesy of Professors H. Duvernoy and P. Rabischong). Note the high density of blood vessels at the level of the gray matter. Calibration bar = 1 mm. <bold>(B)</bold> Detail of human gray matter vascularization showing a dense network of blood vessels at the gray matter, which is thicker at layer IV. Calibration bar = 1 mm. <bold>(C)</bold> Cerebral cortex impregnated with chrome-silver by Luis Simarro (image courtesy of Museum Luis Simarro, Universidad Complutense de Madrid, Madrid, Spain). Arrows indicate some blood vessels among neurons and glial cells. Calibration bar = 100 μm.</p></caption><graphic xlink:href=\"fnins-14-00681-g001\"/></fig><p>In addition, we should also consider the mechanical micromovements between the pulsating neural tissue (due mainly to cardiac pulse and breathing) and the static implants, which can induce different kinds of damage (<xref rid=\"B66\" ref-type=\"bibr\">Polanco et al., 2016</xref>). All of these factors place high demands on the long-term function of any intracortical electrode and also impose unique constrains for the materials, packaging, and insulation of the electronics (<xref rid=\"B59\" ref-type=\"bibr\">Normann and Fernandez, 2016</xref>).</p></sec><sec id=\"S3\"><title>Biotolerability of Neural Electrodes</title><p>The implantation of any intracortical microelectrode into the brain is a traumatic procedure, and all neural electrodes to date, even those considered to be highly biocompatible, induce biological responses characterized by small microhemorrhages and a certain amount of local tissue damage around the electrodes that may impact the stability, performance, and viability of the microelectrodes. Therefore, some authors suggest that instead of biocompatibility, we should talk about biotolerability, highlighting the capacity of the microelectrodes to stay fully functional in the brain without inducing any significant tissue damage for long periods of time (<xref rid=\"B27\" ref-type=\"bibr\">Fernandez and Botella, 2017</xref>).</p><p>While most materials used currently for the fabrication of intracortical electrodes remain relatively inert in the brain, they still induce a foreign-body reaction (FBR) characterized by a neuroinflammatory response of the tissue around the electrodes that may hinder the recording and stimulation of the neurons over time (<xref rid=\"B52\" ref-type=\"bibr\">Marin and Fernandez, 2010</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Fernandez and Botella, 2017</xref>). Often, the FBR starts with the damage to the blood vessels encountered during the implantation of the microelectrodes in the neural tissue (see <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>), which causes small interstitial microhemorrhages. These microhemorrhages stop spontaneously, but there is also increased blood flow to the damaged region, together with increased permeability of local microvasculature, which induces extravasation of fluids, blood cells, and proteins toward the interstitial space. Thus, the microelectrodes become surrounded by many blood cells and plasma proteins that stick to their surface. <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref> shows a representative example. Therefore, blood compatibility should be considered an important issue for improving the long-term performance and viability of any neural electrode.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Utah Electrode Array implanted in a human brain for 10 minutes (the procedure was approved by the Ethics Committee of the Hospital General Universitario of Alicante, Spain). <bold>(A)</bold> Image of several electrode tips surrounded by blood cells and plasma proteins that stick to the surface of the neural electrodes. Calibration bar = 400 μm. <bold>(B)</bold> Scanning electron micrograph showing the surface of several microelectrodes covered by many blood cells. Calibration bar = 100 μm. <bold>(C)</bold> Detail of the tip of a microelectrode. Calibration bar = 10 μm.</p></caption><graphic xlink:href=\"fnins-14-00681-g002\"/></fig><p>On the other hand, as has been reviewed in detail elsewhere (<xref rid=\"B89\" ref-type=\"bibr\">Zhong and Bellamkonda, 2008</xref>; <xref rid=\"B52\" ref-type=\"bibr\">Marin and Fernandez, 2010</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Fernandez and Botella, 2017</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Ferguson et al., 2019</xref>), the inflammatory responses to the implantation of any neural probe into the brain involve a large network of physiological responses including edema, release of cytokines, platelet activation, complement system activation, invasion of blood-borne macrophages, and activation of neighboring astrocytes and microglial cells (<xref rid=\"B45\" ref-type=\"bibr\">Lee et al., 2005</xref>; <xref rid=\"B67\" ref-type=\"bibr\">Polikov et al., 2005</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Biran et al., 2007</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Grill et al., 2009</xref>; <xref rid=\"B54\" ref-type=\"bibr\">Mcconnell et al., 2009</xref>; <xref rid=\"B52\" ref-type=\"bibr\">Marin and Fernandez, 2010</xref>). Subsequently, activated macrophages surround the microelectrodes and fuse into multi-nucleated giant cells that form a barrier, similar to a thin protective membrane, that shields brain tissue from damage (<xref rid=\"B67\" ref-type=\"bibr\">Polikov et al., 2005</xref>). Most of these processes are spontaneously resolved; however, glial scarring and giant cells can be found around many microelectrodes implanted chronically in the brain (<xref rid=\"B67\" ref-type=\"bibr\">Polikov et al., 2005</xref>). This suggests the existence of a chronic inflammation reaction that persists over time and can induce the development of a dense sheath around the microelectrodes, making it difficult to record and stimulate nearby neurons. As a result, long-term biocompatibility or biotolerability is still an unresolved issue, and most intracortical microelectrodes have a maximum <italic>in vivo</italic> lifetime of several months or a few years (<xref rid=\"B80\" ref-type=\"bibr\">Suner et al., 2005</xref>; <xref rid=\"B68\" ref-type=\"bibr\">Prasad et al., 2012</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Barrese et al., 2013</xref>).</p><p>A significant challenge here is to reduce the neuro-inflammatory response. In recent years, several strategies for minimizing trauma and the inflammatory responses have been investigated, for example, the reduction of the cross-sectional area of the electrodes (<xref rid=\"B76\" ref-type=\"bibr\">Seymour and Kipke, 2007</xref>) and the use of more flexible and soft materials that better match the properties of the surrounding tissue (<xref rid=\"B63\" ref-type=\"bibr\">Patel et al., 2016</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Fernandez and Botella, 2017</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Cuttaz et al., 2019</xref>; <xref rid=\"B85\" ref-type=\"bibr\">Wang et al., 2019</xref>). However, these modifications also affect the mechanical properties of the electrodes and could result in a lack of the mechanical strength needed to withstand insertion without buckling and breaking. Another relatively simple way to control the biological responses and improve the long-term biotolerability of neural electrodes is the modification of the chemical composition of the surface of the electrodes by using different polymers and nanomaterials (<xref rid=\"B37\" ref-type=\"bibr\">Hara et al., 2016</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Fernandez and Botella, 2017</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Gulino et al., 2019</xref>). Moreover, we should also consider that the electronics and the connecting pathways to individual microelectrodes must be completely insulated and have to remain perfectly functional over time, which also imposes unique constraints on hermetic packaging (<xref rid=\"B40\" ref-type=\"bibr\">Jiang and Zhou, 2009</xref>; <xref rid=\"B83\" ref-type=\"bibr\">Vanhoestenberghe and Donaldson, 2013</xref>).</p><p>Although it is often not mentioned, an important issue for the long-term success of any neural implant is the quality of the surgical implantation procedures. Thus, we believe that many difficulties encountered in chronic experiments could be directly related to problems during surgery and implantation. Careful implantation seems to increase the biotolerability and long-term longevity of intracortical microelectrode arrays, and there is no way to substitute for good planning and an adequate surgical technique.</p></sec><sec id=\"S4\"><title>Number of Electrodes Required for Functional Vision</title><p>The functional vision that could be restored with an array of intracortical microelectrodes implanted into the brain is a function of many parameters, but it is in part related to the number of implanted electrodes, the interelectrode spacing, and the specific location of each microelectrode in the brain (<xref rid=\"B14\" ref-type=\"bibr\">Cha et al., 1992</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Dagnelie et al., 2006</xref>). However, the assumption that visual perception will improve by increasing only the number of electrodes may be incorrect.</p><p>Although we see with the brain, the input information to the visual system begins at the eye, which catches and focuses light onto the retina. The human retina is approximately 0.5 mm thick and contains both the photoreceptors or sensory neurons that respond to light and intricate neural circuits that perform the first stages of imaging processing. The output neurons of the retina are the ganglion cells, which send their axons (approximately 1–1.5 million per eye) through the optic nerve to the brain (<xref rid=\"B86\" ref-type=\"bibr\">Watson, 2014</xref>). This means that, in order to encode all the features of objects in the visual space (for example, their form, localization, contour, intensity, color, etc.) and the change of these features in time in the same way that the human retina does, we would need at least 1 million parallel channels, which is clearly well beyond the state-of-the-art of current prosthetic technologies.</p><p>Fortunately, despite the above-mentioned figures, the results of several simulation studies suggest that the amount of visual input required to perform basic visually guided tasks is not as great as one might expect. In a series of psychophysical experiments, it has been estimated that 625 electrodes implanted at the primary visual cortex could be enough for reading (although to lower speeds) and to navigate through complex visual environments (<xref rid=\"B14\" ref-type=\"bibr\">Cha et al., 1992</xref>). In this framework, the possibility of providing some degree of functional vision to facilitate the activities of daily living with only around 600–700 electrodes is very encouraging (<xref rid=\"B20\" ref-type=\"bibr\">Dagnelie et al., 2006</xref>). However, this low number of electrodes also usually implies a “tunnel vision”: a restricted visual field that can be a serious problem for orientation and mobility. To cope with this problem, we can implant several arrays of penetrating microelectrodes at different locations of the visual cortex. In this context, multiple microelectrode arrays have already been implanted in monkey visual cortex (<xref rid=\"B15\" ref-type=\"bibr\">Chen et al., 2017</xref>; <xref rid=\"B69\" ref-type=\"bibr\">Roelfsema and Holtmaat, 2018</xref>; <xref rid=\"B82\" ref-type=\"bibr\">Van Vugt et al., 2018</xref>; <xref rid=\"B75\" ref-type=\"bibr\">Self et al., 2019</xref>) and these implants are providing a better understanding of how the brain enhances the representations of visual objects in different visual regions (<xref rid=\"B43\" ref-type=\"bibr\">Klink et al., 2017</xref>; <xref rid=\"B75\" ref-type=\"bibr\">Self et al., 2019</xref>). However, more experiments are still needed, and probably the question of how many electrodes are necessary to restore a limited but useful vision will only be addressable by future experiments in blind subjects.</p></sec><sec id=\"S5\"><title>Engineering a Wireless Intracortical Device With Hundreds of Electrodes</title><p>Although ongoing studies suggest that electrical stimulation via multiple electrodes may give rise to useful vision, extensive efforts are still needed to address the engineering challenges of realizing an intracortical device containing hundreds of electrodes. Furthermore, the device must be wireless, since it is necessary to avoid wires to reduce post-surgical complications such as, for example, the risk of infection. In this context, power and communication constraints, as well as power dissipation in the brain, could pose significant challenges (<xref rid=\"B72\" ref-type=\"bibr\">Sahin and Pikov, 2011</xref>; <xref rid=\"B48\" ref-type=\"bibr\">Lewis et al., 2015</xref>). Other relevant issues in this framework are the so-called “crosstalk” or interference between stimulating electrode sites and the multiplexing of stimulation channels (<xref rid=\"B4\" ref-type=\"bibr\">Barriga-Rivera et al., 2017</xref>). Thus, there is a clear need to develop new implantable technologies optimized for high channel count.</p><p>On the other hand, patients with retinal implants have to undergo long fitting procedures to measure thresholds and fine-tune the stimulation parameters on each individual electrode, but these procedures are not viable if hundreds or thousands of electrode sites need to be tested. Therefore, we need further procedures for fitting devices containing hundreds of electrodes in patients. A possible approach to facilitate the fitting procedures could be to develop bidirectional intracortical devices able to record the neuronal activity in response to electrical stimulation and use the recorded neural activity to optimize the stimulation parameters (<xref rid=\"B70\" ref-type=\"bibr\">Rotermund et al., 2019</xref>). Another possibility could be to use machine learning to find optimal stimulation settings (<xref rid=\"B44\" ref-type=\"bibr\">Kumar et al., 2016</xref>). In any case, more studies are still needed.</p></sec><sec id=\"S6\"><title>Delivery of Information to Implants</title><p>Besides the number of electrodes and the engineering challenges, a key issue for the future success of cortical visual implants is related to how the brain understands artificially encoded information. All visual prostheses developed to date provide very poor vision, with relatively low spatial resolution; therefore, great efforts are still needed to design and develop new systems that can have results similarly successful as those achieved with cochlear implants.</p><p>Part of the success of cochlear implants seems to be related to the development of sophisticated signal-processing techniques and bioinspired coding strategies developed over the years (<xref rid=\"B17\" ref-type=\"bibr\">Clark, 2015</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Boulet et al., 2016</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Jain and Vipin Ghosh, 2018</xref>). Despite these encouraging results, most visual prosthesis devices only try to emulate the phototransducer aspects of the retina and do not consider the complex processes that are found in the mammalian visual system. Some researchers have proposed that performance could be increased significantly by incorporating neural code (<xref rid=\"B58\" ref-type=\"bibr\">Nirenberg and Pandarinath, 2012</xref>), whereas others promote the use of computer vision algorithms and techniques of artificial intelligence (<xref rid=\"B73\" ref-type=\"bibr\">Sanchez-Garcia et al., 2020</xref>). Although more studies are still needed, we expect that bio-inspired visual encoders based on intelligent signal and image-processing strategies, together with new cutting-edge artificial intelligence algorithms running neuromorphic hardware, could have a significant impact in the future to facilitate the interpretation of the processed signals (<xref rid=\"B26\" ref-type=\"bibr\">Fernandez, 2018</xref>).</p><p>On the other hand, whereas there are many relevant aspects in a visual scene (for example, form, color, and motion), most current coding strategies are only aimed at addressing the spatial details. This could be an oversimplification since, for example, the ability to recognize patterns in a scene, or the perceived receptive field size, is critical for many visual tasks. Thus, we can extract complex information, such as identifying human faces, from relatively poor-quality images by using specific cues and multiple visual features (<xref rid=\"B78\" ref-type=\"bibr\">Sinha, 2002</xref>). This suggests that besides image resolution, we should try to pay attention to other relevant visual attributes such as receptive field size, localization, orientation, or movement.</p><p>Another important issue is to focus on the specific needs of the end users. For example, some people may place more demands on object- or person-identification, whereas others could prefer to focus on orientation and mobility. The key issue is to encode and send useful information that can be translated into functional gains for daily life activities (<xref rid=\"B55\" ref-type=\"bibr\">Merabet et al., 2007</xref>). In addition, it is possible that there are subtle differences in the perceived visual field or in coding among subjects. Therefore, future advanced systems to interact with the brain in the blind should allow the customization of the functions to satisfy the particular needs and capabilities of each user.</p></sec><sec id=\"S7\"><title>Neural Plasticity</title><p>The adult visual cortex does not completely lose its functional capacity after years of deprivation of visual input (<xref rid=\"B11\" ref-type=\"bibr\">Brindley and Lewin, 1968a</xref>); however, there is clear clinical evidence showing adaptive neurophysiological changes in the brain, specifically at the occipital lobe. Therefore, a relevant question is whether these adaptive changes could have a significant impact on the success of a cortical visual prosthesis.</p><p>In response to the loss of vision, brain areas normally devoted to the processing of visual information are recruited to process tactile and auditory information and even cognitive functions such as verbal memory and speech processing (<xref rid=\"B29\" ref-type=\"bibr\">Fernandez et al., 2005</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Gilbert et al., 2009</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Legge and Chung, 2016</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Beyeler et al., 2017</xref>; <xref rid=\"B77\" ref-type=\"bibr\">Singh et al., 2018</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Castaldi et al., 2020</xref>). These changes are related to the capability of blind subjects to extract greater information from other senses such as touch and hearing. Thus, neuroplasticity can be viewed as an adaptive and dynamic process able to change the processing patterns of sensory information.</p><p>This neuroplasticity implies that the brain undergoes important remodeling and adaptive changes after the onset of the blindness that could directly impact the success of any cortical prosthesis (<xref rid=\"B34\" ref-type=\"bibr\">Glennon et al., 2019</xref>). Over time, these adaptive changes may lead to the establishment of new connections and functional roles of different brain areas, which is probably influenced by factors such as the cause of the visual loss and the duration of visual deprivation. All these issues may help to define a preferred time window for improving the likelihood of success of any device intended for communicating with the brain in the blind.</p><p>On the other hand, it is unlikely that the re-introduction of the lost sensory input alone will be able to promptly restore sight. Therefore, we should try to develop specific strategies to communicate with the brain of the blind in order to increase the chances of extracting useful information from the artificially encoded stimulation. Furthermore, we should consider the challenges of visual rehabilitation. Thus, improved rehabilitation strategies after the surgical implantation could contribute greatly to ever improving the performance of the neuroprosthetic devices.</p></sec><sec id=\"S8\"><title>Conclusion and Future Perspectives</title><p>The development of new prosthetic technologies for restoring vision to many blind individuals for whose impairment there is currently neither prevention nor cure is a must for the future.</p><p>Cortical prostheses based on penetrating microelectrodes show promise for restoring some limited but useful vision to subjects with certain forms of blindness, but the scientific and technological problems associated with safe and effective communication with the visual brain are very complex, and there are still many unresolved issues delaying its development. We expect that ongoing research on the interactions between intracortical microelectrodes and the local cellular environments, along with a better understanding of neuroplasticity and progress in medical technologies, materials science, neuroelectronic interfaces, neuroscience, and artificial intelligence, will allow advances toward the success envisioned by this technology. Nevertheless, we should go step by step and not create false expectations or underrate the challenges that still remain to be resolved. In this framework, we propose that increased collaborations among clinicians, basic researchers, and neural engineers will enhance our ability to send meaningful information to the visually deprived brain and will help to restore a limited but useful sense of vision to many profoundly blind people.</p></sec><sec sec-type=\"data-availability\" id=\"S9\"><title>Data Availability Statement</title><p>The datasets generated for this study are available on request to the corresponding author.</p></sec><sec id=\"S10\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by the Hospital General Universitario de Alicante. The patients/participants provided their written informed consent to participate in this study.</p></sec><sec id=\"S11\"><title>Author Contributions</title><p>EF, AA, and PG-L contributed to the design and implementation of the research and writing of the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by grant RTI2018-098969-B-100 from the Spanish Ministerio de Ciencia Innovación y Universidades, by PROMETEO/2019/119 from the Generalitat Valenciana, and the Bidons Egara Research Chair of the University Miguel Hernández.</p></fn></fn-group><ack><p>We are grateful to Dr. Lawrence Humphreys (CIBER-BBN) for critical reading of the manuscript.</p></ack><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Artero Castro</surname><given-names>A.</given-names></name><name><surname>Lukovic</surname><given-names>D.</given-names></name><name><surname>Jendelova</surname><given-names>P.</given-names></name><name><surname>Erceg</surname><given-names>S.</given-names></name></person-group> (<year>2018</year>). <article-title>Human induced pluripotent stem cell models of retinitis pigmentosa.</article-title>\n<source><italic>Stem Cells</italic></source>\n<volume>36</volume>\n<fpage>474</fpage>–<lpage>481</lpage>. <pub-id pub-id-type=\"doi\">10.1002/stem.2783</pub-id>\n<pub-id pub-id-type=\"pmid\">29345014</pub-id></mixed-citation></ref><ref id=\"B2\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bak</surname><given-names>M.</given-names></name><name><surname>Girvin</surname><given-names>J. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Behav Neurosci</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Behav Neurosci</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Behav. Neurosci.</journal-id><journal-title-group><journal-title>Frontiers in Behavioral Neuroscience</journal-title></journal-title-group><issn pub-type=\"epub\">1662-5153</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32848657</article-id><article-id pub-id-type=\"pmc\">PMC7431632</article-id><article-id pub-id-type=\"doi\">10.3389/fnbeh.2020.00139</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Neuroscience</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Differential Role of Anterior Cingulate Cortical Glutamatergic Neurons in Pain-Related Aversion Learning and Nociceptive Behaviors in Male and Female Rats</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Jarrin</surname><given-names>Sarah</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/984820/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Pandit</surname><given-names>Abhay</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/23852/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Roche</surname><given-names>Michelle</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/23699/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Finn</surname><given-names>David P.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/281653/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Pharmacology and Therapeutics, National University of Ireland Galway</institution>, <addr-line>Galway</addr-line>, <country>Ireland</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Centre for Pain Research, National University of Ireland Galway</institution>, <addr-line>Galway</addr-line>, <country>Ireland</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Galway Neuroscience Centre, National University of Ireland Galway</institution>, <addr-line>Galway</addr-line>, <country>Ireland</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Centre for Research in Medical Devices (CURAM), National University of Ireland Galway</institution>, <addr-line>Galway</addr-line>, <country>Ireland</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Physiology, School of Medicine, National University of Ireland Galway</institution>, <addr-line>Galway</addr-line>, <country>Ireland</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Hugo Leite-Almeida, University of Minho, Portugal</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Jonathan Cueto-Escobedo, University of Veracruz, Mexico; Eva M. Marco, Complutense University of Madrid, Spain</p></fn><corresp id=\"c001\">*Correspondence: David P. Finn, <email>David.Finn@nuigalway.ie</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Emotion Regulation and Processing, a section of the journal Frontiers in Behavioral Neuroscience</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>14</volume><elocation-id>139</elocation-id><history><date date-type=\"received\"><day>11</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>21</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Jarrin, Pandit, Roche and Finn.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Jarrin, Pandit, Roche and Finn</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Pain is comprised of both sensory and affective components. The anterior cingulate cortex (ACC) is a key brain region involved in the emotional processing of pain. Specifically, glutamatergic transmission within the ACC has been shown to modulate pain-related aversion. In the present study, we use <italic>in vivo</italic> optogenetics to activate or silence, using channelrhodopsin (ChR2) and archaerhodopsin (ArchT) respectively, calmodulin-kinase IIα (CaMKIIα)-expressing excitatory glutamatergic neurons of the ACC during a formalin-induced conditioned place aversion (F-CPA) behavioral paradigm in both female and male adult Sprague-Dawley rats. Expression of c-Fos, a marker of neuronal activity, was assessed within the ACC using immunohistochemistry. Optogenetic inhibition of glutamatergic neurons of the ACC abolished F-CPA without affecting formalin-induced nociceptive behavior during conditioning. In male rats, optogenetic activation of ACC glutamatergic neurons decreased formalin-induced nociceptive behavior during conditioning without affecting F-CPA. Interestingly, the opposite effect was seen in females, where optogenetic activation of glutamatergic neurons of the ACC increased formalin-induced nociceptive behavior during conditioning. The abolition of F-CPA following optogenetic inhibition of glutamatergic neurons of the ACC was associated with a reduction in c-Fos immunoreactivity in the ACC in male rats, but not female rats. These results suggest that excitatory glutamatergic neurons of the ACC play differential and sex-dependent roles in the aversion learning and acute sensory components of pain.</p></abstract><kwd-group><kwd>anterior cingulate cortex</kwd><kwd>glutamate neurons</kwd><kwd>optogenetics</kwd><kwd>inflammatory pain</kwd><kwd>formalin</kwd><kwd>rat</kwd><kwd>c-Fos</kwd><kwd>conditioned place aversion</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Science Foundation Ireland<named-content content-type=\"fundref-id\">10.13039/501100001602</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"5\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"47\"/><page-count count=\"10\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Pain is comprised of both sensory-discriminative and affective-motivational components, which have distinct roles in the pain experience and can often modulate one another. Thus, it is not surprising that chronic pain and anxiety disorders are frequently co-morbid, with approximately 45% of chronic pain patients exhibiting a comorbid anxiety disorder (<xref rid=\"B21\" ref-type=\"bibr\">Lenze et al., 2001</xref>; <xref rid=\"B28\" ref-type=\"bibr\">McWilliams et al., 2003</xref>; <xref rid=\"B16\" ref-type=\"bibr\">Kessler et al., 2005</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Korff et al., 2005</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Roy-Byrne et al., 2008</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Asmundson and Katz, 2009</xref>). Being able to target specifically the affective component of pain would be therapeutically beneficial for patients with chronic pain. In order to develop these improved treatments for chronic pain, there is a need for a better understanding of the neural circuitry involved in pain-related negative affect or aversion.</p><p>The anterior cingulate cortex (ACC) is a key brain region in the affective-motivational component of pain (<xref rid=\"B36\" ref-type=\"bibr\">Price, 2000</xref>). It has been found that lesion of the ACC reduces both formalin-induced conditioned place aversion (F-CPA) and visceral pain-induced CPA, pre-clinical behavioral paradigms used to investigate the affective component of pain, without affecting nociceptive responding (<xref rid=\"B12\" ref-type=\"bibr\">Johansen et al., 2001</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Gao et al., 2004</xref>; <xref rid=\"B46\" ref-type=\"bibr\">Yan et al., 2012</xref>). Glutamatergic transmission and the expression of glutamatergic receptors in the ACC are increased in animal models of pain (<xref rid=\"B45\" ref-type=\"bibr\">Xu et al., 2008</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Chen et al., 2014</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Li W. et al., 2014</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Yi et al., 2014</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Hubbard et al., 2015</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Liu et al., 2015</xref>), as well as clinically in patients with chronic pain conditions (<xref rid=\"B13\" ref-type=\"bibr\">Kameda et al., 2017</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Lv et al., 2018</xref>). Studies have found that optogenetic activation of glutamatergic neurons in the ACC elicits mechanical allodynia in male mice while having no effect on nociceptive responding following an injection of complete Freund’s adjuvant (CFA). Conversely, optogenetic inhibition of glutamatergic neurons in the ACC has an antinociceptive effect of increasing paw withdrawal threshold in the mouse CFA-induced inflammatory pain model (<xref rid=\"B15\" ref-type=\"bibr\">Kang et al., 2015</xref>). It has been found that microinjections of the ionotropic glutamate receptor antagonist, kynurenic acid, into the ACC (<xref rid=\"B11\" ref-type=\"bibr\">Johansen and Fields, 2004</xref>) reduce aversion behavior in an F-CPA paradigm, while microinjection of the excitatory amino acid, homocysteic acid, into the ACC produces avoidance learning in the absence of a noxious stimulus in a CPA paradigm (<xref rid=\"B11\" ref-type=\"bibr\">Johansen and Fields, 2004</xref>). Thus, glutamatergic transmission within the ACC plays an important role in CPA.</p><p>Although chronic pain has a greater prevalence in women than in men (<xref rid=\"B8\" ref-type=\"bibr\">Fayaz et al., 2016</xref>), the vast majority of pre-clinical pain studies have only been conducted in males, with 79% of behavioral non-human animal pain experiments in papers published between 1996 and 2005 using male rodents only (<xref rid=\"B31\" ref-type=\"bibr\">Mogil and Chanda, 2005</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Mogil, 2012</xref>). The inclusion of both sexes in pain studies is important because sex differences in pain have been observed, both in animal models and clinically (<xref rid=\"B2\" ref-type=\"bibr\">Berkley, 1997</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Mogil and Bailey, 2010</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Rhudy et al., 2010</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Mogil, 2012</xref>; <xref rid=\"B40\" ref-type=\"bibr\">Sorge and Totsch, 2017</xref>). Due to the scarcity of pre-clinical pain studies performed in both males and females, little is known about sex differences in the role of glutamatergic neurons in the ACC in regulation of the sensory and affective components of pain.</p><p>In this study, we investigated the hypothesis that the glutamatergic neurons of the ACC have a facilitatory effect on pain-induced aversive behavior, possibly in a sex-dependent manner. The specific aims of the study were (1) to determine the role of glutamatergic neurons of the ACC in both formalin-induced nocifensive and aversive behaviors in female and male rats using optogenetic methodology and (2) to examine if behavioral changes are associated with alterations in expression of the marker of neuronal activity, c-Fos, in the ACC using fluorescent immunohistochemistry.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Animals</title><p>Experiments were carried out on adult male and female Sprague-Dawley rats (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>; Charles River, United Kingdom) maintained at a constant temperature (21 ± 2°C and relative humidity ranged from 36 to 49%) under standard lighting conditions (12:12 h light: dark, lights on from 08.00 to 20.00 h). All surgeries and behavioral trials and testing were carried out during the light phase between 08.00 and 19.00 h. Animals were group housed with three rats per cage until surgery after which they were singly housed. Cages were 42 cm × 26 cm × 13 cm and filled with 3Rs paper bedding (3Rs Lab, United Kingdom). A rectangle plastic insert was placed into the cage under the food hopper allowing animals to access food but preventing animals getting too close to the cage top which may result in damage to the optical fiber implants. Food (14% protein rodent diet, Harlan, United Kingdom) and water were available <italic>ad libitum</italic>. The experimental protocol was carried out following approval (Filing ID: 15/FEB/01) from the Animal Care and Research Ethics Committee, National University of Ireland, Galway, under license (project authorization number AE19125/PO28) from the Health Products Regulatory Authority in the Republic of Ireland and in accordance with EU Directives 86/609 and 2010/63 and were in accordance with ARRIVE guidelines from the National Centre for the Replacement Refinement and Reduction of Animals in Research (<xref rid=\"B17\" ref-type=\"bibr\">Kilkenny et al., 2010</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Summary of experimental groups.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Sex</bold></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Supplier</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Weights</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Age at surgery</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Control (<italic>n</italic>)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>ChR2 (<italic>n</italic>)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>ArchT (<italic>n</italic>)</bold></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Charles River, United Kingdom</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">200–300 <italic>g</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9–10 weeks</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Charles River, United Kingdom</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">400–500 <italic>g</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9–10 weeks</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td></tr></tbody></table></table-wrap></sec><sec id=\"S2.SS2\"><title>Virus Construction and Packaging</title><p>Recombinant adeno-associated viral (AAV) vectors were serotyped with AAV5 coat proteins and packaged by the viral vector core at the University of Pennsylvania, Philadelphia, PA, United States. Viral titer were 5 × 10<sup>12</sup> particles/mL for AAV5.CAMKII.ChR2-mCherry.WPRE.hGH, AAV5.CAMKII. ArchT.eYFP.WPRE.hGH, and AAV5.CAMKII.mCherry. WPRE.hGH. Plasmids were provided by the Deisseroth lab, Stanford, United States.</p></sec><sec id=\"S2.SS3\"><title>Stereotaxic Intracranial Viral Injections and Optical Fiber Implantation</title><p>Following delivery, rats were left to acclimatize to the animal unit for at least 4 days prior to surgery. They were then placed under isoflurane (2–3% in O<sub>2</sub>, 0.5 L/min) anesthesia and 0.5 μL of virus as specified above was bilaterally injected into the ACC (AP: + 1.5 mm; ML: ± 1.3 mm; DV: -1.3 mm at an angle of 12° toward the midline) at a rate of 0.5 μL/min. The microinjection needle was left in place for an additional 3 min prior to its removal. Rats were then bilaterally implanted with optical fibers (0.39 NA, 200 μm core multimode, Thorlabs, Germany) into the ACC (AP: + 1.5 mm; ML: ± 1.3 mm; DV: -1.0 mm at an angle of 12°toward the midline). Optical fiber implants were permanently fixed to the skull using stainless steel screws and glass ionomer dental cement (GC Europe, Kortrijk, Belgium). The non-steroidal anti-inflammatory drug, carprofen (2.5 mg/kg, s.c., Rimadyl, Pfizer, Kent, United Kingdom), was administered before the surgery to manage postoperative analgesia. To prevent postoperative infection, rats received a single daily dose of the antimicrobial agent enrofloxacin (5 mg/kg, s.c., Baytril, Bayer plc, Berkshire, United Kingdom) on the day of surgery and a subsequent 4 days.</p></sec><sec id=\"S2.SS4\"><title>Formalin-Induced Conditioned Place Aversion</title><p>Animals were randomized to treatment groups and an experimenter blind to treatment carried out behavioral scoring. Behavioral testing was carried out 4 weeks after stereotaxic intracranial opsin encoding AAV injections. A two-chamber apparatus (each chamber 30 cm × 30 cm × 40 cm, l × w × h) with distinct odor (peppermint or strawberry) and visual (black and white balanced stripes or black dots on a white background) contexts was used for the F-CPA behavioral testing (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). The apparatus was tested and optimized in pilot experiments prior to the presented study so that there was no statistically significant difference in the time spent in the two chambers. The behavioral paradigm F-CPA combines the formalin test of tonic, persistent pain with the place-conditioning paradigm to measure pain-related aversion learning in rodents (<xref rid=\"B12\" ref-type=\"bibr\">Johansen et al., 2001</xref>). The test was run over 4 days: Day 1: pre-conditioning, Days 2 and 3: conditioning, and Day 4: post-conditioning. The pre-conditioning day consisted of a 20-min trial in which the rat was allowed free access between both chambers and the time spent in each chamber was recorded. The conditioning days consisted of a 60-min trial in which the rat was restricted to one of the chambers on each of the 2 days. On the second conditioning day (formalin conditioning), the preferred chamber from the pre-conditioning trial was paired with an intra-plantar injection of 50 μl formalin (2.5% in 0.89% saline, Sigma, Ireland) into the right hind-paw under brief isoflurane anesthesia (2% in O<sub>2</sub>; 0.5 L/min) as well as bilateral optogenetic stimulation at 10 Hz (15 ms pulse) with 2 s inter-pulse intervals for ChR2 groups and continuous stimulation for ArchT groups with a 465 nm LED light source (Plexon, United States) for the full 60-min conditioning trial. The behavior during the formalin-conditioning trial was recorded. The post-conditioning day consisted of a 20-min trial in which the rat was again allowed free access between the two chambers and the time in each chamber was recorded. The chambers were cleaned with warm soapy water and dried between each animal to remove odor cues. Male and female animals were tested on separate days, in the same apparatus, by the same experimenter, and under identical conditions.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Schematic of F-CPA apparatus and behavioral testing timeline.</p></caption><graphic xlink:href=\"fnbeh-14-00139-g001\"/></fig></sec><sec id=\"S2.SS5\"><title>Behavioral Analysis</title><p>Behavioral trials were recorded and analyzed off-line using the EthoVisionXT11.5 software (Noldus, Netherlands) by a trained observer blind to the experimental conditions. Formalin-evoked nociceptive behavior was scored for the 60-min post formalin administration (day 3) according to the weighted composite pain scoring (CPS) technique (<xref rid=\"B43\" ref-type=\"bibr\">Watson et al., 1997</xref>). According to this method, pain behaviors are categorized as time spent elevating the formalin-injected paw above the floor without contact with any other surface (Pain 1), and holding, licking, biting, shaking or flinching the injected paw (Pain 2) to obtain a CPS [CPS = (Pain 1 + 2(Pain 2))/(duration of trial)]. For F-CPA, time spent in each chamber during the pre- and post-conditioning trials, as well distance moved and durations of rearing and grooming during the 60-min formalin conditioning trial were also assessed using EthoVisionXT11.5. The number of defecation pellets were counted during the 60-min formalin trial. F-CPA was calculated as duration spent in the formalin-paired chamber during the post-conditioning trial (day 4) minus the duration spent in that same chamber during the pre-conditioning trial (day 1). Therefore, a negative F-CPA score indicates an aversion to the formalin-paired chamber.</p></sec><sec id=\"S2.SS6\"><title>Histology</title><p>Animals were euthanized and perfused immediately after completion of the post-conditioning trial on day 4 of testing. Brains were removed and post-fixed in 4% PFA in 0.1 M PBS for 24 h at 4°C before being transferred into 25% sucrose and 1% sodium azide in 0.1 M PBS. The ACC was later coronally sectioned (30 μm) using a freezing microtome and collected in 0.1 M PBS with 1% sodium azide (Sigma-Aldrich, Ireland). The positions of optical fiber tracts were noted during sectioning to locate and confirm placement in the ACC. Fluorophore-tagged opsin expression was confirmed for each brain by mounting sections onto gelatine-coated slides, cover slipping with VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Labs, United Kingdom), and imaging them using an Olympus wide field inverted fluorescence microscope (Olympus, Tokyo).</p></sec><sec id=\"S2.SS7\"><title>Fluorescent Immunohistochemistry</title><p>For confirmation of opsin expression and analysis of c-Fos immunoreactivity in the ACC, immunohistochemical staining was performed on free-floating sections. Sections were given 3 × 10 min washes in PBS, followed by an incubation for 1 h in 20% normal goat serum (Sigma Aldrich, Ireland) in PBS to block non-specific binding of the secondary antibody. Sections were then incubated in polyclonal rabbit anti-c-Fos antibody (Abcam, United Kingdom) at a concentration of 1:2,000 and rat anti-red fluorescent protein (RFP) antibody (Chromotek, Germany) at a concentration of 1:1,000 made up in PBS, 0.2% (v/v) Triton X, and 1% (w/v) normal goat serum for 24 h at room temperature under constant agitation. The sections were then given 3 × 10-min washes in PBS to remove the primary antibody and were then incubated for 3 h in 1:200 goat anti-rabbit secondary antibody in 10 μl/ml NGS in PB (Abcam, United Kingdom), tagged with either Alexa Fluor 488 for mCherry control and mCherry-tagged ChR2 sections or Alexa Fluor 594 for eYFP-tagged ArchT sections in order to distinguish the c-Fos labeling from the fluorophore-tagged opsin. Sections were kept in the dark and washed 3 × 10 min in PB and stored at 4°C until mounted onto gelatin-coated slides cover slipped with VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Labs, United Kingdom).</p><p>Sections were imaged using an Olympus widefield inverted fluorescence microscope (Olympus, Tokyo). The number of c-Fos immunoreactive neurons within a 1 mm<sup>2</sup> area in the ACC were counted for at least 5 sections per rat. The mean number of c-Fos expressing cells was then calculated for each rat that had at least 5 non-damaged sections for analysis followed by the overall group means. Counting was performed with the aid of NIMH Image J software (Bethesda, MD, United States).</p></sec><sec id=\"S2.SS8\"><title>Statistical Analysis</title><p>IBM SPSS Statistics for Windows, version 26.0 (IBM Corp., Armonk, NY, United States) was used to perform two-way repeated measures analysis of variance (ANOVA) and GraphPad Prism statistical package (Graphpad Prism version 8.02 for Windows, GraphPad Software, La Jolla, CA, United States) was used to perform all other analyses, including two-way ANOVAs and <italic>post hoc</italic> pairwise comparisons. Normality and homogeneity of variance were assessed using Shapiro–Wilk and Brown–Forshythe test, respectively. Two-way repeated measures ANOVA was used to analyze CPS in the formalin test and two-way ANOVA was used to analyze F-CPA with sex and optogenetic modulation as factors. Immunohistochemistry results were analyzed with two-way ANOVA. Kruskal–Wallis test was used to analyze non-parametric data. <italic>Post hoc</italic> pairwise comparisons were made using Fisher’s LSD and corrected Dunn’s tests where appropriate. Data were considered significant when <italic>p</italic> < 0.05. Correlation analysis on c-Fos and F-CPA data was performed using Pearson’s correlation. Results are expressed as group means ± standard error of the mean (±SEM).</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Histological Verification of Implant Locations and Opsin Expression</title><p>After histological verification, 90% of males and 88% of females had implant tracts that were found to be within the borders of both the left and the right ACC. The remaining implants were placed in the corpus callosum, or outside the borders of the ACC. Only data from rats where optical fibers were accurately placed in both the left and the right ACC and that had opsin expression within the ACC have been included in the analysis (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Schematic depicting sites of optical fiber implantation in the <bold>left</bold> and <bold>right</bold> ACC of male and female rats, adapted from <xref rid=\"B35\" ref-type=\"bibr\">Paxinos and Watson (2007)</xref>.</p></caption><graphic xlink:href=\"fnbeh-14-00139-g002\"/></fig></sec><sec id=\"S3.SS2\"><title>Formalin-Evoked Nociceptive Behavior</title><p>Intra-plantar administration of formalin into the right hind paw produced robust nociceptive behavior in both male and female SD rats as evidenced by the composite pain score. A two-way repeated measures ANOVA revealed significant effects of time [<italic>F</italic><sub>(</sub><sub>1</sub>,<sub>49</sub><sub>)</sub> = 13.83; <italic>p</italic> < 0.0001], sex × optogenetic modulation interaction [<italic>F</italic><sub>(</sub><sub>2</sub>,<sub>49</sub><sub>)</sub> = 5.56; <italic>p</italic> < 0.01], and time × sex × optogenetic modulation interaction [<italic>F</italic><sub>(</sub><sub>22</sub>,<sub>49</sub><sub>)</sub> = 1.83; <italic>p</italic> < 0.05] on formalin-evoked nociceptive behavior across the 60-min formalin trial but no effects of optogenetic modulation, sex, time × sex, or time × optogenetic modulation interactions (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p><bold>(A)</bold> Effects of optogenetic stimulation (ChR2) or inhibition (ArchT) of glutamatergic neurons in the ACC on formalin-evoked nociceptive behavior of male Sprague-Dawley rats. Data are mean ± SEM (<italic>n</italic> = 8–10 per group). **<italic>p</italic> < 0.01, *<italic>p</italic> < 0.05 ChR2 vs control. <bold>(B)</bold> Effects of optogenetic stimulation (ChR2) or inhibition (ArchT) of glutamatergic neurons in the ACC on formalin-evoked nociceptive behavior of female Sprague-Dawley rats. Data are mean ± SEM (<italic>n</italic> = 7–11 per group). *<italic>p</italic> < 0.05 ChR2 vs control. <sup>+</sup><italic>p</italic> < 0.05 ChR2 female vs ChR2 male, and <sup>&</sup><italic>p</italic> < 0.05 ArchT female vs ArchT male.</p></caption><graphic xlink:href=\"fnbeh-14-00139-g003\"/></fig><p><italic>Post hoc</italic> analysis revealed that optogenetic activation (ChR2) of glutamatergic neurons in the ACC significantly reduced (<italic>p</italic> < 0.05 or 0.01) formalin-evoked nociceptive behavior in male rats compared to controls at time bins 9 and 11 of the formalin trial (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>). By contrast, in female rats, optogenetic activation (ChR2) of glutamatergic neurons significantly increased (<italic>p</italic> < 0.05 or 0.01) formalin-evoked nociceptive behavior compared to controls at time bins 7–9 (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>). Moreover, formalin-evoked nociceptive behavior in the Female-ChR2 group was significantly higher than in Male-ChR2 counterparts at time bins 8 and 9 (<italic>p</italic> < 0.05). Optogenetic inhibition (ArchT) of glutamatergic neurons in the ACC had no significant effect on formalin-evoked nociceptive behavior in male or female rats, however, the Female-ArchT group exhibited significantly less formalin-evoked nociceptive behavior compared to Male-ArchT counterparts at time bins 8, 9, and 11 (<italic>p</italic> < 0.05; <xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>).</p></sec><sec id=\"S3.SS3\"><title>Effects of Optogenetic Modulation on General Locomotor Activity and Defecation in Formalin-Treated Rats</title><p>Two-way ANOVAs revealed that there were significant effects of sex on distance moved [<italic>F</italic><sub>(</sub><sub>1</sub>,<sub>49</sub><sub>)</sub> = 7.858; <italic>p</italic> < 0.01] and grooming [<italic>F</italic><sub>(</sub><sub>1</sub>,<sub>49</sub><sub>)</sub> = 15.82; <italic>p</italic> < 0.01] during the formalin trial, but no effects of optogenetic modulation of glutamatergic neurons in the ACC or sex × optogenetic modulation interaction (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). Distance moved (<italic>p</italic> < 0.01) and duration of grooming (<italic>p</italic> < 0.001) were significantly greater in males than females regardless of optogenetic manipulation. Two-way ANOVAs revealed that there were no effects of optogenetic modulation or sex or their interaction on rearing or defecation (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>).</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Effects of optogenetic modulation of glutamatergic neurons in the ACC on general locomotor behavior and defecation during 60-min formalin conditioning trial in male and female rats.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Grooming (s)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Rearing (s)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Distance moved (cm)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Defecation (pellet number)</bold></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male-Control (<italic>n</italic> = 9)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">207.340.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25.410.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3915719</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.60.5</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male-ChR2 (<italic>n</italic> = 8)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">165.054.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20.99.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50371410</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.50.7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male-ArchT (<italic>n</italic> = 10)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">105.424.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.09.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">48841255</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.80.4</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male-Total (<italic>n</italic> = 27)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15723.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.95.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4606650</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.90.3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female-Control (<italic>n</italic> = 7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">57.215.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19.713.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2633289</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.70.8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female-ChR2 (<italic>n</italic> = 11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">63.318.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.72.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3003265</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.90.4</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female-ArchT (<italic>n</italic> = 10)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">62.313.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14.93.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2475200</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.30.3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female-Total (<italic>n</italic> = 28)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">61.59.3***</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.13.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2722147**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.90.3</td></tr></tbody></table><table-wrap-foot><attrib><italic>Data are mean ± SEM (<italic>n</italic> = 7–11 per group). ***<italic>p</italic> < 0.001, **<italic>p</italic> < 0.01 vs corresponding Male- Total, where Male-Total and Female-Total are the means ± SEM for all rats within each sex.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS4\"><title>Formalin-Induced Conditioned Place Aversion</title><p>Two-way ANOVA revealed an effect of optogenetic modulation of glutamatergic neurons in the ACC [<italic>F</italic><sub>(</sub><sub>2</sub>,<sub>49</sub><sub>)</sub> = 3.910; <italic>p</italic> = 0.03] on F-CPA behavior, but not of sex or sex × optogenetic modulation interaction (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). Optogenetic inhibition (ArchT), but not stimulation (ChR2), of ACC significantly reduced (<italic>p</italic> < 0.01) F-CPA behavior compared to control fluorophore-expressing rats, regardless of sex (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>).</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p><bold>(A)</bold> Effects of optogenetic stimulation (ChR2) or inhibition (ArchT) of glutamatergic neurons in the ACC on formalin-induced conditioned place aversion (F-CPA) in combined male and female Sprague-Dawley rats. Data are mean ± SEM (<italic>n</italic> = 7–11 per group). **<italic>p</italic> < 0.01 vs control. <bold>(B)</bold> Effects of optogenetic stimulation (ChR2) or inhibition (ArchT) of glutamatergic neurons in the ACC on formalin-induced conditioned place aversion (F-CPA) in male Sprague-Dawley rats. Data are mean ± SEM (<italic>n</italic> = 8–10 per group). <bold>(C)</bold> Representative heat map images of post-conditioning trials for each treatment group in male rats. <bold>(D)</bold> Effects of optogenetic stimulation (ChR2) or inhibition (ArchT) of glutamatergic neurons in the ACC on formalin-induced conditioned place aversion (F-CPA) score of female Sprague-Dawley rats. Data are mean ± SEM (<italic>n</italic> = 7–11 per group). <bold>(E)</bold> Representative heat map images of post-conditioning trials for each treatment group in female rats.</p></caption><graphic xlink:href=\"fnbeh-14-00139-g004\"/></fig></sec><sec id=\"S3.SS5\"><title>c-Fos Immunoreactive Cells in the ACC</title><p>Two-way ANOVA revealed a significant effect of optogenetic modulation of glutamatergic neurons in the ACC during the day 3 formalin conditioning trial [<italic>F</italic><sub>(</sub><sub>2</sub>,<sub>24</sub><sub>)</sub> = 6.289; <italic>p</italic> < 0.01] and a significant effect of sex [<italic>F</italic><sub>(</sub><sub>1</sub>,<sub>24</sub><sub>)</sub> = 14.47; <italic>p</italic> < 0.01] on c-Fos-positive immunoreactive cells in the ACC after the day 4 post-conditioning trial (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>). <italic>Post hoc</italic> analysis showed that optogenetic inhibition (ArchT) of glutamatergic neurons in the ACC during formalin conditioning significantly reduced the number of c-Fos-positive immunoreactive cells in the ACC of male rats compared to controls (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>; <italic>p</italic> < 0.05) and that by contrast, optogenetic activation (ChR2) significantly increased the number of c-Fos-positive immunoreactive cells in the ACC of females rats compared to controls (<xref ref-type=\"fig\" rid=\"F5\">Figure 5C</xref>; <italic>p</italic> < 0.01). A correlation analysis was also performed between F-CPA score and the number of c-Fos-positive cells, for groups that exhibited significant differences in c-Fos expression (i.e., the control and ArchT groups for the male rats and the control and ChR2 groups for females). We found that there was a negative correlation between F-CPA score and c-Fos immunoreactivity in the ACC [<italic>r</italic>(9) = -0.59; <italic>p</italic> = 0.05] among the control and ArchT male rats, suggesting that greater formalin-induced aversive behavior was coupled with increased neuronal activity in the ACC in males. However, no correlation between F-CPA score and c-Fos was found in the control and ChR2 female groups [<italic>r</italic>(8) = 0.29; <italic>p</italic> = 0.41].</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Effects of optogenetic stimulation (ChR2) or inhibition (ArchT) of glutamatergic neurons in the ACC on c-Fos positive cells in the ACC of SD rats. <bold>(A)</bold> Representative images of c-Fos (green) expression within the ACC (scale bar = 200 μm). White arrows denote representative c-Fos-positive staining. <bold>(B)</bold> Effects of optogenetic stimulation (ChR2) or inhibition (ArchT) of glutamatergic neurons in the ACC on c-Fos positive cells in the ACC of male SD rats. Data are mean ± SEM (<italic>n</italic> = 5–6 per group). *<italic>p</italic> < 0.05 vs. control. <bold>(C)</bold> Effects of optogenetic stimulation (ChR2) or inhibition (ArchT) of glutamatergic neurons in the ACC on percent of c-Fos positive cells in the ACC of female SD rats. Data are mean ± SEM (<italic>n</italic> = 4–6 per group). *<italic>p</italic> < 0.05 vs. control.</p></caption><graphic xlink:href=\"fnbeh-14-00139-g005\"/></fig></sec></sec><sec id=\"S4\"><title>Discussion</title><p>The results of the present study reveal differential effects of optogenetic modulation on formalin-evoked nociceptive behavior and F-CPA in male and female rats. We found that while optogenetic inhibition did not affect formalin-evoked nociceptive behavior in either sex, optogenetic activation of glutamatergic neurons of the ACC had a differential effect in males and females, reducing formalin-evoked nociceptive behavior in males during the second phase of the formalin trial and increasing formalin-evoked nociceptive behavior in females during this same stage of the trial. One mechanism suggested for the differences in pain observed between males and females is sex-dependent pathways for analgesia and hyperalgesia (<xref rid=\"B34\" ref-type=\"bibr\">Nemmani et al., 2004</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Bryant et al., 2006</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Bliss et al., 2016</xref>). Non-competitive antagonism of NMDA receptors potentiates morphine analgesia in male but not female mice (<xref rid=\"B34\" ref-type=\"bibr\">Nemmani et al., 2004</xref>), and attenuates morphine tolerance in male but not female mice (<xref rid=\"B4\" ref-type=\"bibr\">Bryant et al., 2006</xref>). Human studies have found differences in the functional connectivity between the subgenual ACC (sgACC) and various brain regions of the descending pain pathway in men and women (<xref rid=\"B42\" ref-type=\"bibr\">Wang et al., 2014</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Monroe et al., 2017</xref>). For instance, women have greater functional connectivity between the sgACC and the periaqueductal gray (PAG) than men. It has been suggested that these projections from the ACC to the PAG may be involved in facilitating nociception through the RVM (<xref rid=\"B5\" ref-type=\"bibr\">Calejesan et al., 2000</xref>). If the functional connectivity of the ACC to the PAG is stronger in females compared to males, this difference could explain how optogenetic activation of glutamatergic neurons of the ACC increased formalin-evoked nociceptive behaviors in females and not in males.</p><p>Another possible mechanism underlying sex differences in pain is the influence of sex hormones (<xref rid=\"B27\" ref-type=\"bibr\">Martin, 2009</xref>). In particular, the affective component of pain has been found to be modulated by sex hormones. In female rats, pain-related aversive behavior is attenuated by ovariectomy (<xref rid=\"B7\" ref-type=\"bibr\">Chen et al., 2014</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Li L.-H. et al., 2014</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Hubbard et al., 2015</xref>). Inhibition of estrogen receptors or aromatase androstatrienedione in the ACC blocks F-CPA in both male and female rats, and exogenous estrogen elicits aversive behavior in the absence of a noxious stimulus in both sexes (<xref rid=\"B44\" ref-type=\"bibr\">Xiao et al., 2013</xref>). A limitation of the present study is the lack of estrous cycle stage assessment, however, we did not observe any effect of sex on F-CPA. It has been reported that estrogen enhances glutamatergic excitatory postsynaptic currents (EPSCs) in the ACC, suggesting that the mechanism by which estrogen modulates affective pain may be through modulation of glutamatergic transmission in the ACC (<xref rid=\"B44\" ref-type=\"bibr\">Xiao et al., 2013</xref>). Similarly, female rats in a high estrogen state exhibit increased pain behaviors and reduced excitatory amino acid transporter (EAAT) function in the ACC compared to rats in a low estrogen (<xref rid=\"B32\" ref-type=\"bibr\">Moloney et al., 2016</xref>). These findings may explain why we saw an increase in nociceptive behaviors with optogenetic activation (ChR2) of glutamatergic neurons in the ACC of female, but not male, rats.</p><p>Another key finding of the present study is that the inhibition of glutamatergic neurons of the ACC is sufficient to abolish formalin-induced aversion learning without reducing nociceptive behavior. Our results support and extend previous work which showed that administration of an NMDA receptor antagonist into the ACC abolished F-CPA in SD rats, while not affecting formalin-induced nociceptive behavior (<xref rid=\"B11\" ref-type=\"bibr\">Johansen and Fields, 2004</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Lei et al., 2004a</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Ren et al., 2006</xref>), suggesting that glutamatergic neurons in the ACC are active during pain states and are responsible for the aversive component of pain and that it is possible to dissociate this aversive component of pain from the sensory component. Previous work has also demonstrated that the NMDAR subunits NR2A and NR2B, as well as the NMDAR downstream ERK pathway, within the ACC, are necessary for pain-related aversion learning in rats (<xref rid=\"B6\" ref-type=\"bibr\">Cao et al., 2009</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Li et al., 2009</xref>). Our immunohistochemical analysis revealed a reduction in c-Fos immunoreactive neurons in the ACC during the post-conditioning trial in male rats that received optogenetic inhibition of glutamatergic neurons in the ACC during the formalin conditioning trial, which was not seen in female rats that also received optogenetic inhibition. These results correspond to, and correlate with, the behavioral results observed, in that male rats that received optogenetic inhibition exhibited a reduction in formalin-evoked aversion behavior, which is in agreement with <xref rid=\"B20\" ref-type=\"bibr\">Lei et al. (2004b)</xref> who found that Fos immunoreactivity in the ACC is significantly increased after retrieval of formalin-induced conditioned place avoidance. By contrast, in female rats, optogenetic activation of glutamatergic neurons was associated with an increase in Fos immunoreactivity in the ACC. Interestingly, however, this increase was not coupled with the F-CPA behavior in females, as it was in males. There was no effect of optogenetic modulation on general locomotor activity, suggesting that the effect of optogenetic modulation on formalin-evoked nociceptive behavior was not a result of alterations in motor behavior. Female rats had lower levels of grooming and distance moved compared to males, but as there were no differences between control males and females for formalin-evoked nociceptive behavior, F-CPA or CPS, despite the difference in grooming, and since there were no effects of optogenetic manipulation on grooming, despite its effects on formalin-evoked nociceptive behavior, F-CPA or CPS, it is unlikely that sex differences in grooming have a bearing on the key findings and conclusions of this study.</p><p>A feature of the CPA behavioral paradigm is the necessary learning component, and thus it is possible that the effects of optogenetic modulation could result either from an effect on aversion behavior <italic>per se</italic> and/or an effect on general memory and learning. However, a previous study has found that optogenetic inhibition of glutamatergic neurons in the ACC of mice following induction of complete Freund’s adjuvant-induced arthritic pain induces place preference, suggesting that inhibition of these neurons does not inhibit memory acquisition or learning (<xref rid=\"B14\" ref-type=\"bibr\">Kang et al., 2017</xref>). It has also been found that optogenetic activation of glutamatergic neurons of the ACC in the absence of pain induces CPA to the stimulation-paired chamber (<xref rid=\"B41\" ref-type=\"bibr\">Tan et al., 2017</xref>).</p><p>The present study reveals, for the first time, sex differences in the role of glutamatergic neurons of the ACC in sensory and aversive components of pain. A particularly interesting result was the differential effect of activation of glutamatergic neurons of the ACC on formalin-evoked nociceptive behavior in males and females. The results of the study also support and extend the current understanding that inhibition of glutamatergic neurons of the ACC prevents aversion learning without affecting nociceptive behavior. Our findings identify glutamatergic neurons within the ACC as a potentially important substrate influencing sex differences in pain responding.</p></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p></sec><sec id=\"S6\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by Animal Care and Research Ethics Committee, National University of Ireland Galway.</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>SJ and DF designed the experiments. SJ conducted the behavioral experiments and analysis of the c-Fos expression by immunohistochemistry, analyzed the data, and wrote the manuscript. DF, MR, and AP reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by financial support from Science Foundation Ireland (SFI) and co-funded under the European Regional Development Fund under Grant Number 13/RC/2073. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Genet</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Genet</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Genet.</journal-id><journal-title-group><journal-title>Frontiers in Genetics</journal-title></journal-title-group><issn pub-type=\"epub\">1664-8021</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849838</article-id><article-id pub-id-type=\"pmc\">PMC7431633</article-id><article-id pub-id-type=\"doi\">10.3389/fgene.2020.00867</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Genetics</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Single-Cell Transcriptome Analysis Reveals Six Subpopulations Reflecting Distinct Cellular Fates in Senescent Mouse Embryonic Fibroblasts</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Wei</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/967011/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Xuefei</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1047907/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wei</surname><given-names>Gang</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/990531/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Huang</surname><given-names>Yin</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Shi</surname><given-names>Yufang</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Dan</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Qiu</surname><given-names>Shengnu</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhou</surname><given-names>Bin</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/598356/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Cao</surname><given-names>Junhong</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Meng</given-names></name><xref ref-type=\"aff\" rid=\"aff7\"><sup>7</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Qin</surname><given-names>Pengfei</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Jin</surname><given-names>Wenfei</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c003\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/990554/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ni</surname><given-names>Ting</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c004\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/668913/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, School of Life Sciences, Human Phenome Institute, Fudan University</institution>, <addr-line>Shanghai</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Biology, Southern University of Science and Technology</institution>, <addr-line>Shenzhen</addr-line>, <country>China</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences</institution>, <addr-line>Shanghai</addr-line>, <country>China</country></aff><aff id=\"aff4\"><sup>4</sup><institution>The First Affiliated Hospital of Soochow University and State Key Laboratory of Radiation Medicine and Protection, Institutes for Translational Medicine, Soochow University</institution>, <addr-line>Suzhou</addr-line>, <country>China</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Field Application Department, Fluidigm (Shanghai) Instrument Technology Co., Ltd.</institution>, <addr-line>Shanghai</addr-line>, <country>China</country></aff><aff id=\"aff6\"><sup>6</sup><institution>Division of Biosciences, Faculty of Life Sciences, University College London</institution>, <addr-line>London</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff7\"><sup>7</sup><institution>Eye Institute, Eye & ENT Hospital, Shanghai Medical College, Fudan University</institution>, <addr-line>Shanghai</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Alexey Moskalev, Institute of Biology, Komi Scientific Center (RAS), Russia</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Marco Demaria, University Medical Center Groningen, Netherlands; Argyris Papantonis, University Medical Center Göttingen, Germany</p></fn><corresp id=\"c001\">*Correspondence: Meng Chen, <email>chenmeng_hunter@163.com</email></corresp><corresp id=\"c002\">Pengfei Qin, <email>qinpf@sustech.edu.cn</email></corresp><corresp id=\"c003\">Wenfei Jin, <email>jinwf@sustech.edu.cn</email></corresp><corresp id=\"c004\">Ting Ni, <email>tingni@fudan.edu.cn</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Genetics of Aging, a section of the journal Frontiers in Genetics</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>867</elocation-id><history><date date-type=\"received\"><day>02</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>16</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Chen, Wang, Wei, Huang, Shi, Li, Qiu, Zhou, Cao, Chen, Qin, Jin and Ni.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Chen, Wang, Wei, Huang, Shi, Li, Qiu, Zhou, Cao, Chen, Qin, Jin and Ni</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Replicative senescence is a hallmark of aging, which also contributes to individual aging. Mouse embryonic fibroblasts (MEFs) provide a convenient replicative senescence model. However, the heterogeneity of single MEFs during cellular senescence has remained unclear. Here, we conducted single-cell RNA sequencing on senescent MEFs. Principal component analysis showed obvious heterogeneity among these MEFs such that they could be divided into six subpopulations. Three types of gene expression analysis revealed distinct expression features of these six subpopulations. Trajectory analysis revealed three distinct lineages during MEF senescence. In the main lineage, some senescence-associated secretory phenotypes were upregulated in a subset of cells from senescent clusters, which could not be distinguished in a previous bulk study. In the other two lineages, a possibility of escape from cell cycle arrest and coupling between translation-related genes and ATP synthesis-related genes were also discovered. Additionally, we found co-expression of transcription factor HOXD8 coding gene and its potential target genes in the main lineage. Overexpression of <italic>Hoxd8</italic> led to senescence-associated phenotypes, suggesting HOXD8 is a new regulator of MEF senescence. Together, our single-cell sequencing on senescent MEFs largely expanded the knowledge of a basic cell model for aging research.</p></abstract><kwd-group><kwd>cellular senescence</kwd><kwd>single-cell RNA sequencing</kwd><kwd>mouse embryonic fibroblasts</kwd><kwd>transcriptomic heterogeneity</kwd><kwd>senescence-associated secretory phenotype</kwd><kwd>Hoxd8</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Natural Science Foundation of China<named-content content-type=\"fundref-id\">10.13039/501100001809</named-content></funding-source><award-id rid=\"cn001\">91949107</award-id><award-id rid=\"cn001\">31771336</award-id><award-id rid=\"cn001\">31521003</award-id></award-group></funding-group><counts><fig-count count=\"5\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"62\"/><page-count count=\"16\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Cellular senescence was first discovered by <xref rid=\"B19\" ref-type=\"bibr\">Hayflick and Moorhead (1961)</xref> in normal human fibroblasts during a study in which they observed a finite replicative life span. The mechanism of this phenomenon was subsequently explained as gradual shortening and loss of telomeres in the absence of telomerase (<xref rid=\"B44\" ref-type=\"bibr\">Olovnikov, 1996</xref>). Loss of telomeres triggers the DNA damage response. Proteins involved in DNA damage response (e.g., ATM, Chk1, and Chk2) activates cell cycle proteins p53 and p21, which then inhibit a subset of cyclin-dependent kinases (CDKs) and lead to cell cycle arrest (<xref rid=\"B31\" ref-type=\"bibr\">Leonardo et al., 1994</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Herbig et al., 2004</xref>). In addition to replicative exhaustion, other factors such as DNA damage, oncogene hyperactivation, oxidative stress, and chromatin perturbation can also cause cellular senescence. These factors are considered “stress” and can induce premature cellular senescence. DNA damage can be caused by external agents, such as H<sub>2</sub>O<sub>2</sub>, X-ray or ultraviolet irradiation, and many chemotherapeutic drugs. The pathways activated in this context are similar to those activated during telomere erosion. In oncogene-induced cellular senescence, overexpression (OE) of certain oncogenes causes a DNA damage response or upregulation of alternative reading frame (ARF) (belongs to CDKN2A locus), which activates the p53-p21 pathway or p16 (<xref rid=\"B6\" ref-type=\"bibr\">Campisi and Fagagna, 2007</xref>). In contrast to replicative senescence, oncogene-induced senescence (OIS) is telomere-independent and much more acute (<xref rid=\"B16\" ref-type=\"bibr\">Gorgoulis and Halazonetis, 2010</xref>). In mouse embryonic fibroblasts (MEFs), telomeres are much longer (40–60 kb) than those in human cells (5–15 kb). Cellular senescence that occurs in MEFs is a response to oxidative stress, instead of telomere erosion (<xref rid=\"B49\" ref-type=\"bibr\">Sherr and Dipinho, 2000</xref>; <xref rid=\"B57\" ref-type=\"bibr\">Wright and Shay, 2000</xref>). MEFs cultured in standard conditions (20% oxygen) show substantial DNA damage, whereas physical oxygen concentration (3% oxygen) does not trigger cellular senescence in MEFs (<xref rid=\"B45\" ref-type=\"bibr\">Parrinello et al., 2003</xref>). Exposure of MEFs to oxidative stress activates the p19/ARF-p53 pathway, which leads to cellular senescence (<xref rid=\"B18\" ref-type=\"bibr\">Harvey et al., 1993</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Kamijo et al., 1997</xref>). In summary, replicative senescence is intrinsic and acts as an intracellular mitotic clock in the cell, whereas stress-induced senescence is extrinsic.</p><p>Based on the notion that tumorigenesis is the result of escape of a cell’s regular senescence fate of a cell, replicative senescence can be regarded as an important cancer prevention mechanism (<xref rid=\"B13\" ref-type=\"bibr\">Elkon et al., 2009</xref>). A recent study demonstrated that the <italic>in vivo</italic> induction of senescence in cancer cells attracts natural killer cells to clear the cancer cells; thus, this senescence is beneficial to immunotherapy (<xref rid=\"B47\" ref-type=\"bibr\">Ruscetti et al., 2018</xref>). Senescence-associated secretory phenotype (SASP) components released by senescent cancer cells mediate such clearance by immune cells. Replicative senescence also contributes to individual aging (<xref rid=\"B34\" ref-type=\"bibr\">López-Otín et al., 2013</xref>). Accumulation of senescent cells in aged tissues/organs leads to a considerable release of SASP components into the local environment, which promotes senescence of nearby cells in a paracrine fashion and ultimately results in tissue/organ dysfunction (<xref rid=\"B10\" ref-type=\"bibr\">Dimri et al., 1995</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Muñoz-Espín and Serrano, 2014</xref>). Thus, clearance of senescent cells in the mouse model benefits tissue function and increases health span (<xref rid=\"B3\" ref-type=\"bibr\">Baker et al., 2016</xref>).</p><p>Studies of cellular senescence have been performed using normal human fibroblasts (<xref rid=\"B19\" ref-type=\"bibr\">Hayflick and Moorhead, 1961</xref>), human diploid keratinocytes (<xref rid=\"B46\" ref-type=\"bibr\">Rheinwald and Green, 1975</xref>), human vascular smooth muscle cells (<xref rid=\"B4\" ref-type=\"bibr\">Bierman, 1978</xref>), human lens cells (<xref rid=\"B52\" ref-type=\"bibr\">Tassin et al., 1979</xref>), and human peripheral lymphocyte (<xref rid=\"B55\" ref-type=\"bibr\">Tice et al., 1979</xref>), as well as a variety of other cells. MEFs have a relatively short cultivation time <italic>in vitro</italic> (typically 15–30 population doublings) and thus serve as a time-saving model to study cellular senescence (<xref rid=\"B49\" ref-type=\"bibr\">Sherr and Dipinho, 2000</xref>). Previous studies illustrated that <italic>in vitro</italic> cultivated senescent MEFs manifested upregulation of <italic>Cdkn1a</italic> (encoding p21), <italic>Cdkn2a</italic> (encoding p16), <italic>Lmnb1</italic>, <italic>Mki67</italic>, and other conventional senescence markers (<xref rid=\"B49\" ref-type=\"bibr\">Sherr and Dipinho, 2000</xref>; <xref rid=\"B45\" ref-type=\"bibr\">Parrinello et al., 2003</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Itahana et al., 2004</xref>). This phenotype was similar to that of senescence in human fibroblasts (<xref rid=\"B26\" ref-type=\"bibr\">Kim et al., 2013</xref>). Significant enhancement of senescence-associated β-galactosidase (SA-β-Gal) staining was also detected in senescent MEFs, mimicking the same traditional senescence marker in human cells (<xref rid=\"B10\" ref-type=\"bibr\">Dimri et al., 1995</xref>). Furthermore, primary MEF cells have been used for lentivirus-mediated gene knockdown or OE experiments, facilitating the mechanistic investigation of candidate genes’ impact on senescence (<xref rid=\"B58\" ref-type=\"bibr\">Xu et al., 2019</xref>). Importantly, findings discovered in cultured MEFs can also be extended <italic>in vivo</italic>. For example, the transforming growth factor (TGF)-β/miR-29 pathway that originally revealed in MEFs was further proven to contribute to cardiac aging in a mouse model (<xref rid=\"B35\" ref-type=\"bibr\">Lyu et al., 2018</xref>). Therefore, MEFs can be used as an alternative model for investigation of cellular senescence.</p><p>In the past decade, various single-cell RNA sequencing (scRNA-seq) methods were developed and revealed the heterogeneity of seemingly identical cells, emphasizing minute differences between single cells in immune cells, embryonic cells, neurons, and others (<xref rid=\"B51\" ref-type=\"bibr\">Tang et al., 2010</xref>; <xref rid=\"B59\" ref-type=\"bibr\">Xue et al., 2013</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Deng et al., 2014</xref>). Some studies applied scRNA-seq to aging and/or senescence. One study reported that upon activation, CD4<sup>+</sup> T cells from old (21 months) B6 or CAST mice displayed higher cell-to-cell transcriptomic variability of the core activation program compared with cells from their young (3 months) counterparts; this increasing transcriptomic variability was independent of expression level alteration (<xref rid=\"B39\" ref-type=\"bibr\">Martinez-Jimenez et al., 2017</xref>). Another study illustrated that hematopoietic stem cells from old mice have a higher rate of self-renewal and a lower rate of differentiation, compared with those cells from younger mice, based on single-cell transcriptome analysis (<xref rid=\"B28\" ref-type=\"bibr\">Kowalczyk et al., 2015</xref>). By combining Hi-C, single-cell and bulk RNA-seq, as well as imaging in proliferating and senescent human umbilical vein endothelial cells (HUVECs), <xref rid=\"B62\" ref-type=\"bibr\">Zirkel et al. (2018)</xref> found that high mobility group box 2 (HMGB2) loss during early senescence led to genomic reorganization and senescence-induced CCCTC-Binding Factor (CTCF) clustering. Despite such progress regarding aging-related single-cell RNA-seq analyses, some basic questions remain elusive in replicative senescence models such as MEFs. For example: (1) What is the degree of heterogeneity in MEF senescence? (2) Does SASP contribute to MEF senescence? (3) Could scRNA-seq aid in the discovery of novel senescence regulators in MEFs?</p><p>To address these questions, we performed single-cell full-length RNA-seq in atmospheric (20%) oxygen-cultivated MEFs at a passage where half of the cells were positive in SA-β-Gal staining. We observed considerable variation in gene expression levels, although these cells were obtained from the same passage. Six clusters and three distinct lineages were detected, which exhibited specific molecular features during senescence. In contrast to the finding of a previous bulk study, we discovered that some SASP components were upregulated in a few single MEFs from senescent clusters. Additionally, OE of <italic>Hoxd8</italic> led to several senescence-associated phenotypes. This study provides a new perspective for understanding the basic features of an important senescence model.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Cell Isolation and <italic>in vitro</italic> Cultivation</title><p>Mouse embryos were taken from 12.5∼14.5 days of pregnant C57BL/6, and primary MEF cells were isolated following a previously described protocol (<xref rid=\"B56\" ref-type=\"bibr\">Todaro and Green, 1963</xref>). NIH3T3 cells were provided by the American Type Culture Collection (ATCC, Manassas, VA, United States). Cells were cultivated <italic>in vitro</italic> in Dulbecco’s modified Eagle’s medium (DMEM) medium (Gibco) with 10% fetal bovine serum (FBS; Gibco) in 25-cm<sup>2</sup> flasks, which were placed in an incubator with 5% CO<sub>2</sub> and 37•C. Once the confluence reached 70% in the flask, cells were resuspended by 0.25% trypsin-EDTA (Gibco) and evenly divided into two new flasks. Population doubling (PD) was added by 1 each time MEF cells were subcultured.</p></sec><sec id=\"S2.SS2\"><title>Single-Cell RNA Sequencing</title><p>PD9 MEF cells were collected. Cell counting was performed in Cellometer Mimi (Nexcelom Bioscience). Single cells were added into three 17–25-μm Single-Cell mRNA Seq IFC (Fluidigm C1). After loading into the chip, cells were imaged on the microscope to filter out wells with no cell, cell doublet, or cell debris. Full-length cDNA libraries were auto-constructed in Fluidigm C1 system using SMART-seq v4 kits. Quality control was carried out on each single-cell cDNA library using Qubit 3.0 and Aligent Bioanalyzer 2100 to exclude libraries with abnormal molecular features. Sequencing libraries were constructed using Nextera XT DNA library kit, and another round of quality control was performed. RNA-seq libraries were then pooled and sequenced by Illumina Hiseq 4000 with an average depth of 3 million reads for each single cell.</p></sec><sec id=\"S2.SS3\"><title>Paracrine Experiments</title><p>For total SASP experiments, primary MEF cells were <italic>in vitro</italic> cultivated to PD11. PD11 MEF cells were cultivated with DMEM medium (10% FBS) for 2 days, and senescence-conditioned medium (SCM) was collected. Then, SCM was centrifuged at 3,000 rpm for 5 min and filtered through a 0.45-μm syringe filter. After that, newly thawed primary MEF cells were evenly distributed into two flasks and cultured with normal medium (NM) and SCM simultaneously. For interleukin (IL)6 experiments, we bought recombinant mouse IL6 from R&D Systems (Bio-Techne). Newly thawed primary MEF cells were evenly distributed into three flasks and cultured with DMEM (10% FBS), DMEM (10% FBS and 5 ng/ml IL6), and DMEM (10% FBS and 50 ng/ml IL6) simultaneously. SA-β-Gal staining, cell cycle analysis, RNA extraction, quantitative reverse transcription PCR (qRT-PCR), and RNA-seq were conducted on these three different treated MEFs.</p></sec><sec id=\"S2.SS4\"><title>Senescence-Associated β-Galactosidase Staining</title><p>Proper amount of cells were seeded into multiple wells in a 12-well plate and cultivated for 24 h. Then, SA-β-Gal staining was performed for MEFs with different conditions using Senescence Cells Histochemical Staining Kit (Sigma) following its manual. Photos were taken under an inverted microscope.</p></sec><sec id=\"S2.SS5\"><title>Cell Cycle Analysis</title><p>One fourth of the cells in a flask with 70% confluence was lysed and washed twice with 1 × phosphate buffered saline (PBS). Then, cells were permeabilized with 50 μg/ml Triton-X100 (Sigma) and stained with 0.15% propidium iodide (Thermal Fisher) in the dark for 10 min. Cells were then loaded into Calibur flow cytometer (Becton Dickinson) to measure the fluorescence. Three replicates for each sample were performed. Results were analyzed by ModFit software.</p></sec><sec id=\"S2.SS6\"><title>Ethynyl-2′-Deoxyuridine Incorporation Assay</title><p>The proper amount of cells was seeded into multiple wells in a 24-well plate. Cells were cultivated until the confluence reached 50%. Then, culture medium was aspirated, and cell was incubated at 10 μM 5-ethynyl-2′-deoxyuridine (EdU) for 2 h. Cell fixation, permeation, and staining with fluorescence dyes were implemented following keyFluor488 Click-iT Edu image kit manual (KeyGEN). Fluorescence was examined under IX73 fluorescence microscope (Olympus) with 495 nm (kFluor488) and 350 nm (Hochest 33342) excitation filter. Photos shot on the same field were merged.</p></sec><sec id=\"S2.SS7\"><title>Cell Proliferation Rate Assay</title><p>A total of 2,000 cells were seeded into each well of a 96-well plate. For each sample, five replicates were applied (five wells per sample). Cells were then cultivated at an incubator for 24, 48, 72, and 96 h. After cultivation, cell medium was aspirated, and cells were incubated at 10% Cell Counting Kit-8 solution (Dojindo) at 37•C for 3 h. Absorbance at 450 and 630 nm were detected at Bio-Rad 680 microplate reader.</p></sec><sec id=\"S2.SS8\"><title>Quantitative Reverse Transcription PCR and Bulk Population RNA Sequencing</title><p>Total RNA was extracted by TRIzol reagent (Life Technology) following its manual. For qRT-PCR, genomic DNA depletion and reverse transcription were performed using HiScript II Q RT SuperMix for qPCR (Vazyme) following its manual. qPCR mix was composed of 1 μl 1:5 diluted cDNA product, 0.4 μl 10 μM F/R primer, 10 μl 2 × ChamQ Universal SYBR qPCR Master Mix (Vazyme), and 8.2 μl nuclease-free water. Three replicates were applied for each sample. Then, qPCR was performed in CFX Connect Real-Time PCR Detection System (Bio-Rad) following Vazyme’s program: one cycle of 95•C 30 s, followed by 40 cycles of 95•C 10 s and 60•C 30 s, finally one cycle of 95•C 15 s, 60•C 60 s, and 95•C 15 s. For bulk population RNA-seq, 1 μg total RNA was used as starting material. Libraries were constructed using KAPA RNA HyperPrep Kit (Roche) following its manual. Paired-End 150-bp (PE-150) sequencing was performed at Illumina Hiseq 2500 platform with an average depth of 30 million reads for each sample.</p></sec><sec id=\"S2.SS9\"><title>Hoxd8 Overexpression</title><p>Total RNA from mouse NIH3T3 cell was reversely transcribed by oligo dT(20) primer. The resulting cDNA was amplified by gene-specific PCR primer to obtain full-length cDNA of <italic>Hoxd8</italic> and ligated into pCDH vector to make the OE vector. The vector was sequenced to ensure 100% accuracy of inserted <italic>Hoxd8</italic> cDNA. After vector construction, lentivirus was assembled in 293T cells. Culture medium were centrifuged at 3,000 rpm for 5 min and filtered through a 0.45-μm syringe filter to obtain lentivirus. Then, NIH3T3 cells were infected with lentivirus containing <italic>Hoxd8</italic>. Uninfected cells were filtered out by adding Puro (10 mg/ml, Sangon Biotech) into the culture medium. The OE of <italic>Hoxd8</italic> was confirmed by qRT-PCR, and multiple senescence-associated phenotypes were then examined.</p></sec><sec id=\"S2.SS10\"><title>Bulk-Population RNA Sequencing Data Analysis</title><p>Adapter trimming was performed by cutadapt for RNA sequencing reads in FASTQ-format files. Trimmed sequencing reads were aligned to annotated reference genome (NCBI RefSeq annotations for genomes mm10) by STAR following the default parameters (e.g., “runMode” as genome Generate mode, “allows varying length with parameter sjdbOverhang” as 149, “outFilterMultimapNmax” as 10; <xref rid=\"B11\" ref-type=\"bibr\">Dobin et al., 2013</xref>). The reads per kilobase million (RPKM) of genes were quantified by FeatureCounts (<xref rid=\"B32\" ref-type=\"bibr\">Liao et al., 2014</xref>), which took paired-end reads into consideration, and then normalized by gene length. To compare the difference between bulk population RNA-seq and scRNA-seq, gene expression level of scRNA-seq was averaged for each cluster of cells.</p></sec><sec id=\"S2.SS11\"><title>Dimension Reduction and Clustering Analysis</title><p>RNA sequencing reads were adapter trimmed by cutadapt. Trimmed sequencing reads were further aligned to reference genome using STAR with parameters similar with those as described above. RPKM of genes was quantified by FeatureCounts and normalized by gene length. Cells with extremely low abundance of valid reads were removed. Dimension reduction, namely, principal component analysis (PCA) and T-distributed stochastic neighbor embedding (tSNE), was performed using Seurat v3 (<xref rid=\"B37\" ref-type=\"bibr\">Macosko et al., 2015</xref>). The highly variable genes (HVGs) were selected based on normalized dispersion following <xref rid=\"B37\" ref-type=\"bibr\">Macosko et al. (2015)</xref>. We used around 2,500, 5,000, 8,000 HVGs, respectively, to perform dimension reduction and clustering analysis in order to get reliable results. Because the clustering results were similar, we chose to use around 2,500 HVGs. Outliers were further determined and filtered by PCA projection. About top 50 principal components (PCs) were chosen for tSNE projection (<xref rid=\"B36\" ref-type=\"bibr\">Maaten and Hinton, 2012</xref>). Clustering of single cells was performed by KNN graph construction and Louvain algorithm. Considering the sample size in our case, parameter k in clustering algorithm was set as 10. Parameter of resolution was set from 0.8 to 2 to screen all possible clusters. Differentially expressed gene (DEG) identification of each cluster was performed using all genes following the pipeline of a previous paper (<xref rid=\"B40\" ref-type=\"bibr\">McDavid et al., 2013</xref>). Gene Ontology (GO) analysis was performed in Metascape (<xref rid=\"B61\" ref-type=\"bibr\">Zhou et al., 2019</xref>). Raw clusters with few DEGs were merged to avoid excessive classification. At last perplexity in tSNE was set as 10.</p></sec><sec id=\"S2.SS12\"><title>Expressed Gene Selection of Single-Cell RNA Sequencing and Bulk Population RNA Sequencing Data on Mouse Embryonic Fibroblasts</title><p>The bulk population RNA-seq data of transcriptomes of <italic>in vitro</italic> cultivated MEFs from PD6 to PD11 was obtained from our previous paper (<xref rid=\"B7\" ref-type=\"bibr\">Chen et al., 2018</xref>). The expressed genes in scRNA-seq were filtered by criteria of log (exp) > 0.01, which is the 10% quantile of expression level. For bulk RNA data, we only considered genes of > 1 fragment per kilobase million (FPKM) in all the <italic>in vitro</italic> cultivation passages from PD6 to PD11.</p></sec><sec id=\"S2.SS13\"><title>Gene Set Enrichment Analysis</title><p>Gene set enrichment analysis (GSEA) measures the enrichment of <italic>a priori</italic> defined set of genes. We implemented a GSEA approach for single cells, following steps as below: (1) Genes were ranked according to their expression level for each cell. (2) Recovery curve was created by walking down <italic>a priori</italic> defined gene list, and steps were increased when we encounter a gene in the gene set. The area under the curve (AUC) was computed as the indicator of enrichment for a certain gene set. And only AUC of top 3,000 ranked genes was considered. AUC values were normalized across cells.</p></sec><sec id=\"S2.SS14\"><title>Cell Cycle-Related Genes, Immune and Senescence-Related Genes, and Translation-Related Genes</title><p>The list of cell cycle-related genes in mouse was obtained from the previous paper “Highly Parallel Genome Wide Expression Profiling of Individual Cells Using Nanoliter Droplets” (<xref rid=\"B37\" ref-type=\"bibr\">Macosko et al., 2015</xref>). Immune and senescence-related genes were chosen from immune and senescence-related GO terms enriched by DEGs of clusters 3 and 4. Translation-related genes were all the genes of GO term “translation” enriched by DEGs of cluster 6.</p></sec><sec id=\"S2.SS15\"><title>Inference of Aging Lineage and Cell Pseudotime Along Lineages</title><p>In order to identify the continuous senescence of MEFs, we implemented Slingshot (<xref rid=\"B25\" ref-type=\"bibr\">Kelly et al., 2018</xref>) on the single-cell sequencing data. The cell clusters inferred by PCA and Louvain algorithm were used as input for Slingshot to infer the lineage relationship. Cluster 1 was set as the starting cells according to the expressing trend of aging genes we found in bulk RNA-seq data and previous studies. Pseudotime of each single cell along lineages was projected by principal curve implemented in Slingshot. Weight of projection was filtered by cutoff of 0.6. Lineage inference was performed on three panels of HVGs, which were around 2,500, 5,000, and 8,000, respectively. Three lineages, namely, the main lineage (1-2-3-4), duplication-regained lineage (1-2-5), and translation-elevated lineage (1-2-6), were supported by analysis using 2,500, 5,000, or 8,000 HVGs.</p></sec><sec id=\"S2.SS16\"><title>Transcription Factor Network Discovery and Lineage Committed Transcription Factor Inference in the Main Lineage</title><p>We constructed co-expression modules between transcription factors (TFs) and candidate target genes (TGs). Motif rankings which indicate motif–target affinity were drawn from SCENIC database (<xref rid=\"B2\" ref-type=\"bibr\">Aibar et al., 2017</xref>). Co-expressed genes in the TF modules where treated as TF targets if they are enriched in the top motif rankings. Enrichment analysis of TF regulons in each single cell was implemented in each cell by calculating the AUC value of recovery curve. Pseudotime of each cell on certain lineage was inferred by principal curve implemented in Slingshot. Lineage committed TF networks were identified by examining the correlation between enrichment of TF network and the increase of cell pseudotime. AUC values of the recovery curve of each TF regulon were normalized across cells.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Full-Length Single-Cell RNA Sequencing for Senescent Mouse Embryonic Fibroblasts</title><p>To explore the heterogeneity and potential cell–cell interactions during replicative senescence of MEFs, we cultured MEFs in atmosphere (20%) oxygen condition until nearly half of the cells exhibited SA-β-Gal positive staining (<xref ref-type=\"fig\" rid=\"F1\">Figures 1A,B</xref>). The advantage of this stage is that it includes both senescent and younger cells; thus, it is relatively cost-effective compared with select populations containing excesses of pro-senescent or senescent cells. Cell shape and viability were also evaluated prior to loading in the Fluidigm C1 system. Distinct differences among single cells were observed in terms of cellular shape (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1</xref>), suggesting that cells might undergo different stages of senescence process during a single passage. Single-cell transcriptome libraries were constructed in the C1 system and submitted to high-throughput sequencing. After quality control (including removal of potential contaminants and cell doublets), 175 single cells with an average sequencing depth of 3.96 million (M) reads per cell were selected for further analyses (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S2</xref>). A total of 26,121 genes in total and an average of nearly 10,000 genes per cell were detected (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S2</xref>), suggesting the high sensitivity with this full-length RNA-seq strategy. Subsequently, 2,556 genes that were more likely to reflect real biological differences among these single cells were selected based on gene expression dispersion, in accordance with previous publications (<xref rid=\"B37\" ref-type=\"bibr\">Macosko et al., 2015</xref>); these genes were subjected to further analysis (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S3</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Single-cell RNA sequencing (RNA-seq) for senescent mouse embryonic fibroblasts (MEFs) revealed six clusters with distinct expression patterns. <bold>(A,B)</bold> Senescence-associated β-galactosidase (SA-β-Gal) staining of PD6 and PD9 MEFs. The left panel shows representative staining, and the right panel shows the quantitative evaluation. <sup>∗∗</sup><italic>p</italic>-value < 0.01, <italic>t</italic>-test. <bold>(C)</bold> T-distributed stochastic neighbor embedding (tSNE) visualization of six color-coded clusters of PD9 MEFs. <bold>(D)</bold> Heatmap of significantly enriched Gene Ontology (GO) terms using differentially expressed genes (DEGs) of each cluster. Clusters representing GO terms are highlighted in red color. <bold>(E)</bold> Cricos showing connections of DEGs of the six clusters. Purple lines link the identical DEGs between clusters. PD, population doubling.</p></caption><graphic xlink:href=\"fgene-11-00867-g001\"/></fig></sec><sec id=\"S3.SS2\"><title>Clustering Analysis Reveals Six Subpopulations of Mouse Embryonic Fibroblasts With Distinct Senescence Features</title><p>PCA coupled with clustering is widely used in single-cell transcriptome analysis (<xref rid=\"B60\" ref-type=\"bibr\">Zheng et al., 2017</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Han et al., 2018</xref>). We thus applied it to our single-cell data; the 2,556 HVGs mentioned above were included in PCA. Because the top 50 PCs explained more than 90% of single-cell variance (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S4</xref>), we chose them to cluster 175 MEFs by Seurat package (<xref rid=\"B37\" ref-type=\"bibr\">Macosko et al., 2015</xref>). One hundred seventy-five MEFs were split into six clusters and displayed in two-dimensional tSNE (<xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S5</xref>). DEG identification, followed by GO analysis, was carried out on these six clusters. Upregulated DEGs for each cluster were analyzed as compared to combined remaining five clusters to define the molecular features of this cluster. Overall, each cluster had characteristic GO terms (<xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>), reinforcing the reliability of the clustering analysis. Notably, several clusters also showed overlapped GO terms. For example, the senescence-associated GO term inflammatory response existed in both clusters 3 and 4 (<xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>), suggesting that these two clusters of cells were senescent but may have been at different stages of senescence.</p><p>We next analyzed these six clusters in a sequential manner. Cluster 1 was enriched in GO terms such as mitotic cell cycle, cell division, and DNA replication (<xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>); this suggested a normal replication status for the cells in the cluster. Cluster 2 had weak enrichment of normal function such as cell–cell junction organization and a few senescence-related GO terms such as inflammatory response (<xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>), implying that this group of cells was undergoing a transition state to senescence. Clusters 3 and 4 both showed signs of senescence (e.g., inflammatory response; <xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>). In addition, cluster 3 exhibited DEGs enriched in positive regulation of cell death and migration, while cluster 4 exhibited DEGs enriched in acute inflammatory response and apoptotic signaling pathway. As noted above, the results suggest that clusters 3 and 4 are at two different stages of senescence, as in previous reports where cell death-related genes were highly expressed in senescent cell populations (<xref rid=\"B26\" ref-type=\"bibr\">Kim et al., 2013</xref>). Notably, cluster 5 was a small but unique cluster in which upregulated DEGs were enriched in both cell duplication-associated GO terms (e.g., mitotic cell cycle and cell division) and SASP-associated GO terms (e.g., inflammatory response and positive regulation of cytokine production) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>). There are two possible reasons to explain the appearance of these cells. One possibility is that these cells might be affected by SASP components in the culture, which were produced by adjacent senescent cells (e.g., cells in clusters 3 and 4) through a paracrine effect. The other possibility is that these cells might escape from cell cycle arrest (<xref rid=\"B49\" ref-type=\"bibr\">Sherr and Dipinho, 2000</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Krones-Herzig et al., 2003</xref>). The exact origin of this small number of cells deserves further experimental validation. Cluster 6, another small but distinct cluster, exhibited OE of translation and ATP synthesis-associated GO terms based on enrichment analysis of upregulated DEGs (<xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>); this finding implied mass production of proteins coordinated with higher energy supply because protein translation is a biological process with a high energy demand (<xref rid=\"B30\" ref-type=\"bibr\">Lane and Martin, 2010</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Gonskikh and Polacek, 2017</xref>). There were some shared GO terms between two clusters, such as mitotic cell cycle and cell division in both clusters 1 and 5, as well as ribosome biogenesis in both clusters 1 and 6. Thus, we examined whether these shared GO terms reflected similar or different DEGs in the same GO category. To systematically address this question, we analyzed all possible genes overlapping among these six clusters. We found that clusters 3 and 4 shared a considerable proportion of DEGs (<xref ref-type=\"fig\" rid=\"F1\">Figure 1E</xref>). Certain DEGs in cluster 1 were also present in cluster 5 or 6 (<xref ref-type=\"fig\" rid=\"F1\">Figure 1E</xref>), suggesting that these genes were dynamically regulated. Taken together, we defined these six clusters of single cells with signature genes and speculated that (1) cluster 1 was presumably proliferating cells that replicated normally; (2) cluster 2 could be an intermediate stage between clusters 3 and 4; (3) clusters 3 and 4 were presumably senescent cells with cell cycle arrest and SASP (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S6</xref>); (4) cluster 5 represented a subset of MEFs that might be affected by SASP components in the culture or might escape from cell cycle arrest; (5) cluster 6 suggested that a subset of MEFs could coordinate protein translation with ATP synthesis.</p><p>To further confirm the senescence status of each cluster, we applied our previously published bulk population MEF RNA-seq data (<xref rid=\"B7\" ref-type=\"bibr\">Chen et al., 2018</xref>), which contain transcriptomes of <italic>in vitro</italic>-cultivated MEFs from PD6 to PD11 to the current single-cell data. As expected, two data sets shared a large proportion of expressed genes (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>). To further investigate shared genes between two data sets, we overlapped those shared genes with literature-supported senescence-associated genes (from CellAge<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup>). Approximately 60% (168 out of 279) senescence-associated genes were simultaneously expressed in two data sets (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). Among expressed genes that were shared in these two datasets, we individually displayed expression levels of a subset of well-known senescence marker genes in the tSNE map. Cyclin-dependent kinase inhibitor (CKDI) coding genes like <italic>Cdkn1a</italic> and <italic>Cdkn2b</italic> had relatively higher expression levels in clusters 3 and 4 than in cluster 1 (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>), as did the senescence-associated TF coding gene <italic>Egr1</italic>, SASP-related genes like <italic>Cxcl1</italic> and <italic>Ccl2</italic> (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>). In contrast, the cell proliferation marker gene <italic>Mki67</italic> showed a higher expression level in cluster 1 than in clusters 3 and 4. We also performed expression plots of gene pairs (<italic>Cdkn1a</italic> and <italic>Cdkn2b</italic>, <italic>Cdkn1a</italic> and <italic>Egr1</italic>, <italic>Cdkn1a</italic> and <italic>Hif1a, Cdkn1a</italic> and <italic>Pcna</italic>) to further explore the co-expression at the single-cell level. Expression levels of <italic>Cdkn1a</italic> and <italic>Cdkn2b</italic>, expression levels of <italic>Cdkn1a</italic> and <italic>Egr1</italic>, and expression levels of <italic>Cdkn1a</italic> and <italic>Hif1a</italic> were positively correlated. Although expression levels of <italic>Cdkn1a</italic> and <italic>Pcna</italic> were not negatively correlated, the expression level of <italic>Cdkn1a</italic> in most cells was higher than 1 FPKM, whereas the expression level of <italic>Pcna</italic> in most cells was lower than 1 FPKM (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S7</xref>). The result above suggested that cluster 1 exhibited transcriptome features of pro-senescent cells, whereas clusters 3 and 4 exhibited transcriptome features of senescent cells.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Comparison of single-cell RNA sequencing (scRNA-seq) data and bulk population data on mouse embryonic fibroblasts (MEFs). <bold>(A)</bold> Venn diagram presented the overlap of expressed genes between scRNA-seq data and our previous bulk population data on <italic>in vitro</italic> cultivated MEFs’ transcriptomes. <bold>(B)</bold> Venn diagram presented the overlap of expressed genes from scRNA-seq data, expressed genes from bulk population data, and senescence-associated genes from CellAge. <bold>(C)</bold> T-distributed stochastic neighbor embedding (tSNE) plots presenting the expression level of 10 well-known senescence markers in each single cell; these 10 genes were among the overlap of scRNA-seq data and bulk population data. <bold>(D,E)</bold> Overall expression levels of continuously upregulated <bold>(D)</bold> and downregulated <bold>(E)</bold> genes in our previous PD6 to PD11 MEF bulk population RNA-seq data in each single cell were presented in tSNE plot (<xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref>). The overall expression level was displayed in normalized area under the curve (AUC) level (Z-score). Z-score, value calculated by Z normalization using AUC value; Bulk-seq, PD6 to PD11 MEF bulk population RNA-seq; Exp, expression level.</p></caption><graphic xlink:href=\"fgene-11-00867-g002\"/></fig><p>Next, we selected the genes that were consecutively upregulated (1,531 genes) or downregulated (1,993 genes) during the course of <italic>in vitro</italic> cellular senescence in MEFs (from PD6 to PD11; <xref rid=\"B7\" ref-type=\"bibr\">Chen et al., 2018</xref>) and displayed their overall expression level (displayed by Z score) in the two-dimensional tSNE map of single cells (<xref ref-type=\"fig\" rid=\"F2\">Figures 2D,E</xref>). As expected, upregulated genes in bulk population data demonstrated higher expression levels in clusters 3 and 4 (<xref ref-type=\"fig\" rid=\"F2\">Figure 2D</xref>), in which DEGs were enriched in senescence-associated GO terms. Similarly, downregulated genes in bulk population data demonstrated high expression levels in cluster 1 (<xref ref-type=\"fig\" rid=\"F2\">Figure 2E</xref>), in which DEGs were enriched in duplication-associated GO terms. Cells from cluster 2 expressed subsets of both upregulated and downregulated genes (<xref ref-type=\"fig\" rid=\"F2\">Figures 2D,E</xref>). This result further supported that MEFs in cluster 1 were younger than that in clusters 3 and 4; cluster 2 exhibited intermediate features. To further illustrate this point, we plotted the expression of literature-supported senescence-associated genes (from CellAge<sup>1</sup>, within those consecutively upregulated or downregulated genes on the two-dimensional tSNE map. The conclusion remained consistent with the finding above (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S8</xref>). Together, we provided three types of evidence (including GO analysis of DEGs for each cluster, classical senescence marker genes, and bulk RNA-seq from senescent MEFs) to support the notion that these six clusters reflect distinct stages of MEF senescence.</p></sec><sec id=\"S3.SS3\"><title>Trajectory Analysis Reveals Three Distinct Lineages During Mouse Embryonic Fibroblast Senescence</title><p>The above results indicated that MEFs were at different stages of senescence, although they had been cultured simultaneously. Different clusters might reflect different senescence statuses of MEFs through the senescence paths. To explore whether such paths exist, we analyzed the development trajectories of single-cell transcriptome to investigate relationships among these six clusters. Along the pseudotime, there were three indicated lineages, all of which began with cluster 1, followed by cluster 2, and then diverged into three different branches (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S9</xref>). This result suggested that MEFs might undergo different biological processes during cellular senescence. The main lineage contained clusters 1, 2, 3, and 4, which possessed a large proportion of all single cells and could be a major senescence path. Compared with cluster 2, cluster 1 exhibited upregulation of DEGs that were enriched in duplication-associated GO terms, while cluster 3 exhibited upregulation of DEGs that were enriched in senescence-associated GO terms (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>); these findings suggested that cluster 1 was younger than cluster 2, while cluster 3 exhibited greater senescence than cluster 2. Next, when conducting DEG analysis between clusters 3 and 4, we found that GO terms such as apoptotic signaling pathway and positive regulation of cell death were exclusively displayed in cluster 4, consistent with the hypothesis that apoptosis and cell death is the next step of senescence (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Kim et al., 2013</xref>). These results implied that MEFs in this lineage first underwent cell cycle arrest (from cluster 1 to cluster 2), and then acquired SASP and inflammation-related traits (from cluster 2 to cluster 3), and finally underwent apoptosis or cell death (from cluster 3 to cluster 4). This lineage was also supported by the finding of a previous study that showed senescent human diploid fibroblasts (HDFs, another commonly used cell line in studying senescence) first exhibited features of reduced cell cycle-related genes, followed by elevated expression of inflammation and apoptosis-associated genes and ultimate elevation of cell death-related genes (<xref rid=\"B26\" ref-type=\"bibr\">Kim et al., 2013</xref>). The second lineage contained clusters 1, 2, and 5. To understand this lineage further, we conducted DEG analysis between clusters 5 and 2. The result revealed that upregulated DEGs in cluster 5 were enriched in both cell duplication-associated GO terms and senescence-associated GO terms, suggesting a possibility of escape from cell cycle arrest and acquisition of SASP for cells in cluster 5 (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>). Furthermore, we selected cell cycle-related genes described in a previous publication (<xref rid=\"B37\" ref-type=\"bibr\">Macosko et al., 2015</xref>), as well as immune and senescence genes from clusters 3 and 4 DEGs and plotted them on the clusters. We found that cells in cluster 5 expressed higher levels of both cell cycle and immune, senescence-related genes (<xref ref-type=\"fig\" rid=\"F3\">Figure 3D</xref>), further supporting the distinct status of this cluster. Previous studies demonstrated that a small amount of <italic>in vitro</italic> cultured MEFs may be immortal because of mutations in the p53-MDM2-p19ARF pathway (<xref rid=\"B29\" ref-type=\"bibr\">Krones-Herzig et al., 2003</xref>). That might explain how cells in cluster 5 were able to escape cell cycle arrest. The third lineage contained clusters 1, 2, and 6. When compared with cluster 2, cluster 6 did not demonstrate an upregulation of inflammation-related genes, instead upregulated DEGs were enriched in translation and ATP synthesis-associated GO terms (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>), suggesting coordinated expression of these two biologically relevant gene types. Intriguingly, the overall downregulation of translation-related ribosomal proteins in the main lineage (1-2-3-4) was not detected in this lineage (1-2-6) (<xref ref-type=\"fig\" rid=\"F3\">Figure 3E</xref>). A previous study also observed that ribosomal protein levels and overall translation were elevated in premature aging (<xref rid=\"B5\" ref-type=\"bibr\">Buchwalter and Hetzer, 2017</xref>). The underlying mechanism by which enhanced translation and ATP synthesis occurred during <italic>in vitro</italic> cultivation of MEFs is unclear and merits further investigation. Together, trajectory analysis of these single cells revealed three distinct lineages of MEFs during cellular senescence, namely, the main lineage (1-2-3-4), duplication-regained lineage (1-2-5), and translation-elevated lineage (1-2-6).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Distinct biological processes of senescence uncovered by trajectory inference. <bold>(A)</bold> Trajectory analysis by Slingshot revealed three development lineages during senescence. <bold>(B,C)</bold> Heatmaps of main Gene Ontology (GO) terms enriched in Metascape using differentially expressed genes (DEGs) between two adjacent clusters in lineage 1 <bold>(B)</bold> and lineages 2 and 3 <bold>(C)</bold>. Only highly expressed DEGs were used for GO terms enrichment. <bold>(D)</bold> T-distributed stochastic neighbor embedding (tSNE) plots of overall expression level of cell cycle-related genes (upper panel) and immune and senescence-related genes (lower panel) in each single cell. <bold>(E)</bold> Graphs showing overall expression levels of translation-related genes selected from cluster 6 DEGs in each single cell along the pseudotimes of lineages 3 (left panel) and lineage 1 (right panel). Z-score, value calculated by Z normalization using the AUC value.</p></caption><graphic xlink:href=\"fgene-11-00867-g003\"/></fig></sec><sec id=\"S3.SS4\"><title>Upregulation of Some Senescence-Associated Secretory Phenotype Components Was Observed in Subsets of Cells During Senescence of Mouse Embryonic Fibroblasts Cultivated in Atmospheric Oxygen</title><p>Factors that contributed to the main lineage (1-2-3-4) were next examined. We noticed that some SASP components were upregulated as cells progressed through this lineage (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>). However, only a subset of cells exhibited upregulation of these factors (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>), whereas other cells did not change. Notably, <italic>Nfkb1</italic> (p50) and <italic>Cebpb</italic>, two well-known TF coding genes that have been reported to regulate SASP (<xref rid=\"B14\" ref-type=\"bibr\">Freund et al., 2010</xref>), were also upregulated in a subset of cells in the lineage 1-2-3-4 (<xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>), implying that certain TF-SASP signal signaling interactions contribute to senescence of some cells in this lineage. This result was initially considered contradictory to the findings of a previous study, which reported that the secretion of SASP factors from atmospheric oxygen cultivated MEFs was not detected during the progression of senescence (<xref rid=\"B8\" ref-type=\"bibr\">Coppé et al., 2010</xref>). However, because the discovered SASP-related genes such as <italic>Il6</italic> and <italic>Cxcl1</italic> were only upregulated in a small percentage of cells (<xref ref-type=\"fig\" rid=\"F4\">Figures 4A,B</xref>), those genes presumably could not be detected by bulk cell analysis due to a dilution effect, whereas they could be detected by single-cell analysis. To further confirm the existence of SASP in MEFs, we simultaneously cultivated MEFs with NM and SCM collected from senescent MEFs. Enhancement of SA-β-Gal staining and G1-phase cell cycle arrest was observed in SCM-cultivated MEFs compared with NM-cultivated cells (<xref ref-type=\"fig\" rid=\"F4\">Figures 4D–G</xref>). Additionally, upregulation of <italic>Cdkn2b</italic> and <italic>Cdkn1a</italic> was observed in SCM-cultivated cells by reverse transcription followed by qRT-PCR (<xref ref-type=\"fig\" rid=\"F4\">Figure 4H</xref>). Because SCM contains SASP mediators such as IL6 that are released by senescent cells (<xref rid=\"B8\" ref-type=\"bibr\">Coppé et al., 2010</xref>), the above results support the notion that atmospheric oxygen-cultivated MEFs can be induced to senescence through an SASP mechanism.</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Upregulation of some senescence-associated secretory phenotypes (SASPs) was observed in atmospheric oxygen-cultivated mouse embryonic fibroblasts (MEFs). <bold>(A)</bold> Representative SASP components were upregulated along lineage 1 in <xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>. Z-score, value calculated by Z normalization using expression level; corr, correlation score along lineage 1. <bold>(B,C)</bold> T-distributed stochastic neighbor embedding (tSNE) plots presenting expression levels of upregulated SASPs <bold>(B)</bold> and their upstream transcription factors (TFs) <bold>(C)</bold> in each single cell. As shown in the picture, SASPs and two SASP-related TFs were upregulated in clusters 3 and 4 in a scattered matter. <bold>(D–H)</bold> Senescence-associated β-galactosidase (SA-β-Gal) staining <bold>(D)</bold> and its quantitative evaluation <bold>(E)</bold>, cell cycle analysis <bold>(F)</bold> and its quantitative evaluation <bold>(G)</bold>, and quantitative reverse transcription PCR (qRT-PCR) of several senescence markers <bold>(H)</bold> were compared between normal medium (NM)- and senescence-conditioned medium (SCM)-cultivated MEFs. <italic>Gapdh</italic> was used as internal control in qRT-PCR. *, **, and *** represent <italic>p</italic>-values less than 0.05, 0.01, and 0.001 by <italic>t</italic>-test, respectively. <bold>(I)</bold> Cells cultivated with NM, SCM, medium without interleukin (IL)6, medium with 5 ng/ml IL6, and medium with 50 ng/ml IL6 were collected and subjected to RNA sequencing (RNA-seq). Principal component analysis (PCA) was performed with bulk RNA-seq data and single-cell RNA-seq data (cells in the same cluster were combined together) using their overlapped genes. PC1 likely represents the data type difference between bulk and single-cell RNA-seq. NM, normal medium-cultivated MEFs; SCM, senescence-conditioned medium-cultivated MEFs; -IL6, MEFs cultivated in medium without IL6; + IL6 (5 ng/ml)/ + IL6 (50 ng/ml), MEFs cultivated in medium with 5 or 50 ng/ml IL6. Two replicates (_1 and _2) of samples with NM, SCM, and 50 ng/ml IL6 were included for analysis. Exp, expression level.</p></caption><graphic xlink:href=\"fgene-11-00867-g004\"/></fig><p>To explore if a single SASP factor such as IL6, for which expression was upregulated in a few of the senescent MEFs, can lead to senescence by means of paracrine signaling, we added IL6 into culture medium and examined senescence-associated phenotypes in MEFs by comparison with control medium. The impact of IL6 was less striking than the addition of SCM, which contains multiple SASP factors, however, a paracrine tendency was observed, including a higher percentage of SA-β-Gal staining and elevated <italic>Cdkn2b</italic> expression upon IL6 treatment in younger MEFs (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S10</xref>). This supported the notion that a few MEFs could secrete SASP mediators such as IL6 to promote senescence of nearby cells in a paracrine manner. To examine the similarity of cellular senescence induced by SCM (or IL6) and the senescent path in the main lineage, we performed bulk population RNA-seq on MEFs with and without SCM or IL6 treatment. PCA was applied to compare bulk RNA-seq with single-cell clusters. Because PC1 apparently differed on the basis of RNA-seq data type (bulk vs. single cell), we used PC2 for comparison of real biological differences. Consistent with the enhanced SA-β-Gal staining upon SCM treatment, we found that SCM-cultivated MEFs were close to clusters 3 and 4, while NM-cultivated cells were close to clusters 1 and 2 with respect to whole transcriptome expression (<xref ref-type=\"fig\" rid=\"F4\">Figure 4D</xref>). Consistent with the weak impact observed regarding <italic>Cdkn2b</italic> (<xref ref-type=\"fig\" rid=\"F4\">Figure 4H</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S10</xref>), IL6-treated MEFs also demonstrated a moderate transcriptome shift to senescence compared with NM treatment (<xref ref-type=\"fig\" rid=\"F4\">Figure 4I</xref>). Overall, the above data showed that some SASP components were upregulated in a subset of cells during senescence of atmospheric oxygen-cultivated MEFs, which might be related to paracrine senescence.</p></sec><sec id=\"S3.SS5\"><title>Network Analysis Shows HOXD8 Is a Novel Regulator for Mouse Embryonic Fibroblast Senescence in the Main Lineage</title><p>We hypothesized that dynamically changed TF may regulate multiple TGs including SASP to promote senescence in the main lineage. To screen potential TF–TG networks, we selected and filtered TFs and their co-expression genes using a pipeline similar to SCENIC (<xref rid=\"B2\" ref-type=\"bibr\">Aibar et al., 2017</xref>). We identified lineage-committed TF–TG networks on the basis of the correlation between enrichment of TGs in cells (measured by AUC of recovery curve of the TGs) and cell pseudotime of the main lineage inferred by Slingshot (correlation > 0.3 or < -0.3; <xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>). Notably, several TFs (e.g., JUNB, CEBPB, MEOX2, and TFDP) have been shown to inhibit cell proliferation and induce cellular senescence (<xref rid=\"B27\" ref-type=\"bibr\">Konishi et al., 2008</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Douville et al., 2011</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Ohdaira et al., 2012</xref>; <xref rid=\"B54\" ref-type=\"bibr\">Thomsen et al., 2015</xref>), suggesting that these discovered TF–TG networks reliably contributes to senescence. To further investigate TFs that have not been identified as regulators of cellular senescence, we overexpressed <italic>Nr1d1</italic> and <italic>Hoxd8</italic>, for which corresponding TGs exhibited positive correlations with cell pseudotime, and knocked down <italic>Ssrp1</italic> and <italic>Nfyc</italic>, for which corresponding TGs exhibited negative correlations with cell pseudotime (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S11</xref>). We discovered that OE of <italic>Hoxd8</italic> could promote senescence-associated phenotypes (<xref ref-type=\"fig\" rid=\"F5\">Figures 5B–J</xref>). HOXD8 is a TF that has been reported to regulate cell proliferation and differentiation and to determine regional identity during embryogenesis (<xref rid=\"B41\" ref-type=\"bibr\">Mcginnis and Krumlauf, 1992</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Liu et al., 2016</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Mansour and Senga, 2017</xref>). OE of <italic>Hoxd8</italic> in NIH3T3 (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>), a mouse fibroblast cell line derived from MEFs, led to multiple senescence-associated phenotypes, including reduced cell proliferation rate (<xref ref-type=\"fig\" rid=\"F5\">Figure 5C</xref>), G1 phase arrest (<xref ref-type=\"fig\" rid=\"F5\">Figures 5D,E</xref>), enhanced SA-β-Gal staining (<xref ref-type=\"fig\" rid=\"F5\">Figures 5F,G</xref>), and reduced DNA replication activity (<xref ref-type=\"fig\" rid=\"F5\">Figures 5H,I</xref>). Upregulation of senescence markers including <italic>Cdkn1a</italic>, <italic>Cdkn1b</italic>, <italic>Ccnd1</italic>, and <italic>Ckdn2d</italic> was also observed by qRT-PCR (<xref ref-type=\"fig\" rid=\"F5\">Figure 5J</xref>). Notably, the expression of <italic>Il6</italic> was also enhanced in <italic>Hoxd8</italic>-OE cells (<xref ref-type=\"fig\" rid=\"F5\">Figure 5J</xref>). Transcriptomic analysis revealed DEGs between <italic>Hoxd8</italic>-OE and normal NIH3T3 cells that were enriched in senescence-related GO terms such as cellular response to DNA damage stimulus, cell division, and regulation of translation (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S12</xref>). We also observed a tendency of cellular senescence in <italic>Hoxd8</italic>-OE MEFs (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S13</xref>). These results indicate that HOXD8 could be a novel regulator in regulating MEF senescence, according to both single-cell analysis and experimental validation.</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Transcription factor (TF) HOXD8 is a new senescence regulator in mouse embryonic fibroblasts (MEFs). <bold>(A)</bold> Co-expression modules of TFs and their predicted target genes (TGs) were filtered using TF-motif database in RcisTarget along lineage 1 (<xref rid=\"B2\" ref-type=\"bibr\">Aibar et al., 2017</xref>). Pearson correlation was calculated between the overall expression of co-expression genes and lineage 1 pseudotime. Modules with correlation coefficient (corr.) either more than 0.3 or less than -0.3 are displayed in heatmap. Z-score, value calculated by Z normalization using area under the curve (AUC) value. <bold>(B)</bold> Overexpression (OE) of Hoxd8 in NIH3T3 cells was confirmed by quantitative reverse transcription PCR (qRT-PCR). <italic>Gapdh</italic> was used as internal control. <bold>(C)</bold> Cell proliferation rate was evaluated by Cell Counting Kit-8 (CCK-8) assay. <bold>(D,E)</bold> Cell cycle analysis between Hoxd8-OE and control NIH3T3 cells. <bold>(D)</bold> Shows representative cell cycle distribution, and <bold>(E)</bold> shows the quantitative evaluation. <bold>(F,G)</bold> Senescence-associated β-galactosidase (SA-β-Gal) staining <bold>(F)</bold> and its quantitative evaluation <bold>(G)</bold> were compared between Hoxd8-OE and control NIH3T3 cells. <bold>(H,I)</bold> 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay <bold>(H)</bold> and its quantitative evaluation <bold>(I)</bold> were compared between Hoxd8-OE and control NIH3T3 cells. Blue, 4′,6-diamidino-2-phenylindole (DAPI) staining; green, EdU incorporation. <bold>(J)</bold> qRT-PCR analysis for cell cycle and senescence-related genes in both Hoxd8-OE and control cells. <italic>Gapdh</italic> was used as internal control. <sup>∗</sup>, <sup>∗∗</sup>, and <sup>∗∗∗</sup> represent <italic>p</italic>-values less than 0.05, 0.01, and 0.001 by <italic>t</italic>-test, respectively.</p></caption><graphic xlink:href=\"fgene-11-00867-g005\"/></fig></sec></sec><sec id=\"S4\"><title>Discussion</title><p>To the best of our knowledge, this study is the first single-cell transcriptome analysis of replicative senescent MEFs, a basic and valuable cellular aging model. Although it has been known that senescent cells are not identical, even at the same passage during <italic>in vitro</italic> cultivation, there has been no clear understanding of this heterogeneity among single MEFs. By sequencing each single cell at a half SA-β-Gal staining passage of MEFs with sufficient sequencing coverage, we identified dramatic differences among these single cells. Six clusters were discovered, and each represents a distinct status with signature genes associated with different stages of MEF senescence. Furthermore, these six clusters reflected three lineages, indicating three distinct cell fates that each single cell may choose. Whereas a previous bulk population study suggested that senescent MEFs cultivated in atmospheric oxygen did not secrete higher levels of any SASP factors, compared with pro-senescent MEFs (<xref rid=\"B8\" ref-type=\"bibr\">Coppé et al., 2010</xref>), our single-cell transcriptome analysis revealed that at least a few MEFs exhibited a higher expression of SASPs and their upstream TFs. By integrative analysis and experimental validation, we also identified the TF HOXD8 as a novel regulator for MEF senescence. Thus, this study thus provides a new perspective for this important senescence model.</p><p>A previous study performed extensive analyses of the transcriptome dynamics of HDFs over the course of <italic>in vitro</italic> replicative senescence, however, the heterogeneity of these senescent cells remained unclear (<xref rid=\"B26\" ref-type=\"bibr\">Kim et al., 2013</xref>). Using single-cell RNA-seq, we detected three distinct lineages in replicative MEFs. The main lineage manifested features on SASPs, inflammation, and cell death, similar to the pattern of HDFs (<xref rid=\"B26\" ref-type=\"bibr\">Kim et al., 2013</xref>). However, the duplication-regained lineage indicated a possibility of recovery of cell replication ability and persistent expression of SASPs; the translation-elevated lineage showed a coupled enhancement of translation and ATP synthesis. These two lineages only comprised a small percentage of single cells and may be overwhelmed by bulk transcriptome analysis. Thus, single-cell analysis of MEFs provided us a complex but intriguing perspective of gene expression in senescence. It revealed that single cells may choose different cell fates and exhibit distinct gene expression features upon exposure to replicative stress. Notably, the relationship between global translation level and cellular senescence/aging has been reported in other studies with various study materials. A previous study demonstrated enhancement of global translation level and ribosome synthesis in a premature aging mouse model (<xref rid=\"B5\" ref-type=\"bibr\">Buchwalter and Hetzer, 2017</xref>). Additionally, multiple studies showed life span extension in flies or worms in knockout mutants with certain ribosomal protein genes (<xref rid=\"B50\" ref-type=\"bibr\">Steffen et al., 2008</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Janssens and Veenhoff, 2016</xref>). Because cellular senescence is known to promote individual aging (<xref rid=\"B34\" ref-type=\"bibr\">López-Otín et al., 2013</xref>), the higher translation activity in cluster 6, compared with cluster 2, suggested a possible link between protein synthesis and senescence, which merits further investigation.</p><p>To deeper understand the driving force of senescence in the main lineage, we investigated the SASP components. Some SASPs were detected to be upregulated in the senescent clusters in a scattered pattern (only upregulated in some cells). A previous study did not detect significantly higher secretion levels of any SASP factor in senescent MEFs (e.g., cultivated in atmospheric oxygen) compared with pro-senescent MEFs (<xref rid=\"B8\" ref-type=\"bibr\">Coppé et al., 2010</xref>). This disparity might be related to differences in the experimental approach between these two studies; in particular, upregulation of SASPs in a small proportion of cells might not change the overall expression level. That is another insight that can only be observed by single cell-based experiments. Consistent with the findings of previous studies (<xref rid=\"B21\" ref-type=\"bibr\">Hubackova et al., 2012</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Acosta et al., 2013</xref>), we observed the induction of paracrine senescence in MEFs by SASP components when cells were exposed to conditioned medium; IL6-treated MEFs displayed weaker senescence features compared with SCM-cultivated MEFs.</p><p><italic>Hoxd8</italic> belongs to the <italic>Hox</italic> gene family, an important regulator family that functions in embryogenesis (<xref rid=\"B41\" ref-type=\"bibr\">Mcginnis and Krumlauf, 1992</xref>). Recent studies suggested that reactivation of this developmentally critical gene family played an unexpected role during aging (<xref rid=\"B48\" ref-type=\"bibr\">Schwörer et al., 2016</xref>). For example, <italic>Hoxa9</italic> was discovered to be reactivated in aged individuals and impaired the function of the muscle stem cell (<xref rid=\"B48\" ref-type=\"bibr\">Schwörer et al., 2016</xref>). As for <italic>Hoxd8</italic>, it has been reported to regulate the cell cycle and oncogenesis in several carcinomas (<xref rid=\"B33\" ref-type=\"bibr\">Liu et al., 2016</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Mansour and Senga, 2017</xref>). By single-cell analysis of MEFs, we first showed that <italic>Hoxd8</italic> could also be a regulator of senescence, extending the link between this gene family and aging. ChIP-seq coupled with RNA-seq analysis need to be performed in a future investigation to fully elucidate the direct TGs of this TF with respect to senescence.</p><p><xref rid=\"B62\" ref-type=\"bibr\">Zirkel et al. (2018)</xref> carried out scRNA-seq on ∼8,300 proliferating and ∼5,200 replicative senescent HUVECs on a 10X Genomics platform. We found multiple senescent marker genes [<italic>CDKN1A</italic> (p21), <italic>PCNA</italic>, <italic>LMNB1</italic>, <italic>EZH2</italic>, <italic>CCNA2</italic>, <italic>IL6</italic>] had similar heterogeneity between replicative MEFs and HUVECs (<xref ref-type=\"fig\" rid=\"F2\">Figures 2B,D</xref> from <xref rid=\"B62\" ref-type=\"bibr\">Zirkel et al., 2018</xref>, and <xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref> from our study). For example, <italic>PCNA</italic> showed higher expression in proliferating cells while reduced expression in senescent cells (in both MEFs and HUVECs). IL6 exhibited increased cell number of IL6<sup>high</sup> cells in senescent HUVECs compared with younger cells, but the overall cell proportion is not very high (Figure 2B in <xref rid=\"B62\" ref-type=\"bibr\">Zirkel et al., 2018</xref>), consistent with our finding in replicative MEFs that a few of the senescent MEFs had high expression of <italic>Il6</italic> (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>). <italic>CDKN1A</italic> showed an increased expression trend along the pseudotime course of HUVECs (<xref ref-type=\"fig\" rid=\"F2\">Figure 2D</xref> from <xref rid=\"B62\" ref-type=\"bibr\">Zirkel et al., 2018</xref>), in line with the elevated expression trend of <italic>Cdkn1a</italic> along the main lineage (1-2-3-4) of MEFs (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>, <xref ref-type=\"fig\" rid=\"F2\">Figure 3</xref>). In <xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref> of our MEF data, we showed that immune and senescence-related genes had an increased expression trend along the main lineage. However, SASP-related genes such as <italic>CXCL1</italic> and <italic>IL8</italic> did not show an increased expression trend along the pseudotime course of HUVECs (<xref ref-type=\"fig\" rid=\"F2\">Figure 2D</xref> from <xref rid=\"B62\" ref-type=\"bibr\">Zirkel et al., 2018</xref>). One possible explanation is that <xref rid=\"B62\" ref-type=\"bibr\">Zirkel et al. (2018)</xref> sequenced senescence entry cells while some of the MEFs we sequenced were at a later stage of senescence, which is featured by immune-senescence cross talk-related genes. Since the study of <xref rid=\"B62\" ref-type=\"bibr\">Zirkel et al. (2018)</xref> focused on the function and mechanism of candidate gene <italic>HMGB2</italic>, after discovering the eight senescent cell states along the main path, they did not perform further analysis on scRNA-seq data. We thus did not know the dynamic changes of translation-related genes in HUVECs, and this point deserves further study.</p><p><xref rid=\"B53\" ref-type=\"bibr\">Teo et al. (2019)</xref> performed scRNA-seq (Smart-Seq2 protocol) in an H-RasG12V-induced IMR90 fibroblast model. They obtained transcriptome data of hundreds of single cells. The main discovery of their study is that two cell senescence endpoints were identified, and the primary endpoint is featured by Ras while the secondary one is marked by Notch signaling activation. Interestingly, cell cycle-related genes such as <italic>CDKN1A</italic> and <italic>CDKN2B</italic> showed distinct expression profiling in these two different senescence states (<xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref> from <xref rid=\"B53\" ref-type=\"bibr\">Teo et al., 2019</xref>). In our MEF model, we also observed dynamic changes of cell cycle-related genes along the three lineages identified (<xref ref-type=\"fig\" rid=\"F2\">Figures 2C</xref>, <xref ref-type=\"fig\" rid=\"F2\">Figures 3A,D</xref>). One common feature between replicative MEFs and oncogene-induced IMR90 senescence is elevated expression of <italic>CEBPB</italic>. We found increased <italic>Cebpb</italic> along the main lineage of senescent MEFs (<xref ref-type=\"fig\" rid=\"F4\">Figures 4A,C</xref>, <xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>), and <xref rid=\"B53\" ref-type=\"bibr\">Teo et al. (2019)</xref> also discovered upregulated <italic>CEBPB</italic> in both primary and secondary senescent states in IMR90 (see Figure 2D from <xref rid=\"B53\" ref-type=\"bibr\">Teo et al., 2019</xref>). We also found that a few SASP genes such as CXCL and IL family members showed a similar upregulation in senescent MEFs and IMR90 cells (<xref ref-type=\"fig\" rid=\"F1\">Figures 1D</xref>, H from <xref rid=\"B53\" ref-type=\"bibr\">Teo et al., 2019</xref>; <xref ref-type=\"fig\" rid=\"F2\">Figures 2–4</xref>).</p><p>There were some limitations in our study. We observed different senescence stages of single cells from PD9 MEFs and reconstructed the senescence process by lineage analysis. It would be preferable to chronologically sequence the single-cell transcriptomes of cells from the first <italic>in vitro</italic> cultivation passage to the last passage, which would allow construction of the authentic temporal lineage of each cluster during cell passages. That will help us understand more about the mechanisms involved in senescence (for example, when does the cluster with upregulated translation genes and ATP synthesis genes appear, and whether it will disappear in the end). Another limit is that we provided some new insights about how paracrine effects and <italic>Hoxd8</italic> functioned in the senescence process of mouse fibroblasts, but we still did not investigate whether and how they function in other replicative senescence models, premature aging models, or aged tissues/organisms. Future investigations following this study are warranted to fully understand the whole picture of senescence at the single-cell level.</p></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://bigd.big.ac.cn/gsa/browse/CRA002582\">https://bigd.big.ac.cn/gsa/browse/CRA002582</ext-link>, CRA002582.</p></sec><sec id=\"S6\"><title>Author Contributions</title><p>WC conducted the single-cell RNA-seq and cellular experiments and drafted the manuscript. PQ, XW, and GW analyzed the data. YH and DL directed the experiments of single-cell RNA-seq. YS provided the support of Fluidigm C1 system. SQ and JC conducted the partial cellular experiments. BZ separated the MEFs from mouse. MC directed the cellular experiments. WJ directed the data analysis. TN designed the project, directed result analysis, and revised the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>DL was employed by the Fluidigm (Shanghai) Instrument Technology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This research was funded by the National Key R&D Program of China (Grant No. 2018YFC1003500), National Natural Science Foundation of China (Grant Nos. 91949107, 31771336, and 31521003), and Shenzhen Science and Technology Program (Grant Nos. KQTD20180411143432337 and JCYJ20170817111841427).</p></fn></fn-group><ack><p>We thank Qi Jia for her help on cell cultivation experiments, Suqin Shen for her support on flow cytometry experiment, and Sibo Zhu for his advice on the manuscript organization. We also thank Ryan Chastain-Gross, Ph.D., from Edanz Group of China, for editing the English text of a draft of this manuscript. The sequencing service is provided by Mayorbio.</p></ack><fn-group><fn id=\"footnote1\"><label>1</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://genomics.senescence.info/cells/\">http://genomics.senescence.info/cells/</ext-link></p></fn></fn-group><sec id=\"S9\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fgene.2020.00867/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fgene.2020.00867/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"FS1\"><media xlink:href=\"Image_1.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS1\"><media xlink:href=\"Table_1.xls\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Acosta</surname><given-names>J. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Neurosci</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Neurosci</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Neurosci.</journal-id><journal-title-group><journal-title>Frontiers in Neuroscience</journal-title></journal-title-group><issn pub-type=\"ppub\">1662-4548</issn><issn pub-type=\"epub\">1662-453X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32848563</article-id><article-id pub-id-type=\"pmc\">PMC7431634</article-id><article-id pub-id-type=\"doi\">10.3389/fnins.2020.00790</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Neuroscience</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Electroencephalographic Cross-Frequency Coupling as a Sign of Disease Progression in Patients With Mild Cognitive Impairment: A Pilot Study</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Musaeus</surname><given-names>Christian Sandøe</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/689312/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Nielsen</surname><given-names>Malene Schjønning</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Musaeus</surname><given-names>Jørgen Sandøe</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Høgh</surname><given-names>Peter</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Neurology, Danish Dementia Research Centre (DDRC), Rigshospitalet, University of Copenhagen</institution>, <addr-line>Copenhagen</addr-line>, <country>Denmark</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Neurology, Regional Dementia Research Centre, Zealand University Hospital</institution>, <addr-line>Roskilde</addr-line>, <country>Denmark</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Niels Bohr Institute, University of Copenhagen</institution>, <addr-line>Copenhagen</addr-line>, <country>Denmark</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Clinical Medicine, University of Copenhagen</institution>, <addr-line>Copenhagen</addr-line>, <country>Denmark</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Gianfranco Spalletta, Santa Lucia Foundation (IRCCS), Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Marco Onofrj, School of Medicine and Health Sciences, G. D’Annunzio University of Chieti–Pescara, Italy; Fernando Maestú, Complutense University of Madrid, Spain</p></fn><corresp id=\"c001\">*Correspondence: Christian Sandøe Musaeus, <email>christian.sandoee.musaeus@regionh.dk</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>14</volume><elocation-id>790</elocation-id><history><date date-type=\"received\"><day>20</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>06</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Musaeus, Nielsen, Musaeus and Høgh.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Musaeus, Nielsen, Musaeus and Høgh</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Mild cognitive impairment (MCI) refers to mild objective cognitive deficits and is associated with the later development of Alzheimer’s disease (AD). However, not all patients with MCI convert to AD. EEG spectral power has shown promise as a marker of progression, but brain oscillations in different frequencies are not isolated entities. Coupling between different frequency bands, so-called cross-frequency coupling (CFC), has been associated with memory function and may further contribute to our understanding of what characterizes patients with MCI who progress to AD. In the current study, we wanted to investigate the changes in gamma/theta CFC in patients with AD and MCI compared to HC and in patients with pMCI compared to patients with sMCI. Furthermore, we wanted to investigate the association with cognitive test scores. EEGs were included at baseline for 15 patients with AD, 25 patients with MCI, and 36 older HC, and the participants were followed for up to 3 years. To investigate CFC, we calculated the modulation index (MI), which has been shown to be less affected by noisy data compared to other techniques. We found that patients with pMCI showed a significantly lower global gamma/theta CFC compared to patients with sMCI. In addition, global gamma/theta CFC was significantly correlated with Addenbrooke’s Cognitive Examination (ACE) score (<italic>p</italic>-value = 0.030, rho = 0.527). Although not significant, patients with AD and MCI showed a lower gamma/theta CFC compared to HC. These findings suggest that gamma/theta CFC is important for proper cognitive functioning and that a decrease in gamma/theta CFC in patients with MCI may be a sign of progression. Gamma/theta CFC may therefore serve as a progression marker in MCI, but larger studies are needed to validate these findings.</p></abstract><kwd-group><kwd>mild cognitive impairment</kwd><kwd>Alzheimer’s disease</kwd><kwd>cross-frequency coupling</kwd><kwd>phase-amplitude coupling</kwd><kwd>gamma</kwd><kwd>theta</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Velux Fonden<named-content content-type=\"fundref-id\">10.13039/100008397</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"4\"/><table-count count=\"3\"/><equation-count count=\"0\"/><ref-count count=\"44\"/><page-count count=\"10\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Alzheimer’s disease (AD) is a progressive neurodegenerative disease leading to a disruption of normal brain oscillations (<xref rid=\"B5\" ref-type=\"bibr\">Coben et al., 1985</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Jeong, 2004</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Musaeus et al., 2018a</xref>). The disruption of brain oscillations has even been seen in the prodromal phase of a disease known as mild cognitive impairment (MCI) (<xref rid=\"B26\" ref-type=\"bibr\">Musaeus et al., 2018b</xref>). MCI refers to mild objective cognitive deficits and is associated with the later development of AD (<xref rid=\"B32\" ref-type=\"bibr\">Petersen et al., 1999</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Petersen, 2004</xref>), but not all patients with mild MCI convert to AD (<xref rid=\"B31\" ref-type=\"bibr\">Petersen, 2004</xref>). Attempts have been made to use electroencephalography (EEG) as a marker of whether a patient with MCI will progress (pMCI) or remain stable (sMCI) using spectral power (<xref rid=\"B16\" ref-type=\"bibr\">Jelic et al., 2000</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Poil et al., 2013</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Musaeus et al., 2018b</xref>). However, brain oscillations of different frequencies are not isolated entities, and the coupling between different frequency bands may further our understanding of the pathophysiological processes (<xref rid=\"B3\" ref-type=\"bibr\">Buzsáki and Watson, 2012</xref>). This coupling between different frequencies, a phenomenon called cross-frequency coupling (CFC) (<xref rid=\"B4\" ref-type=\"bibr\">Canolty and Knight, 2010</xref>), has shown a variety of functional roles, including memory in both rodents and humans.</p><p>The most commonly studied coupling is between the amplitude in the gamma band and the phase in the theta range (gamma/theta). This has been associated with activity in the hippocampus and performance in memory tasks in rodents (<xref rid=\"B38\" ref-type=\"bibr\">Tort et al., 2009</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Canolty and Knight, 2010</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Lisman and Jensen, 2013</xref>) and has been shown to be vital for working memory in humans (<xref rid=\"B1\" ref-type=\"bibr\">Axmacher et al., 2010</xref>). In rodent models of AD, studies have found that impaired gamma/theta coupling arises before amyloid beta accumulation (<xref rid=\"B12\" ref-type=\"bibr\">Goutagny et al., 2013</xref>) and attenuated gamma/theta coupling in knock-out mice (<xref rid=\"B44\" ref-type=\"bibr\">Zhang et al., 2016</xref>; <xref rid=\"B2\" ref-type=\"bibr\">Bazzigaluppi et al., 2018</xref>). Studies investigating patients with AD have found diverging results (<xref rid=\"B34\" ref-type=\"bibr\">Poza et al., 2017</xref>; <xref rid=\"B40\" ref-type=\"bibr\">Wang et al., 2017</xref>). One study found a decrease in CFC (<xref rid=\"B34\" ref-type=\"bibr\">Poza et al., 2017</xref>), while another found an increased CFC (<xref rid=\"B40\" ref-type=\"bibr\">Wang et al., 2017</xref>) in AD compared to healthy controls (HC). Furthermore, one study found that CFC was decreased in both AD and MCI compared with HC and associated with working memory deficits (<xref rid=\"B11\" ref-type=\"bibr\">Goodman et al., 2018</xref>). The underlying reason for the diverging results may be due to differences in the methods applied to calculate CFC. Furthermore, to our knowledge, CFC has not been examined in patients with pMCI compared to patients with sMCI, which may give valuable knowledge on the underlying pathophysiology.</p><p>When considering the CFC in different frequency bands, besides gamma/theta CFC, studies have found that CFC coupling between alpha, beta, and gamma may play an important role in coordination in perception, consciousness, and working memory (<xref rid=\"B30\" ref-type=\"bibr\">Palva and Palva, 2007</xref>). Furthermore, beta–delta CFC has been associated with neuropsychiatric symptoms, which is also prevalent in patients with AD (<xref rid=\"B23\" ref-type=\"bibr\">Miskovic et al., 2010</xref>). Therefore, couplings between other frequencies besides gamma/theta CFC may also be disrupted in both AD and MCI.</p><p>In the current study, we investigated the differences in gamma/theta CFC between AD, MCI, and HC and the differences in patients with pMCI compared to patients with sMCI. Our hypothesis was that patients with AD showed lower gamma/theta CFC compared to both MCI and HC and that pMCI showed lower gamma/theta CFC than sMCI. In an exploratory manner, we also examined the changes in CFC in multiple frequency bands. Lastly, we investigated the association between gamma/theta CFC and both cognitive function and cerebrospinal fluid (CSF) markers in AD.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Subjects</title><p>Data have also been presented in other studies using other types of analyses (<xref rid=\"B26\" ref-type=\"bibr\">Musaeus et al., 2018b</xref>, <xref rid=\"B27\" ref-type=\"bibr\">2019a</xref>,<xref rid=\"B28\" ref-type=\"bibr\">b</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Schjonning Nielsen et al., 2018</xref>), and the data were in part collected as a large multicenter study (<xref rid=\"B9\" ref-type=\"bibr\">Engedal et al., 2015</xref>). This cohort study was conducted at two Danish memory clinics and involved patients who were diagnosed with either MCI or mild AD and a baseline Mini-Mental State Examination (MMSE) score of ≥22. The following exclusion criteria were used: (1) no close relatives who wished to participate, (2) participating in intervention studies or (3) suffered from other neurological, psychiatric, or other severe diseases, (4) received sedative medication, and (5) had any past or current addiction to alcohol or medications.</p><p>The HC were volunteers and recruited through public advertisements at memory clinics, at local associations for elders, or through an online recruitment site. The inclusion criteria were (1) between 50 and 90 years old, (2) MMSE score >26, (3) ACE > 85, (4) normal neurological and clinical examination, (5) normal or age-related brain atrophy measured on a computed tomography scan, and (6) normal routine blood tests. The exclusion criteria were (1) an inability to participate (including impaired vision or hearing), (2) cognitive symptoms including memory complaints, (3) major neurological, psychiatric, or other severe diseases, which could cause cognitive impairments including major depression or a geriatric depression scale score >7, (4) pregnancy, (5) had undergone general anesthesia, (6) received electroconvulsive therapy in the last 3 months, (7) use of sedatives, or (8) past or current addiction to alcohol or medications.</p><p>In total, we included 17 patients with AD, 27 patients with MCI, and 38 HC in the study. The study was approved by the Regional Ethical Committee.</p><p>Due to excessive artifacts, we excluded the following number of EEGs: two from patients with AD, two from patients with MCI, and one from HC. When comparing pMCI and sMCI, one EEG from MCI was excluded due to clinical progression to vascular dementia. One HC was excluded from the CFC analysis due to fewer than 70 1-s epochs.</p></sec><sec id=\"S2.SS2\"><title>Diagnostic Assessment</title><p>All the patients underwent a full physical and neurological examination, routine blood analysis, brain CT or MRI scan as well as cognitive screening. The cognitive screening included MMSE, Addenbrooke’s Cognitive Examination (ACE), and Digit Symbol Substitution Test (DSST), including Clinical Dementia Rating (CDR). In addition, the patients and the relatives underwent NeuroPsychiatric Inventory (NPI), Major Depression Inventory (MDI), and Activities of Daily Living Inventory (ADCS-ADL). All CT and MRI scans were examined by a neuro-radiologist. Except for two patients with MCI and six HC, all patients had a lumbar puncture performed. If it was considered relevant for the diagnostic evaluation, the patients underwent a neuropsychological evaluation by a clinical neuropsychologist. Diagnoses were settled by consensus of a multidisciplinary team based on all examination results. The included MCI patients fulfilled the Winblad consensus criteria (<xref rid=\"B43\" ref-type=\"bibr\">Winblad et al., 2004</xref>) and the AD patients fulfilled the NIA-AA criteria (<xref rid=\"B22\" ref-type=\"bibr\">McKhann et al., 2011</xref>).</p><p>All HC underwent the standardized diagnostic assessment at the time of inclusion, which included cognitive tests (ACE, MMSE, and DSST) and MDI. Lumbar puncture and subsequent CSF analysis was performed on almost all HC. At the baseline visit, all HC were referred for a standardized EEG, and the EEG recordings were not used in the assessment.</p></sec><sec id=\"S2.SS3\"><title>Study Design</title><p>All patients were recruited within 6 months after the diagnosis and tests were repeated at inclusion. Follow-up visits were carried out on a yearly basis as part of the routine visits, with serial cognitive tests, which included MMSE and ACE and the NPI, MDI, ADCS-ADL, and CDR scales. Clinical progression of MCI to AD was determined based on whether the patient clinically fulfilled the NIA-AA criteria (<xref rid=\"B22\" ref-type=\"bibr\">McKhann et al., 2011</xref>). If a patient with MCI progressed to another diagnosis than AD, they were excluded from the comparison between pMCI and sMCI.</p><p>The primary investigator performing the tests was blinded from the results of the EEG, imaging, and CSF analysis during the study period. This was done to ensure that the investigator was blinded for the potential presence of underlying AD pathology.</p><p>The reason for the dropout from the study without having a yearly follow-up for 3 years was mostly that they were recruited later and therefore could not complete all 3 years (six AD and eight MCI) or were not able to be tested in a proper way in a follow-up session (three AD and one MCI), wanted to drop out (one AD, two MCI, and three HC) or died (one AD and two MCI).</p></sec><sec id=\"S2.SS4\"><title>Electroencephalography Recording</title><p>The EEG recordings were performed at the two participating centers and performed using NicoletOne EEG Systems (Natus<sup>®</sup>), with a sampling rate of either 500 or 1,000 Hz. Nineteen electrodes were positioned according to the International 10–20 system. Most EEGs were recorded with alternating closed eyes, and the eyes were open for 3 min each, but some of the recordings only had closed eyes segments. The participants were alerted if they became visibly drowsy. The neurophysiology assistant recording the EEG made notations in the EEG when the participant closed and opened their eyes. After the recording, the files were exported as raw EEG.</p></sec><sec id=\"S2.SS5\"><title>Collection and Analysis of Cerebrospinal Fluid</title><p>Lumbar puncture was performed between the L3/L4 or the L4/L5 intervertebral space, and the subsequent CSF was collected in polypropylene tubes. The analyses included routine parameters and the core AD biomarkers, i.e., Aβ<sub>42</sub>, T-tau, and P-tau. The AD biomarkers were quantified with sandwich ELISAs [INNOTEST amyloid-β<sub>42</sub>, hTau, and Phospho-Tau (181P), respectively; Fujirebio Europe, Ghent, Belgium]. The AD biomarker analyses from both clinics were all carried out at one central laboratory.</p><p>In some of the patients, the CSF concentration of the measured AD biomarker was above the detection rate. In these cases, the value was excluded from further analysis.</p></sec><sec id=\"S2.SS6\"><title>Preprocessing of EEG</title><p>For the pre-processing steps, MATLAB (Mathworks, v2016a) and EEGLAB toolbox (<xref rid=\"B6\" ref-type=\"bibr\">Delorme and Makeig, 2004</xref>) were used. The closed eyes segments were selected and the electrodes were computationally located on the scalp using the dipfit toolbox (<xref rid=\"B29\" ref-type=\"bibr\">Oostenveld et al., 2011</xref>) using the standard 10–20 electrode model. Afterward, the data were bandpass-filtered from 1 to 70 Hz and bandstop-filtered from 45 to 55 Hz using the <italic>pop_firws</italic> function in MATLAB, with a filter order of 2, and the Kaiser window parameter beta was estimated using a maximum passband ripple of 0.001; the data were downsampled to 200 Hz. Afterward, the data were divided into 1-s epochs, and the epochs with excessive noise or artifacts as judged by visual inspection were removed. Spherical interpolation was applied for channels with excessive noise, drift, or bad connection. The EEG had to have less than or equal to three electrodes with excessive artifact; otherwise, the EEG was excluded from the analysis. Afterward, the EEGs were re-referenced to average reference, and independent component analysis was performed using the extended infomax algorithm (<xref rid=\"B18\" ref-type=\"bibr\">Lee et al., 1999</xref>) for each file. The components that contained eye blinks, eye movement, or specific line noise artifacts were removed. Lastly, the EEGs were inspected visually again, and the epochs with excessive noise or artifacts were removed. The investigator performing the preprocessing was blinded to the diagnosis.</p></sec><sec id=\"S2.SS7\"><title>Cross-Frequency Coupling</title><p>To calculate CFC, we used the modulation index (MI) (<xref rid=\"B39\" ref-type=\"bibr\">Tort et al., 2008</xref>) since it has been shown to be less affected by noisy data recorded at a lower sampling compared with other techniques (<xref rid=\"B14\" ref-type=\"bibr\">Hülsemann et al., 2019</xref>). Since MI is affected by small segments, we concatenated the 70 1-s epochs of EEG from each participant, thereby having one epoch of 70 s. The number of epochs was selected based on the largest number of epochs being included while excluding the least number of participants. First, raw data are filtered at the two frequency ranges using the <italic>pop_eegfiltnew</italic> function from EEGLAB (<xref rid=\"B6\" ref-type=\"bibr\">Delorme and Makeig, 2004</xref>). Afterward, the Hilbert transform was applied to extract the time series of the amplitude of the higher frequency band and the phase of the lower frequency band. The composite time series is then constructed, which gives the amplitude of the higher frequency band oscillation at each phase of the lower frequency band rhythm. Next, the phases of the lower band were binned into 18 frequency bins, and the mean of the amplitude of the higher frequency band over each phase bin was calculated. We then normalize the mean amplitudes by dividing each bin value by the sum of the mean amplitudes over all the bins. Lastly, we calculated the associated entropy measure for the normalized mean amplitudes and used them to compute the MI (see the <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Material</xref> for the MATLAB code). First, we calculated the gamma/theta CFC with gamma between 30 and 40 Hz and theta between 4 and 7 Hz. We did not include 40–50 Hz gamma due to contamination with line noise. For the exploratory analysis, we performed CFC for gamma–beta, gamma–alpha, gamma–delta, beta–alpha, beta–theta, beta–delta, alpha–theta, alpha–delta, and theta–delta. The MATLAB script used for calculating CFC in the current study can be found in the <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Material</xref>.</p></sec><sec id=\"S2.SS8\"><title>Statistics</title><p>All statistics were performed in MATLAB (vR2017b). When comparing the demographics and the cognitive scores for AD, MCI, and HC, we performed one-way ANOVAs, and <italic>t</italic>-tests were used to compare AD and HC, AD and MCI, and MCI and HC. The <italic>t</italic>-tests were used when comparing baseline demographics, cognitive test scores, and CSF measurements for pMCI and sMCI and for comparing baseline and 2nd-year follow-up for HC, MCI, and AD.</p><p>The CFC data were log-transformed before any of the subsequent analyses between groups due to the non-normal distribution. For comparing CFC between all three groups, we performed an ANCOVA (<xref rid=\"B13\" ref-type=\"bibr\">Gruner, 2020</xref>) with age, gender, education, and current medication as covariates. For comparing pMCI vs. sMCI based on the 2nd-year follow-up, we used a general linear model with the same covariate as mentioned above. In addition, we also compared AD and pMCI using the same technique as mentioned above. We performed correction for multiple comparisons for the electrode-to-electrode comparisons for each frequency band separately using false discovery rate. The comparisons between three groups and two groups for global CFC were computed as described above. For the correlation between gamma/theta CFC and CSF biomarkers (amyloid, total tau, and phosphorylated tau), ACE, and MMSE in patients with MCI, we used partial correlation with the same covariates as described above due to variability between subjects. The same analysis was performed to correlate ACE and total tau.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Demographics</title><p>See <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> for characterization of the patients included in the analysis and the comparisons between groups. We found a significantly lower age for HC compared to MCI (<italic>p</italic>-value = 0.002, <italic>t</italic>-value = 3.187), and the mean MMSE was significantly lower in patients with AD (<italic>p</italic>-value < 0.001, <italic>t</italic>-value = −4.840) and MCI (<italic>p</italic>-value < 0.001, <italic>t</italic>-value = −4.646) compared to HC. Regarding medications, patients with MCI received significantly more antidepressants than HC (<italic>p</italic>-value < 0.05), while patients with AD received significantly more cholinesterase inhibitors (<italic>p</italic>-value < 0.001) compared to both HC and MCI. In AD, we found that CSF amyloid was significantly lower than in both MCI (<italic>p</italic>-value = 0.015) and HC (<italic>p</italic>-value < 0.001) and that it was significantly lower in MCI compared to HC (<italic>p</italic>-value 0.022). Furthermore, AD had a significantly higher concentration of CSF total tau compared to both MCI (<italic>p</italic>-value = 0.004) and HC (<italic>p</italic>-value < 0.001), and MCI showed a higher CSF total tau compared to HC (<italic>p</italic>-value = 0.012). The performance on cognitive screening instruments for baseline and at follow-up visit after 2 years with comparison between the scores can be seen in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Characteristics of the participants included in the analysis.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HC (<italic>n</italic> = 36)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">AD (<italic>n</italic> = 15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MCI (<italic>n</italic> = 25)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic>-value</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mean age (SD), years</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">65.8 (7.1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70.1 (7.8)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">71.4 (6.0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.008*</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female gender, <italic>n</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.133</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Education, years (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.8 (3.6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.1 (4.0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.6 (3.4)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.080</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MMSE, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.1 (1.0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.3 (3.2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.6 (1.5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.001*</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Antidepressants</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.046*</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cholinesterase Inhibitors</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.001*</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pain killers</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.555</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CSF amyloid, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">997.5 (320.2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">550.7 (141.2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">782.3 (319.8)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.001*</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CSF total tau, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">303.3 (144.7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">618.4 (186.0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">419.6 (173.9)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.001*</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CSF phosphorylated tau, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">68.5 (103.4)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">93.0 (33.3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">59.4 (21.5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.384</td></tr></tbody></table><table-wrap-foot><attrib><italic>HC, healthy controls; AD, Alzheimer’s disease; MCI, mild cognitive impairment; SD, standard deviation; MMSE, Mini-Mental State Examination; CSF, cerebrospinal fluid. The asterisk indicates a significant p-value (<0.05).</italic></attrib></table-wrap-foot></table-wrap><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>The cognitive scores, number of participants that dropped out, and number of patients with mild cognitive impairment (MCI) that progressed during follow-up in year 2.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Baseline</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2nd-year follow-up</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>t</italic>-value</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic>-value</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"5\" rowspan=\"1\"><bold>HC</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dropout/total (<italic>n</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/36</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Progression/no-progression</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MMSE, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.37 (0.84)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.08 (1.00)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.313</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.193</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ACE, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">95.66 (3.34)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">94.72 (3.33)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.181</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.242</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MDI, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.86 (3.13)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.53 (2.85)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.464</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.644</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"5\" rowspan=\"1\"><bold>MCI</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dropout/total (<italic>n</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6/25</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Progression/no-progression</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12/13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MMSE, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.60 (1.50)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.00 (3.33)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.138</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.038*</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ACE, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">84.13 (8.17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">79.67 (11.59)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.464</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.151</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MDI, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.13 (5.91)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.22 (7.75)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.450</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.155</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NPI, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.38 (3.49)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.24 (2.49)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.844</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.073</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ADL, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70.71 (4.84)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">66.59 (9.87)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.544</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.133</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"5\" rowspan=\"1\"><bold>AD</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dropout/total (<italic>n</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7/15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Progression/no-progression</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MMSE, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.27 (3.17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.50 (5.53)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.537</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.139</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ACE, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">77.60 (12.87)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">67.14 (18.85)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.532</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.141</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MDI, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.67 (4.70)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.17 (4.62)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.664</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.515</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NPI, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.5 (1.24)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.00 (2.45)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–4.235</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.000*</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ADL, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70.86 (8.16)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">67.38 (8.67)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.942</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.358</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missing values (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic>In addition, the percentage of missing values for the cognitive scores can be seen. All cognitive scores have been compared over time using a paired <italic>t</italic>-test. The asterisk indicates a significant <italic>p</italic>-value (<0.05).</italic></attrib></table-wrap-foot></table-wrap><p>When comparing the scores at baseline between pMCI and sMCI, we found that ACE was significantly different (see <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). When looking at the ACE subscores, we found that the ACE subscore comprehension was significantly different between the two groups [mean pMCI (SD) = 3.57 (0.79), mean sMCI (SD) = 4 (0), <italic>p</italic>-value = 0.035, <italic>t</italic>-stat = −2.249] and a trend for anterograde memory [mean pMCI (SD) = 16.14 (7.71), mean sMCI (SD) = 21.50 (4.68), <italic>p</italic>-value = 0.051, <italic>t</italic>-stat = −2.070]. The rest of the subscores were not significantly different between pMCI and sMCI (<italic>p</italic>-value > 0.05).</p><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>Demographics, baseline cognitive scores, and cerebrospinal fluid results for stable mild cognitive impairment (sMCI) and progressed mild cognitive impairment (pMCI).</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Baseline – sMCI (<italic>n</italic> = 13)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Baseline – pMCI (<italic>n</italic> = 11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic>-value</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mean age (SD), years</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">72.38 (6.06)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70.27 (6.63)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.424</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female gender, <italic>n</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.500</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Education, years (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.69 (3.84)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.55 (3.36)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.922</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CSF amyloid, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">820.08 (348.64)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">695.75 (309.90)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.419</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CSF total tau, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">398.25 (162.10)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">461.56 (206.29)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.440</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CSF phosphorylated tau, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60.54 (24.54)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">59.89 (19.28)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.948</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MMSE, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.92 (1.38)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.09 (1.58)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.182</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ACE, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">87.54 (6.08)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">79.00 (8.36)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.010*</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MDI, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.67 (6.89)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.00 (4.14)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.297</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NPI, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.09 (3.96)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.00 (2.24)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.952</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CDR, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.50 (0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.56 (0.17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.281</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ADL, mean (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70.60 (6.06)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70.86 (2.73)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.918</td></tr></tbody></table><table-wrap-foot><attrib><italic>The <italic>t</italic>-tests were performed to compare the two groups for each score separately. The asterisk indicates a significant (<italic>p</italic>-value < 0.05) difference. One patient with mild cognitive impairment showed up during follow-up to fulfill the criteria for vascular dementia and was not included in the comparison between pMCI and sMCI.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS2\"><title>Gamma/Theta Cross-Frequency Coupling</title><p>We found a significantly lower global gamma/theta CFC in pMCI compared with sMCI (<italic>p</italic>-value = 0.004, <italic>t</italic>-value = −11.09; see <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). After adjusting for multiple comparisons using false discovery rate, no significant differences in the electrode-to-electrode comparisons between pMCI and sMCI were found. The strongest correlation was found between ACE and gamma/theta CFC (<italic>p</italic>-value = 0.030, rho = 0.527; see <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref> for the scatterplot). No significant correlations were found between gamma/theta CFC and amyloid (<italic>p</italic>-value = 0.971, rho = 0.010), total tau (<italic>p</italic>-value = 0.592, rho = −0.151), or phosphorylated tau (<italic>p</italic>-value = 0.824, rho = −0.060) in MCI. Since total tau is a marker for neurodegeneration and found to be related to progression from MCI to AD (<xref rid=\"B8\" ref-type=\"bibr\">Diniz et al., 2008</xref>), we investigated the partial correlation between ACE and total tau in patients with MCI, which was not significant (<italic>p</italic>-value = 0.107, rho = −0.449).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Bar graph showing the gamma/theta cross-frequency coupling for progressed mild cognitive impairment (pMCI) or stable mild cognitive impairment (sMCI). The star indicates a significant difference between pMCI and sMCI (<italic>p</italic>-value = 0.004, <italic>t</italic>-value = –11.09).</p></caption><graphic xlink:href=\"fnins-14-00790-g001\"/></fig><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Scatterplots showing the significant correlation between gamma/theta cross-frequency coupling and Addenbrooke’s Cognitive Examination for patients with mild cognitive impairment.</p></caption><graphic xlink:href=\"fnins-14-00790-g002\"/></fig><p>When looking at AD and MCI compared to HC, we found a significantly lower gamma/theta CFC for patients with AD and MCI compared with HC (adjusted <italic>p</italic>-value < 0.05). Although not significant (<italic>p</italic>-value = 0.170, <italic>F</italic>-value = 1.821), we found a lower gamma/theta CFC in AD and MCI compared with that in HC (see <xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Bar graph showing the gamma/theta cross-frequency coupling for healthy controls and for mild cognitive impairment and Alzheimer’s disease patients.</p></caption><graphic xlink:href=\"fnins-14-00790-g003\"/></fig><p>When comparing the global gamma/theta CFC between AD and pMCI, we did not find a significant difference (<italic>p</italic>-value = 0.555, <italic>F</italic>-value = 0.360).</p></sec><sec id=\"S3.SS3\"><title>Exploratory Cross-Frequency Coupling Analyses</title><p>When examining the difference in CFC between multiple frequency bands, we did not find any significant changes in the electrode-to-electrode comparisons after correcting for multiple comparisons using false discovery rate (<italic>p</italic>-value < 0.05). The largest difference in global CFC was found for gamma/alpha CFC (<italic>p</italic>-value = 0.076, <italic>F</italic>-value = 2.678; see <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref> for the bar plots of global CFC). When comparing the global CFC for the rest, the differences were not as pronounced (<italic>p</italic>-value > 0.1).</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Bar graph showing the global scores for healthy controls and for mild cognitive impairment and Alzheimer’s disease patients for the exploratory cross-frequency coupling analyses.</p></caption><graphic xlink:href=\"fnins-14-00790-g004\"/></fig></sec></sec><sec id=\"S4\"><title>Discussion</title><p>In the current study, we found that global gamma/theta CFC was significantly lower in pMCI compared to that in sMCI. Furthermore, gamma/theta CFC was strongly correlated with ACE (<italic>p</italic>-value = 0.030, rho = 0.527). In the exploratory analysis of CFC, global gamma/alpha CFC showed the largest difference between the groups.</p><p>Only a few studies have investigated the changes in CFC in patients with AD. Here, two of the studies have found decreased CFC (<xref rid=\"B34\" ref-type=\"bibr\">Poza et al., 2017</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Goodman et al., 2018</xref>), while one study found increased CFC (<xref rid=\"B40\" ref-type=\"bibr\">Wang et al., 2017</xref>) in patients with AD compared to HC using the amplitude at a high-frequency band and the phase at a low-frequency band. In the current study, we found, although not significant, that gamma/theta CFC was decreased in both patients with AD and MCI compared to that in HC. This finding suggests that patients with AD show a disruption of the coupling between the frequency bands. More interestingly, we found that gamma/theta CFC was decreased in pMCI compared to sMCI. Gamma/theta CFC has been associated with activity in the hippocampus and performance in memory tasks in rodents (<xref rid=\"B38\" ref-type=\"bibr\">Tort et al., 2009</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Canolty and Knight, 2010</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Lisman and Jensen, 2013</xref>) and has been shown to be vital for working memory in humans (<xref rid=\"B1\" ref-type=\"bibr\">Axmacher et al., 2010</xref>). This could suggest a disruption of brain areas known to be affected in AD. We hypothesize that the decreased gamma/theta CFC in pMCI is associated with a network dysfunction but possibly not directly associated with atrophy since no correlation was found with total tau. The lack of any electrode-specific changes between pMCI and sMCI could indicate that this is more related to global dysfunction and less specifically to the temporal lobes. In addition, studies implementing either an N-back task (<xref rid=\"B11\" ref-type=\"bibr\">Goodman et al., 2018</xref>) or an auditory oddball paradigm (<xref rid=\"B7\" ref-type=\"bibr\">Dimitriadis et al., 2015</xref>) found more pronounced results, which may indicate that the difference in CFC is larger during a task. As a diagnostic tool in AD and MCI, this suggests that gamma/theta CFC should be implemented during a task. Other EEG markers of progression have been suggested, and decreased spectral beta power has been the most commonly reported (<xref rid=\"B16\" ref-type=\"bibr\">Jelic et al., 2000</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Poil et al., 2013</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Musaeus et al., 2018b</xref>). This study suggests that gamma/theta CFC may serve as a marker of progression but has some limitations for clinical application due to low signal-to-noise ratio in the gamma band and the possible necessity to conduct the EEG during a task. More studies are needed to understand the applicability of gamma/theta CFC, which may, however, be beneficial in research on the pathophysiology of progressive neurodegenerative disorders vs. non-progressive conditions and potentially also in clinical trials.</p><p>When investigating the relationship between global gamma/theta CFC and clinical presentation, we found that it was correlated with ACE (<italic>p</italic>-value = 0.030, rho = 0.527). Previous studies have found that gamma/theta CFC has been associated with dysfunction in the temporal lobe structures. However, since we calculated the global gamma/theta CFC score, it may be the reason for a significant correlation with ACE since it is a score for multiple cognitive domains (<xref rid=\"B21\" ref-type=\"bibr\">Mathuranath et al., 2000</xref>). Therefore, gamma/theta CFC may reflect decreased functional coupling between the temporal lobes and the rest of the brain. Total tau has previously been shown to be a marker progression from MCI to AD (<xref rid=\"B8\" ref-type=\"bibr\">Diniz et al., 2008</xref>) and associated with neurodegeneration regardless of the underlying condition (<xref rid=\"B15\" ref-type=\"bibr\">Jack and Holtzman, 2013</xref>) and may therefore be related to gamma/theta CFC. However, we did not find a significant correlation between total tau and gamma/theta CFC nor total ACE test score. This may be attributed to the low sample size or delays between neurodegeneration and subsequent changes in cognition. If gamma/theta CFC is to be considered as an early marker of progressive symptoms, it may also be abnormal in patients with progressive pathophysiology with yet no neurodegeneration, parallel to what is observed in, for instance, PET biomarker studies.</p><p>The majority of studies have focused on gamma/theta CFC, but coupling between other frequency bands has been shown to play an important role for coordination in perception, consciousness, and working memory (<xref rid=\"B30\" ref-type=\"bibr\">Palva and Palva, 2007</xref>) and has been associated with neuropsychiatric symptoms (<xref rid=\"B23\" ref-type=\"bibr\">Miskovic et al., 2010</xref>), which are also prevalent in patients with AD. However, we did not find any significant changes for CFC between AD, MCI, and HC in the other frequency bands. The largest difference was found for gamma/alpha coupling (<italic>p</italic>-value = 0.076, <italic>F</italic>-value = 2.678). The lack of findings suggests that gamma/theta coupling may be the most important measure in AD. However, larger studies are needed to fully understand the pathophysiological role of CFC in AD.</p><p>When examining the neuropsychological test scores between pMCI and sMCI, we found that patients with pMCI had a significantly lower ACE compared to patients with sMCI (see <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). Studies have previously found that neuropsychological tests (<xref rid=\"B20\" ref-type=\"bibr\">Maioli et al., 2007</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Rozzini et al., 2008</xref>) and ACE (<xref rid=\"B10\" ref-type=\"bibr\">Galton et al., 2005</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Mitchell et al., 2009</xref>) are able to predict the progression from MCI to AD. Furthermore, we found that global gamma/theta CFC was not significantly different between pMCI and AD. These findings may indicate that the included patients with pMCI were further downstream in the disease compared to patients with sMCI at the time of inclusion or that pMCI and sMCI comprised a fundamentally different underlying etiology. More studies are needed to investigate the potential of CFC as a marker of progression and potentially include patients at a very early stage of the disease.</p><p>The current study has some limitations. Firstly, we acknowledge the relatively small sample size, but these changes suggest an overall decoupling between the frequency bands even in the early stages of AD. Furthermore, we found a significant difference in the age between HC and MCI and have therefore used age as a covariate in the analysis. In addition, the follow-up time was short and, according to previous studies, the annual clinical progression rate is 15% (<xref rid=\"B32\" ref-type=\"bibr\">Petersen et al., 1999</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Saxton et al., 2009</xref>), which means that only 30% of the patients with MCI should have progressed to AD in the present cohort. However, we found that 48% progressed, which may in part be due to the patients with MCI being at a more advanced stage of the disease at inclusion. Furthermore, we concatenated 1-s epochs and, due to the discontinuity, this may have induced some additional noise to the calculations. Due to line noise, we calculated the amplitude of gamma between 30 and 40 Hz, which is a narrow range compared to the previous studies analyzing CFC (<xref rid=\"B4\" ref-type=\"bibr\">Canolty and Knight, 2010</xref>). In addition, the gamma band for standard scalp EEG has been suggested to be highly influenced by muscle activity (<xref rid=\"B42\" ref-type=\"bibr\">Whitham et al., 2007</xref>, <xref rid=\"B41\" ref-type=\"bibr\">2008</xref>), and this poses some limitations in implementing this technique as a clinical tool. Nevertheless, our findings in this small pilot study with decreased global gamma/theta CFC in patients with pMCI compared to patients with sMCI may be able to guide larger studies.</p></sec><sec id=\"S5\"><title>Conclusion</title><p>In conclusion, our findings suggest that decreased global gamma/theta CFC is associated with patients with MCI, who over time progress to AD. Furthermore, global gamma/theta CFC may be related to global cognitive function as assessed with ACE. Gamma/theta CFC may therefore serve as a progression marker in MCI. However, larger studies are needed to validate these findings.</p></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>The datasets supporting the conclusions of this article will be made available by the authors to any qualified researcher. However, due to regulations, we are not able to share the EEG files. Requests to access the datasets should be directed to CM, <email>christian.sandoee.musaeus@regionh.dk</email>.</p></sec><sec id=\"S7\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by the Regional Committee on Health Research Ethics. The patients/participants provided their written informed consent to participate in this study.</p></sec><sec id=\"S8\"><title>Author Contributions</title><p>PH, MN, and CM conceived the project idea of using quantitative EEG. PH and MN conducted the experiments. CM and JM conducted the data analyses. CM drafted the manuscript. PH, MN, JM, and CM contributed to revising the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was funded by the Velux Foundation.</p></fn></fn-group><ack><p>We would like to thank the study nurses for all their help in conducting this study.</p></ack><sec id=\"S11\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fnins.2020.00790/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fnins.2020.00790/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Table_1.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Axmacher</surname><given-names>N.</given-names></name><name><surname>Henseler</surname><given-names>M. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Endocrinol.</journal-id><journal-title-group><journal-title>Frontiers in Endocrinology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2392</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849314</article-id><article-id pub-id-type=\"pmc\">PMC7431635</article-id><article-id pub-id-type=\"doi\">10.3389/fendo.2020.00550</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Fructose Consumption During Pregnancy Influences Milk Lipid Composition and Offspring Lipid Profiles in Guinea Pigs</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Smith</surname><given-names>Erin Vanessa LaRae</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/936852/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Dyson</surname><given-names>Rebecca Maree</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/461406/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Berry</surname><given-names>Mary Judith</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/464764/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Gray</surname><given-names>Clint</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/223526/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Paediatrics and Child Health, University of Otago</institution>, <addr-line>Wellington</addr-line>, <country>New Zealand</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Centre for Translational Physiology, University of Otago</institution>, <addr-line>Wellington</addr-line>, <country>New Zealand</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Sarah Glastras, Royal North Shore Hospital, Australia</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Alberto Camacho-Morales, Autonomous University of Nuevo León, Mexico; Ma. Cecilia Opazo, Andres Bello University, Chile</p></fn><corresp id=\"c001\">*Correspondence: Clint Gray <email>clint.gray@otago.ac.nz</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Translational Endocrinology, a section of the journal Frontiers in Endocrinology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>550</elocation-id><history><date date-type=\"received\"><day>09</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>06</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Smith, Dyson, Berry and Gray.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Smith, Dyson, Berry and Gray</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Excess dietary fructose is a major public health concern (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>–<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Evidence shows increased fructose intake can cause insulin resistance, hepatic <italic>de novo</italic> lipogenesis, hypertriglyceridemia, obesity and non-alcoholic fatty liver disease (NAFLD) (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>–<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). However, little is known about the effects of fructose during pregnancy and its influence on offspring development and predisposition to later-life disease. To determine whether moderately increased maternal fructose intake could have health consequences on offspring, we have investigated the effects of 10% w/v fructose water intake during preconception and pregnancy. Female Dunkin Hartley guinea pigs were fed a control diet (CD) or fructose diet (FD;10% kcal from fructose) <italic>ad-libitum</italic> 60 days prior to mating and throughout gestation. Offspring were culled at weaning, day 21 (d21). Compared to CD dams, FD dams had altered glucose metabolism and increased milk free fatty acid content. Matsuda-DeFronzo insulin sensitivity index (M-ISI) from OGTT plasma showed no significant difference in whole-body insulin sensitivity between FD and CD dams 60 days post-dietary intervention and during midgestation. Fetal exposure to increased maternal fructose resulted in offspring with significantly altered serum free fatty acids at days 0, 7, 14, and 21 [including pentadecanoic acid (15:0), dma16:0, margaric acid (17:0) palmitoleic acid, total omega-7 and total saturates], increased levels of uric acid and triglycerides were also observed at d21. We have demonstrated that increased fructose intake during pregnancy can cause significant changes in maternal metabolic function and milk composition, which alters offspring metabolism. Taken together, these changes in pregnancy outcomes and feto-maternal condition may underlie their offspring's predisposition to metabolic dysfunction during later-life.</p></abstract><kwd-group><kwd>maternal fructose</kwd><kwd>developmental programming</kwd><kwd>milk composition</kwd><kwd>hepatic lipids</kwd><kwd>free fatty acids</kwd></kwd-group><counts><fig-count count=\"4\"/><table-count count=\"6\"/><equation-count count=\"1\"/><ref-count count=\"64\"/><page-count count=\"12\"/><word-count count=\"9056\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Excess dietary fructose intake is a major public health concern (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>–<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Under normal physiological conditions, when the liver metabolizes fructose, it favors lipogenesis. It has also been shown that when the liver metabolizes fructose excess, it can result in metabolic dysregulation resulting in hyperlipidemia, insulin resistance, obesity, diabetes, cardiovascular disease and non-alcoholic fatty liver disease (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>–<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). The World Health Organization (WHO) recommends simple sugars from processed foods and sugar-sweetened beverages should be <10% of total daily caloric intake (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). It is currently estimated that 18–25% of total daily calorie intake comes from simple sugars in a Westernized diet (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). However, there is a paucity of data examining the impact of increased fructose intake before and during pregnancy and subsequent adverse effects on lactation, fetal development and offspring metabolic function.</p><p>Metabolic adaptations during pregnancy are necessary for appropriate metabolic demands supporting appropriate growth and development of the fetus and preparation for lactation. Human epidemiological studies and animal models have shown that poor quality nutrition during fetal growth can predispose the offspring to long-term health consequences (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Unbalanced maternal nutrition, in particular, overnutrition, can have permanent effects on her offspring's organ structure and function, predisposing them to adult-onset of non-communicable disease such as; obesity, diabetes, NAFLD and cardiovasclar risk factors (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>–<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). The contemporary western women consuming high levels of fructose before and/or during pregnancy may alter critical phases of pregnancy, such as embryogenesis, fetal-placental development, and milk production and quality (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>).</p><p>Previous research in rodents has demonstrated that maternal consumption of 10% fructose in water during pregnancy and lactation resulted in maternal hyperglycemia, hyperinsulinemia and hypertriglyceridemia, which were associated with significantly elevated plasma insulin in offspring at weaning, suggesting offspring susceptibility to diabetes during adulthood (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Further rodent studies demonstrated that 20% of caloric intake from fructose during gestation resulted in maternal hyperinsulinemia and sex-specific effects in offspring with female offspring having higher plasma leptin and glucose and displaying greater vulnerability to metabolic disturbances in neonatal life than male offspring (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). Additionally, we have previously shown sex-specific cardio-metabolic differences in the offspring of maternal 10% w/v fructose-fed dams (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Some studies have reported that high fructose intake alters beta-oxidation, increases free fatty acids (FFAs) and triglycerides, causing dyslipidemia, hepatic lipid accumulation and insulin resistance (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>, <xref rid=\"B21\" ref-type=\"bibr\">21</xref>, <xref rid=\"B22\" ref-type=\"bibr\">22</xref>). It is essential to determine how the vertical transmission of such deleterious metabolic effects might increase <italic>de novo</italic> lipogenesis, fatty acid acylation and subsequent metabolic programming in offspring. Therefore, the current study aimed to characterize the phenotypic effects of excess maternal fructose consumption on maternal physiology, metabolism, milk quality and determine the impact upon physiology and metabolic status in young offspring.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Ethics</title><p>All studies were conducted with the prior ethical approval of the University of Otago Wellington Animal Ethics Committee (AEC 7-15) and performed in line with the standards described in the Guide for the Care and Use of Laboratory Animals, 8th Edition, and the National Animal Ethics Advisory Commission, New Zealand (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). Results are reported according to the ARRIVE guidelines (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>).</p></sec><sec><title>Animal Model</title><p>All animals were housed in polypropylene cages (Tecniplast, Australia) lined with untreated pine wood shavings in a sound-insulated room under a 12 h day/night light cycle within a temperature (18–23°C) and humidity controlled facility. Nineteen Virgin Dunkin Hartley females were randomly allocated to two different experimental groups: (1) Control diet (CD; <italic>n</italic> = 10) and (2) Fructose-diet (FD; <italic>n</italic> = 9). All guinea pigs received standard guinea pig chow (guinea pig pellets; Sharpes Stock Feeds, NZ) (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>), hay ~10 g, half of fresh silverbeet and ~18 g carrots daily. Millipore filtered water supplemented with 1.2 g/L of ascorbic acid was available <italic>ad libitum</italic>. FD dams water was additionally supplemented with 10 g/ml of D-fructose (Sigma Aldrich, USA) to create 10% fructose water from 12 weeks (w) to term delivery. Dam and offspring weights, water and food intake, as well as caloric intake, were calculated daily. Kcal intake from food was calculated daily using the manufacturer's feed formulation guide (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Guinea pig pellet nutrition facts and ingredients from Nutritech (Nutritech International Ltd., NZ).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Feedstuff</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Fresh weight</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>(%)</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Nutrient</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Amount</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>DM 100%</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"6\" style=\"background-color:#bbbdc0\" rowspan=\"1\"><bold>FORMULATION SUMMARY</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Broll</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">381.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">38.10</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bulk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.14</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lucerne pellets</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">300.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.00</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dry Matter (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">88.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.00</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Barley</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">220.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22.00</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RUM_MER (MJ/kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.03</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11.40</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Soya, extr, 47%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">55.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.50</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Crude Protein (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.90</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18.06</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Molasses, cane</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.00</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Oil A (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.61</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.96</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Limestone</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.00</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Crude Fiber (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14.05</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Salt</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.20</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ADF (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.31</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17.39</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rabbit Premix</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.20</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NDF (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.87</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.93</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rabbit Premix</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.20</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NDF (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.87</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.93</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,000.00 kg</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.00</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Starch (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20.92</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.77</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Starch + Sugar (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.57</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.19</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Calcium (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.50</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phosphorus (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.62</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Magnesium (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.27</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sodium (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.13</td></tr></tbody></table></table-wrap></sec><sec><title>Mating and Postnatal Care</title><p>At 6-months of age, dams were housed in a non-lineage harem with a single boar during estrous for a maximum of 72 h to ensure timed mating. Following mating, dams returned to individual cages and continued their experimental diets throughout gestation. After mating, abdominal ultrasounds were performed by experienced technicians to confirm pregnancy, detectable 3 weeks post-mating. Dams delivered spontaneously, and all litters were maintained at four pups following delivery. Total groups included CD males (<italic>n</italic> = 7); FD males (<italic>n</italic> = 7); CD females (<italic>n</italic> = 7); and FD females (<italic>n</italic> = 7). Excess pups were humanely euthanized by intraperitoneal injection of 0.5 ml of Pentobarbital. Each dam was housed with her pups until weaning (d21). Dams were weighed weekly, signs of lactation tracked daily and all pups weighed daily.</p></sec><sec><title>Dam and Offspring Oral Glucose Tolerance Tests and Blood Glucose Analysis</title><p>Oral glucose tolerance tests (OGTT) were performed on dams and offspring. In brief, all animals were fasted for 14 h overnight with <italic>ad libitum</italic> access to water. The animals were wrapping in a towel to limit movement and stress. A baseline blood collection (250 μl) was collected from the auricular vein. A standardized oral dextrose load (1,000 mg/kg) was measured then administered orally using a 5 ml syringe. Serial blood collections for glucose and insulin at 15, 30, 45, 60, 75, 90, 120, and 180 min for dams and 30, 60, 120, and 180 min for the offspring, due to their smaller size. In addition, offspring blood samples (50 μl) were taken for glucose at day 0 (prior to suckling), 7 and 14, following a 4 h fast during which time food, but not water, was withdrawn. All blood samples were collected via the auricular vein, placed on ice, then centrifuged at 4,000 rpm for 15 min. The plasma layer was extracted and stored at −80°C pending analysis. The Matsuda-DeFronzo Insulin Sensitivity Index (M-ISI) was used to evaluate whole-body insulin sensitivity in dams (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B26\" ref-type=\"bibr\">26</xref>). The Matsuda Index\n<disp-formula id=\"E1\"><mml:math id=\"M1\"><mml:mrow><mml:mtable columnalign=\"left\"><mml:mtr columnalign=\"left\"><mml:mtd columnalign=\"left\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>,</mml:mo><mml:mn>000</mml:mn><mml:mo>/</mml:mo><mml:mo>√</mml:mo><mml:mo stretchy=\"false\">[</mml:mo><mml:mo stretchy=\"false\">(</mml:mo><mml:mtext>fasting plasma glucose </mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:mtext>mg/dl</mml:mtext><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mtd></mml:mtr><mml:mtr columnalign=\"left\"><mml:mtd columnalign=\"left\"><mml:mrow><mml:mtext>               </mml:mtext><mml:mo>×</mml:mo><mml:mtext>fasting plasma insulin </mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>μ</mml:mi><mml:mtext>U/ml</mml:mtext><mml:mo stretchy=\"false\">)</mml:mo><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mtd></mml:mtr><mml:mtr columnalign=\"left\"><mml:mtd columnalign=\"left\"><mml:mrow><mml:mtext>               </mml:mtext><mml:mo>×</mml:mo><mml:mo stretchy=\"false\">(</mml:mo><mml:mtext>mean glucose </mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:mtext>mg/dl</mml:mtext><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mtd></mml:mtr><mml:mtr columnalign=\"left\"><mml:mtd columnalign=\"left\"><mml:mrow><mml:mtext>               </mml:mtext><mml:mo>×</mml:mo><mml:mtext>mean insulin </mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>μ</mml:mi><mml:mtext>U/ml</mml:mtext><mml:mo stretchy=\"false\">)</mml:mo><mml:mo stretchy=\"false\">]</mml:mo><mml:mo>.</mml:mo></mml:mrow></mml:mtd></mml:mtr></mml:mtable></mml:mrow></mml:math></disp-formula></p></sec><sec><title>Dam Milk Free Fatty Acid Analysis</title><p>Dam's milk was extracted within 4 h of the birth of the last pup. Approximately 40 μl of milk was applied to a Dried Milk Spot (DMS) PUFAcoat? card (University of Adelaide, Australia). PUFAcoat? cards were dried at room temperature for 1 h and stored at room temperature until further analysis (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B28\" ref-type=\"bibr\">28</xref>).</p></sec><sec><title>Offspring Serum Free Fatty Acids Analysis</title><p>At d0 (prior to feeding), d7, d14, and d21 ~40 μl of blood was collected from the auricular vein and placed on a Dried Blood Spot (DBS) PUFAcoat? card (University of Adelaide, Australia), dried at room temperature for ~1 h and stored at room temperature until further analysis.</p></sec><sec><title>PUFAcoat Milk and Offspring Serum Free Fatty Acid Analysis</title><p>Both milk and offspring serum measurement for free fatty acids were performed using a modified direct transesterification method (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). Fatty acids were transmethylated to fatty acid methyl esters (FAME) using 2 ml of 1% (v/v) sulphuric acid H<sub>2</sub>SO<sub>4</sub> in anhydrous methanol in a 5 ml sealed vial and heated for 3 h at 70°C. This procedure allows for fatty acids to be released from structural lipids into a total fatty acid pool. The sequential FAME was extracted into heptanes for further gas chromatography analysis (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B28\" ref-type=\"bibr\">28</xref>, <xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Sample FAME identification and quantification were achieved by comparing the retention times and peak area values to those of the commercial lipid standards (Nu-Chek-Prep, USA) using the Hewlett-Packard Chemstation data system (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B28\" ref-type=\"bibr\">28</xref>, <xref rid=\"B30\" ref-type=\"bibr\">30</xref>, <xref rid=\"B31\" ref-type=\"bibr\">31</xref>).</p></sec><sec><title>d21 Offspring Tissue Collection</title><p>Immediately prior to weaning (d21), offspring were fasted for 14 h. Guinea pigs were euthanised by intracardiac injection of sodium pentobarbital (>100 mg/kg) (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>) and brain, plasma, urine, liver, quadracep muscle, visceral and subcutaneous fat depots were collected, weighed, snap-frozen in liquid nitrogen and stored at −80°C or fixed in 10% formalin phosphate buffer for later analysis.</p></sec><sec><title>Plasma Metabolic Analysis</title><p>Maternal and offspring plasma were analyzed by Cobas c311 autoanalyzer (Roche) according to manufacturer instructions for cholesterol (CHOL), triglycerides (TAG) low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL) and uric acid (UA). Maternal and offspring plasma were analyzed for insulin by ELISA (Abclonal, USA) according to the manufacturer's instructions.</p></sec><sec><title>Statistical Analysis</title><p>Statistical analyses were performed using IBM SPSS Statistics 25 software (IBM, USA). All data were graphed using Prism 8 software (GraphPad Software Inc., USA). Dam growth, caloric intake, stillbirth/death during delivery to live birth ratio, litter size, Matsuda-DeFronzo Insulin Sensitivity Index (M-ISI) during OGTT, plasma biochemistry and milk free fatty acids analyses were analyzed by independent <italic>T</italic>-test in dams. Offspring weight, base blood glucose, serum free fatty acids and plasma biochemistry were analyzed using two-way ANOVA with diet and sex as factors and Bonferroni <italic>post-hoc</italic> test was performed where indicated for multiple comparisons testing between groups. Differences between groups were considered significant at <italic>P</italic> < 0.05. All data are presented as mean ± SEM.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>Pre-pregnancy and Pregnancy Water and Caloric Intake</title><p>To identify specific differences in caloric intake between groups, 60 days prior to pregnancy and during pregnancy, water, food and caloric intake was measured daily. Total caloric intake in FD dams was significantly increased, an effect of diet, 60 days prior to pregnancy and the 69 days during pregnancy (CD, 57.94 ±2.58 vs. FD, 85.58 ± 1.98 Kcals; <italic>P</italic> < 0.0001) when compared to CD dams. Caloric intake from pellet food in CD and FD dams were not different. Ten percentage fructose water intake during the 60 day pre-pregnancy period and 69 days of pregnancy, on average, contributed an extra 34.0 ± 4.2 Kcal/day in fructose-fed dams. CD dams consumed, on average, 172.2 Kcal/day throughout pre-gestation and pregnancy, whereas FD dams consumed, on average, 206.2 Kcal/day with 10% fructose (w/v) contributing an extra 16.5% of total daily caloric intake in the fructose group (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p></sec><sec><title>Effects of Fructose During Pre-pregnancy and Pregnancy Weight Gain</title><p>To observe any differences in weight gain during pre-pregnancy and pregnancy animals were weighed twice weekly. No significant differences in body weight were observed between FD and CD dams prior to (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>) or during pregnancy (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Pre-pregnancy and pregnancy weight gain during fructose-feeding <bold>(A)</bold> dam pre-pregnancy and <bold>(B)</bold> pregnancy weight gain. FD dams were fructose-fed for 60 d prior to mating and throughout pregnancy. CD (<italic>n</italic> = 10); FD (<italic>n</italic> = 9). Graph <bold>(A)</bold> represents average pre-pregnancy weight gain (12–20 wk), graph <bold>(B)</bold> represents average pregnancy weight gain (1–11 wk). All data were analyzed as a 2x2 factorial design with diet<sup>*</sup>time<sup>*</sup>interaction included (generalized linear model analysis) using IBM SPSS statistics 25 (IBM, USA). Data shown as standard error of the mean (SEM).</p></caption><graphic xlink:href=\"fendo-11-00550-g0001\"/></fig></sec><sec><title>Effects of Fructose on Maternal Plasma Glucose and Insulin</title><p>OGTT's were performed to examine any potential effect of fructose on maternal glucose and insulin response following 60 days of consumption prior to pregnancy and during pregnancy at 35 days. A whole body insulin sensitivity index, Matsuda-DeFronzo Insulin Sensitivity Index (M-ISI), was also calculated. There was non-significant increase (<italic>P</italic> = 0.06)following 60 day pre-pregnancy fructose feeding in OGTT blood glucose and insulin AUC (<xref ref-type=\"fig\" rid=\"F2\">Figures 2A,B</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Table 1</xref>) At midgestation (d35) there was no significant effect between CD and FD dams in OGTT blood glucose and insulin AUC (<xref ref-type=\"fig\" rid=\"F2\">Figures 2C,D</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Table 1</xref>).</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Dam pre-pregnancy and midgestation OGTT glucose AUC and insulin AUC <bold>(A)</bold> dam blood glucose AUC in response to OGTT pre-pregnancy 60 days post-dietary intervention. <bold>(B)</bold> Dam plasma insulin AUC in response to OGTT pre-pregnancy 60 days post-dietary intervention. <bold>(C)</bold> Dam blood glucose AUC in response to OGTT midgestation. <bold>(D)</bold> Dam plasma insulin AUC in response to OGTT midgestation.CD (<italic>n</italic> = 10); FD (<italic>n</italic> = 9). All data were analyzed as a 2x2 factorial design with diet*time*interaction included (generalized linear model analysis) using IBM SPSS statistics 25 (IBM, USA). Data shown as standard error of the mean (SEM).</p></caption><graphic xlink:href=\"fendo-11-00550-g0002\"/></fig><p>A non-significant increase was observed 60 days post-dietary intervention between CD and FD dams M-ISI (CD, 2.64 ± 0.27 vs. FD, 4.94 ± 1.04; <italic>P</italic> = 0.06) (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Table 1</xref>).</p></sec><sec><title>Pre-pregnancy and Pregnancy Plasma Biochemistry</title><p>To examine the effects of fructose following 60 days of consumption prior to pregnancy and consumption during pregnancy at 35 days, plasma biochemistry was analyzed. Fructose was observed to have significant effects on dam plasma biochemistry prior to and during pregnancy. High density lipoprotein (HDL) concentrations were significantly increased, an effect of diet (CD, 0.03 ± 0.01 vs. FD, 0.15 ± 0.01 mmol/L; <italic>P</italic> < 0.0001), during pre-pregnancy following 60 days fructose feeding in FD dams compared to CD dams. No significant differences between FD and CD dams were observed during pregnancy. Triglycerides (TAG) were significantly increased in FD dams following 60 days of fructose feeding (CD, 0.52 ± 0.03 vs. FD, 0.69 ±0.05 mmol/L; <italic>P</italic> = 0.03), compared to CD dams, but this was no longer significantly different between FD and CD dams during pregnancy. No significant differences in Uric acid (UA) were observed between FD and CD dams during the 60 days of fructose feeding prior to pregnancy, whereas during midgestation there was a significant increase in uric acid of FD dams when compared to CD dams(CD, 18.66 ± 2.76 vs. FD, 21.00 ±2.76 μmol/L; <italic>P</italic> = 0.007) (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>).</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Dam plasma biochemistry at pre-pregnancy 60 days post-dietary intervention and midgestation.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Dam plasma biochemistry</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Dietary group</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>60 days Post-dietary intervention</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Midgestation</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CHOL (mmol/L)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Control</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.64 ± 0.10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.48 ± 0.09</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fructose</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.64 ± 0.05</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.37 ± 0.03</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">LDL (mmol/L)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Control</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.71 ± 0.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.19 ± 0.01</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fructose</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.72 ± 0.05</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.22 ± 0.02</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HDL (mmol/L)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Control</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.03 ± 0.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.02 ± 0.00</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fructose</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.15 ± 0.01<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>**</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.03 ± 0.00</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TAG (mmol/L)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Control</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.52 ± 0.03</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.53 ± 0.35</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fructose</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.69 ± 0.05<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.43 ± 0.29</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UA (μmol/L</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Control</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18.66 ± 2.76</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21.55 ± 1.36</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fructose</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21.00 ± 2.67</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.75 ± 1.88<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>**</sup></xref></td></tr></tbody></table><table-wrap-foot><p><italic>CD (n = 10); FD (n = 9). All data was analyzed using an independent T-test using IBM SPSS statistics 25. Data presented as group mean ± SEM</italic>.</p><fn id=\"TN1\"><label>*</label><p>denotes significance of p < 0.05;</p></fn><fn id=\"TN2\"><label>**</label><p><italic>denotes significance of p < 0.001</italic>.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Pregnancy Outcomes</title><p>To measure the effect of fructose consumption on general pregnancy and developmental outcomes littersize, stillbirths and birth weight were measured at day of delivery. There was no significant difference between CD and FD dams in litter size, rate of stillbirth/death at delivery or offspring birth weight (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>).</p><table-wrap id=\"T3\" position=\"float\"><label>Table 3</label><caption><p>Dam pregnancy outcomes.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Dam pregnancy outcomes</bold></th><th valign=\"top\" align=\"center\" colspan=\"2\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Day 0</bold></th></tr><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\"><bold>Control</bold></th><th valign=\"top\" align=\"center\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\"><bold>Fructose</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Litter size</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.90 ± 0.23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.11 ± 0.20</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Stillbirth/Death at delivery</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.44 ± 0.29</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male birth weight</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">90.08 ± 1.23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91.38 ± 1.23</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female weight</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">90.56 ± 1.23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">88.71 ± 1.23</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Litter birth weight</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">90.34 ± 1.23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">90.15 ± 1.23</td></tr></tbody></table><table-wrap-foot><p><italic>Control (n = 10) and fructose (n = 9). Litter size and stillbirth/death at delivery data was analyzed using an independent T-test while birth weight was analyzed using a 2x2 factorial design with diet, sex and interaction include (general analysis of variance) using IBM SPSS statistics 25. Data presented as group mean ± SEM</italic>.</p></table-wrap-foot></table-wrap></sec><sec><title>Maternal Milk Free Fatty Acid Composition</title><p>To examine the effects of fructose consumption on milk lipid composition a dried milk spot and lipidomic analysis were performed at day of delivery. Maternal fructose intake was observed to have significant effects on dams' milk lipid composition. From the 33 fatty acids analyzed, significant increases in FD dam milk were observed in myristic acid (CD, 1.00 ± 0.05 vs. FD, 1.16 ± 0.05 mg/100g; <italic>P</italic> = 0.04), total trans fatty acids (CD, 0.33 ± 0.03 vs. FD, 0.46 ± 0.036 mg/100 g; <italic>P</italic> = 0.02), vaccenic acid (CD, 0.15 ± 0.01 vs. FD, 0.22 ± 0.00 mg/100 g; <italic>P</italic> = <0.001), linoelaidic Acid (CD, 0.04 ± 0.00 vs. FD, 0.12 ± 0.02 mg/100 g; <italic>P</italic> = 0.002), cis-vaccenic (CD, 1.14 ± 0.06 vs. FD, 1.31 ±0.04 mg/100 g; <italic>P</italic> = 0.04), total omega-7 (CD, 2.39 ± 0.12 vs. FD, 2.71 ±0.08 mg/100 g; <italic>P</italic> = 0.05) and gamma-linolenic acid (CD, 0.06 ± 0.00 vs. FD, 0.08 ±0.00 mg/100 g; <italic>P</italic> = 0.05). There were no differences between CD and FD in any of the other 26 FFA concentrations measured in maternal milk (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>).</p><table-wrap id=\"T4\" position=\"float\"><label>Table 4</label><caption><p>Free fatty acids in dams' milk at day of delivery.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Dam milk</bold><break/>\n<bold>Free fatty acid (mg/100 g)</bold></th><th valign=\"top\" align=\"center\" colspan=\"2\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Day 0</bold></th></tr><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Control</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Fructose</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Myristic acid (14:0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.00 ± 0.05</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.16 ± 0.05<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total trans fatty acids</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.33 ± 0.03</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.46 ± 0.03<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Vaccenic acid (t18:1n-7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.15 ± 0.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.22 ± 0.00<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Linoelaidic acid (t18:2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.04 ± 0.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.12 ± 0.02<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cis-Vaccenic acid (18:1n-7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.14 ± 0.06</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.31 ± 0.04<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total omega-7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.39 ± 0.12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.71 ± 0.08<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gamma-Linolenic acid (18:3n-6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.06 ± 0.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 ± 0.00<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total saturates</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.23 ± 1.31</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.64 ± 1.09</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lauric acid (12:0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.03 ± 0.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.03 ± 0.00</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pentadecanoic acid (15:0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.46 ± 0.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.52 ± 0.02</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Palmitic acid (16:0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21.35 ± 0.96</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.44 ± 1.06</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Margaric acid (17:0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.86 ± 0.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.91 ± 0.03</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lignoceric acid (24:0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.02 ± 0.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.02 ± 0.00</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Elaidic acid (t18:1n-9)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 ± 0.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.09 ± 0.00</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total monos</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">34.49 ± 0.43</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35.49 ± 0.49</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Palmitoleic acid (16:1n-7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.24 ± 0.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.39 ± 0.05</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Oleic acid (18:1n-9)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.11 ± 0.55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.28 ± 0.42</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total Omega-9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.62 ± 0.55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.77 ± 0.44</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total Omega-3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.08 ± 0.34</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.34 ± 0.29</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Arachidonic acid (20:4n-6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.22 ± 0.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.23 ± 0.03</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Capric acid (10:0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.05 ± 0.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.04 ± 0.00</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Stearic acid (18:0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.49 ± 0.32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.48 ± 0.34</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Arachidic acid (20:0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.12 ± 0.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.11 ± 0.00</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Palmitelaidic acid (t16:1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.06 ± 0.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.04 ± 0.00</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gondoic acid (20:1n-9)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.48 ± 0.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.48 ± 0.03</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Alpha-Linolenic acid (18:3n-3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.35 ± 0.46</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.18 ± 0.30</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Docosapentaenoic acid (22:5n-3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.10 ± 0.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.10 ± 0.01</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Docosahexaenoic acid (22:6n-3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.04 ± 0.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.04 ± 0.00</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total Omega-6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.61 ± 1.51</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.06 ± 0.85</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Linoleic acid (18:2n-6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.42 ± 1.41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25.86 ± 0.77</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Eicosadienoic acid (20:2n-6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.67 ± 0.05</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.64 ± 0.05</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dihomo-y-linolenic acid (20:3n-6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.17 ± 0.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.12 ± 0.02</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Adrenic acid (22:4n-6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.10 ± 0.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.09 ± 0.015</td></tr></tbody></table><table-wrap-foot><p><italic>Control (n = 10) and fructose (n = 9). All data was analyzed using an independent T-test using IBM SPSS statistics 25. Data presented as group mean ± SEM</italic>.</p><fn id=\"TN3\"><label>*</label><p><italic>denotes significance of p < 0.05</italic>.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Postnatal Offspring Weight Gain</title><p>Offspring weights were taken daily from days 1 to 21 of age to determine any effect of dams maternal fructose intake on growth trajectory. Maternal fructose intake was observed to have an overall significant effect of diet (<italic>P</italic> = 0.05), on fructose offspring weight gain in both males and females. Following repeated measures analysis, FD offspring gained less weight than CD offspring from days 12 to 21of age (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>).</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Weanling offsprings' weight gain from days 0 to 21. Control males (<italic>n</italic> = 7); fructose males (<italic>n</italic> = 7); control females (<italic>n</italic> = 7) and fructose females (<italic>n</italic> = 7). Significant effects were shown in diet of fructose offspring weight gain. All data was analyzed using a 2x2 factorial design with repeated measures, diet andsex as factors (general analysis of variance) using IBM SPSS statistics 25. Data presented as group mean ± SEM. *denotes significance of <italic>p</italic> < 0.05.</p></caption><graphic xlink:href=\"fendo-11-00550-g0003\"/></fig></sec><sec><title>Offspring Effects of Maternal Fructose on Plasma Biochemistry</title><p>To examine the effects of maternal fructose on offspring plasma biochemistry markers of fructose and lipid metabolism were analyzed at day 21 of age. Fructose offspring had significantly increased plasma TAG concentrations at d21 compared to CD offspring(<italic>P</italic> = 0.01) (CDM, 0.51 ± 0.01 vs. FDM, 0.58 ± 0.01 vs. CDF, 0.56 ± 0.01 vs. FDF, 0.68 ± 0.01 mmol/L; <italic>P</italic> = 0.01). A sex effect (<italic>P</italic> = 0.05) was also observed, with CD and FD female offspring having increased TAG when compared to CD and FD male offspring. Similarly, plasma UA concentrations were significantly increased in FD offspring when compared to their CD counterparts (CDM, 42.33 ± 3.90 vs. FDM, 69.40 ± 3.90 vs. CDF, 46.50 ± 3.90 vs. FDF, 62.75 ± 3.90 μmol/L; <italic>P</italic> = 0.004). A significant effect of sex was observed in female offspring with an increase in CHOL (CDM, 0.87 ± 0.07 vs. FDM, 1.05 ± 0.07 vs. CDF, 1.50 ± 0.07 vs. FDF, 1.35 ± 0.07 mmol/L; <italic>P</italic> = 0.001), LDL (CDM, 0.79 ± 0.06 vs. FDM, 1.04 ± 0.06 vs. CDF, 1.25 ± 0.06 vs. FDF, 1.22 ± 0.06 mmol/L; <italic>P</italic> = 0.005) and HDL (CDM, 0.08 ± 0.00 vs. FDM, 0.10 ± 0.00 vs. CDF, 0.12 ± 0.00 vs. FDF, 0.13 ± 0.00 mmol/L; <italic>P</italic> = 0.001) (<xref rid=\"T5\" ref-type=\"table\">Table 5</xref>).</p><table-wrap id=\"T5\" position=\"float\"><label>Table 5</label><caption><p>Weanling offspring plasma biochemistry.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Weanling offspring plasma biochemistry</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Sex</bold></th><th valign=\"top\" align=\"center\" colspan=\"2\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Day 21</bold></th></tr><tr><th rowspan=\"1\" colspan=\"1\"/><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Control</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Fructose</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CHOL (mmol/L)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.87 ± 0.07</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.05 ± 0.07</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.50 ± 0.07<xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.35 ± 0.07<xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">LDL (mmol/L)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.79 ± 0.06</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.04 ± 0.06</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.25 ± 0.06<xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.22 ± 0.06<xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HDL (mmol/L)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 ± 0.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.10 ± 0.00</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.12 ± 0.00<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>**</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.13 ± 0.00<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>**</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TAG (mmol/L)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.51 ± 0.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.58 ± 0.01<xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.56 ± 0.01<xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.68 ± 0.01<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>**</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UA (μmol/L</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">42.33 ± 3.90</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">69.40 ± 3.90<xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">46.50 ± 3.90</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">62.75 ± 3.90<xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td></tr></tbody></table><table-wrap-foot><p><italic>Control males (n = 7); fructose males (n = 7); control females (n = 7) and fructose females (n = 7). All data was analyzed using a 2x2 factorial design with diet and sex (general analysis of variance) using IBM SPSS statistics 25. Data presented as group mean ± SEM</italic>.</p><fn id=\"TN4\"><label>*</label><p>denotes significance of p < 0.05;</p></fn><fn id=\"TN5\"><label>**</label><p><italic>denotes significance of p < 0.001</italic>.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Weanling Serum Free Fatty Acid Composition</title><p>To examine the effects of maternal fructose consumption on offspring plasma lipid composition a dried blood spot and lipidomic analysis were performed at days 0, 7, 14, and 21 in offspring. Maternal fructose intake was observed to have significant effects on offspring free fatty acid composition, such that, out of the 33 serum FFAs analyzed, a significant diet effect was observed in six specific fatty acids in males and females of FD dams consistently across d0, d7, d14, and d21. Fructose males and females had significantly increased pentadecanoic acid (15:0) compared to control males and females. In fructose offspring there was an effect of diet (<italic>P</italic> < 0.0001) at d0, in addition, there was an interaction effect of diet and sex (<italic>P</italic> < 0.0001) in fructose males compared to any other group or sex. An effect of diet continued in fructose offspring at d7 (<italic>P</italic> = 0.02), d14 (<italic>P</italic> = 0.01), and d21 (<italic>P</italic> < 0.0001). A significant effect of diet was observed in fructose offspring dma16:0 at d0 (<italic>P</italic> < 0.0001), d7 (<italic>P</italic> < 0.0001), d14 (<italic>P</italic> = 0.001), and d21 (<italic>P</italic> < 0.0001). Margaric acid (17:0) was significantly increased in fructose offspring, an effect of diet, at d0 (<italic>P</italic> = 0.02), d7 (<italic>P</italic> = 0.02), and d14 (<italic>P</italic> = 0.02) and d21 (<italic>P</italic> = 0.005). Palmitoleic acid (16:1n-7) in fructose male and female offspring were also observed to be significantly increased, an effect of diet, compared to control at d0 (<italic>P</italic> = 0.02), day 7 (<italic>P</italic> = 0.007), d14 (<italic>P</italic> = 0.002), and d21 (<italic>P</italic> < 0.0001). A significant increase in total omega-7 (ω-7) was observed, an effect of diet, at d0 (<italic>P</italic> = 0.05), d7 (<italic>P</italic> = 0.003), d14 (<italic>P</italic> < 0.0001), and d21 (<italic>P</italic> < 0.0001) in fructose male and female offspring compared to control. In addition, a significant diet effect was observed in both FD male and female total saturates at d0 (<italic>P</italic> = 0.002) as well as a sex effect (<italic>P</italic> = 0.02) which was increased in FD females. An effect of diet continued in fructose offspring at d7 (<italic>P</italic> = 0.02), d14 (<italic>P</italic> = 0.05), and d21 (<italic>P</italic> < 0.0001) (d0, <xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>; d7, <xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>; d14, <xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>; and d21, <xref ref-type=\"fig\" rid=\"F4\">Figure 4D</xref>).</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Free fatty acids in weanling offspring serum. Control males (<italic>n</italic> = 7); fructose males (<italic>n</italic> = 7); control females (<italic>n</italic> = 7) and fructose females (<italic>n</italic> = 7). <bold>(A)</bold> Represents offspring day 0 serum free fatty acids. <bold>(B)</bold> Represents offspring day 7 serum free fatty acids. <bold>(C)</bold> Represents offspring day 14 serum free fatty acids. <bold>(D)</bold> Represents offspring day 21 serum free fatty acids. All data was analyzed using a 2x2 factorial design with diet and sex as factors (general analysis of variance) using IBM SPSS statistics 25. Data presented as group mean ± SEM. *denotes significance of <italic>p</italic> < 0.05.</p></caption><graphic xlink:href=\"fendo-11-00550-g0004\"/></fig><p>From the remaining 27 free fatty acids analyzed, total omega-6 free fatty acids were shown to be significantly increased, an effect of diet, in control male and female offspring consistently across d0 (<italic>P</italic> = 0.02), d7 (<italic>P</italic> = 0.01), d14 (<italic>P</italic> = 0.05), and d21(<italic>P</italic> < 0.0001). The remaining free fatty acids were either non-significant or not consistently significant across all time-points.</p></sec><sec><title>Effects of Fructose on Offspring Plasma Glucose</title><p>To assess the effects of maternal fructose consumption on baseline blood glucose levels analysis were performed at days 0, 7, 14, and 21 in offspring. Maternal fructose intake had a significant effect on blood glucose concentrations on d7, with an increase in FD male and female offspring (CDM, 7.40 ± 0.21 vs. FDM, 8.40 ± 0.21 vs. CDF, 7.20 ± 0.21 vs. FDF, 8.08 ± 0.21 mmol/L; <italic>P</italic> = 0.03) compared to their CD counterparts. However, no significant differences between CD and FD offspring were observed on d0, d14, and d21 (<xref rid=\"T6\" ref-type=\"table\">Table 6</xref>).</p><table-wrap id=\"T6\" position=\"float\"><label>Table 6</label><caption><p>Plasma biochemistry of base blood glucose in weanling offspring.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Weanling offspring plasma biochemistry</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Sex</bold></th><th valign=\"top\" align=\"center\" colspan=\"2\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Day 0</bold></th><th valign=\"top\" align=\"center\" colspan=\"2\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Day 7</bold></th><th valign=\"top\" align=\"center\" colspan=\"2\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Day 14</bold></th><th valign=\"top\" align=\"center\" colspan=\"2\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Day 21</bold></th></tr><tr><th rowspan=\"1\" colspan=\"1\"/><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Control</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Fructose</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Control</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Fructose</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Control</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Fructose</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Control</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Fructose</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Base Blood Glucose (mmol/L)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Male</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.73 ± 0.16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.02 ± 0.16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.40 ± 0.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.40 ± 0.21<xref ref-type=\"table-fn\" rid=\"TN6\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.50 ± 0.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.48 ± 0.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.97 ± 0.12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.71 ± 0.12</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.00 ± 0.16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.65 ± 0.16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.20 ± 0.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.08 ± 0.21<xref ref-type=\"table-fn\" rid=\"TN6\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.83 ± 0.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.00 ± 0.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.68 ± 0.12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.22 ± 0.12</td></tr></tbody></table><table-wrap-foot><p><italic>Control males (n = 7); fructose males (n = 7); control females (n = 7) and fructose females (n = 7). All data was analyzed using a 2x2 factorial design with diet, sex and interaction include (general analysis of variance) using IBM SPSS statistics 25. Data presented as group mean ± SEM</italic>.</p><fn id=\"TN6\"><label>*</label><p><italic>denotes significance of p < 0.05</italic>.</p></fn></table-wrap-foot></table-wrap></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>We have investigated the effects of moderate fructose intake on the metabolic status of dams prior to and during pregnancy and subsequent offspring metabolic status up to weaning. We hypothesized that changes in maternal nutritional status following fructose intake would alter dams metabolic profiles and milk lipid composition and that offspring metabolic profiles also display signs of hyperlipidemia. We show that an intake of fructose which closely resembles average human consumption, contributing 16.5% of total caloric intake during pregnancy, has a significant impact upon a pregnant dams' metabolic status and negatively impacts milk lipid composition. We also provide the first evidence that offspring born from fructose-fed dams displaying a very specific pattern of increased FFAs at multiple time points (d0, d7, d14, & d21). Similar to previous studies, increases in triglycerides and placental uric acid were also observed in male and female offspring (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>). These potentially deleterious changes in offspring metabolic status and weight gain may likely be an effect of <italic>in utero</italic> exposure to maternal fructose consumption.</p><p>With similar placental characteristics to humans, relatively long gestation and comparable developmental maturation of organ systems before birth (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>), the guinea pig is a relevant translational model to study the effects of maternal fructose intake on offspring development. In the current study and previously shown in the rat model, we show that water intake increased in fructose mothers, due to the addition of 10% fructose (w/v), making the water sweeter and more palatable (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B35\" ref-type=\"bibr\">35</xref>). All mothers consumed, on average, 172 Kcal/day from pelleted food while fructose-fed mothers ingested an extra 34 Kcal/day (16.5% total daily Kcal) from 10% fructose (w/v). Recent data has shown that human average caloric intake from fructose can be between ~10 and 20% (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). In a human context, consuming a basal diet of 2,000 kcal/day with the addition of one 16 oz sugar-sweetened beverage would equate to an increase of up to 10% of total kcal/day intake. It has been recently reported that consumption of sugar by pregnant women equates to around 14% of their average daily caloric intake (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). Animal studies of fructose intake have ranged from the supraphysiological (30–70%); (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B36\" ref-type=\"bibr\">36</xref>, <xref rid=\"B37\" ref-type=\"bibr\">37</xref>) to the more comparable ranges of 10–20% fructose (w/v) (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B20\" ref-type=\"bibr\">20</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>, <xref rid=\"B39\" ref-type=\"bibr\">39</xref>).</p><p>Animal and human studies have shown that continued excessive dietary fructose intake increases activity of lipogenic liver enzymes, lipid synthesis and circulating LDL, VLDL, HDL and triglyceride concentrations in blood as fructose favors lipogenesis (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>, <xref rid=\"B9\" ref-type=\"bibr\">9</xref>, <xref rid=\"B40\" ref-type=\"bibr\">40</xref>, <xref rid=\"B41\" ref-type=\"bibr\">41</xref>). We show that prior to pregnancy, fructose intake increased levels of triglycerides and HDL without an increase in LDL but these differences were abolished during pregnancy. Similar to the current study, others have also shown no change in plasma triglycerides, HDL and LDL during pregnancy following fructose-feeding (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>). The relationship between dietary fructose and insulin resistance in humans is uncertain as excess dietary fructose does not invariably cause elevated insulin concentrations (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>). Bezerra et al. showed increased insulin in response to fructose-feeding and Vickers et al. published reports of fructose increasing insulin in rats (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>, <xref rid=\"B44\" ref-type=\"bibr\">44</xref>). In contrast, we show non-significant increase in blood glucose AUC and reduced insulin response AUC in dams exposed to fructose enriched diets. This has been shown previously (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>) and may involve reduced insulin response due to higher concentrations of FFAs, triglycerides, HDLs and LDLs (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>, <xref rid=\"B47\" ref-type=\"bibr\">47</xref>). Our study uses a more physiologically relevant fructose intake (10%) and thus, the increases in plasma lipids observed in our non-pregnant dams could have been ameliorated by blood volume expansion, increased metabolic demand that is placed upon the expectant mother, increased hepatic gluconeogenesis and FFA production, typically observed during pregnancy (<xref rid=\"B48\" ref-type=\"bibr\">48</xref>).</p><p>Of particular interest to our study, is milk FFA composition and the potential negative impact of altered milk composition on the vertical transmission of FFAs from mother to offspring. Research has shown that milk composition can be selectively affected by maternal nutritional status and even more so during lactation. Two recent studies have shown that fructose is transferred from mother to infant during breastfeeding (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>, <xref rid=\"B50\" ref-type=\"bibr\">50</xref>), and a positive association between level of fructose in breast milk and infant adiposity at 6 months of age. In the present study, significantly increased FFAs in maternal milk included myristic acid (14:0), total trans fatty acids, vaccenic acid (t18:1n-7), linoelaidic acid (t18:2) cis-vaccenic acid (18:1n-7) total omega-7 and gamma-linolenic acid (18:3n-6), the majority of which are trans-fats. Studies have demonstrated that trans fatty acid content in lactating mothers is both directly associated with diet and independent of diet through maternal adipose tissue (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>, <xref rid=\"B52\" ref-type=\"bibr\">52</xref>).</p><p>Despite dams not having access to fructose during lactation in the current study, we also show a reduced pup weight at d12–21. Zou et al. reported a reduction in pup weight over time from mothers that were fed fructose (63%) during pregnancy and lactation (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>). Similarly, Rawana et al. reported reduced body weight and total litter weight at weaning in offspring of dams consuming 10% fructose (w/v) during pregnancy and lactation (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). In contrast, Alzamendi et al. reported that at d20 of gestation fetal:placental ratio was increased in dams fed 10% (w/v) fructose solution throughout pregnancy, suggesting that fructose offspring were heavier than the control group (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>). There is evidence to suggest that fructose intake is linked with inappropriate vascularisation of the placenta and further studies have shown irregularities in the zonal regions of the placenta (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). This may suggest a potential mechanism by which maternal fructose intake has deleterious effects on vascularisation in the placenta and nutrient transfer, contributing to reducing neonatal growth trajectory. We would suggest that following breastfeeding, specific changes in milk FFA composition, as a result of gestational fructose intake, may further contribute to the already increased circulating FFA profile observed in fructose offspring. This may indicate an additional route by which offspring are further exposed to the deleterious effects of maternal fructose-feeding despite the mothers never receiving fructose during lactation.</p><p>The deleterious effects of elevated plasma uric acid concentrations following fructose consumption in non-pregnant subjects has been well established (<xref rid=\"B55\" ref-type=\"bibr\">55</xref>, <xref rid=\"B56\" ref-type=\"bibr\">56</xref>). Some research, but not all, has shown excessive maternal fructose intake to induce increased uric acid production during pregnancy by increasing adenosine triphosphate (ATP) degradation to adenosine monophosphate (AMP), a uric acid precursor (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>, <xref rid=\"B56\" ref-type=\"bibr\">56</xref>, <xref rid=\"B57\" ref-type=\"bibr\">57</xref>). Similar effects of maternal fructose intake are evident in our pre-pregnant and pregnant dams, with a significant increase in uric acid before pregnancy and during pregnancy. Moreover, we also show significantly increased plasma uric acid and triglycerides in fructose offspring at weaning, despite these pups never consuming fructose themselves. These results are likely to be a consequence of (a) fructose dams and offspring producing high concentrations of uric acid due to the metabolic effects of higher circulating FFAs, which is causal of increased intracellular ATP resulting from excessive fructose (<xref rid=\"B56\" ref-type=\"bibr\">56</xref>, <xref rid=\"B57\" ref-type=\"bibr\">57</xref>), and/or (b) uric acid is transferred to the fetus during pregnancy from the mother via the placenta. Uric acid has been shown to cross the placenta freely and increased exposure to uric acid <italic>in utero</italic> may potentially affect physiological set-points in the developing fetus. In the current study, increased uric acid observed in offspring are likely to also be a consequence of dyslipidemia and unregulated circulating FFAs and potentially, dysfunctional beta-oxidation and increased accumulation of intracellular ATP in the offspring. However, which pathway is more likely to be playing the primary role in the current study is unknown at this stage and further work to examine the molecular nature of such significant findings must be performed.</p><p>FFAs during fasting or in the absence of glucose are released from adipose tissue and are primarily metabolized by skeletal muscle and the liver (<xref rid=\"B58\" ref-type=\"bibr\">58</xref>). It is well established that chronic increased levels of circulating FFAs can increase the risk of obesity, hepatic lipid deposition, insulin resistance, type 2 diabetes and cardiovascular disease (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>) and increased fatty acid synthesis has been shown to occur following fructose consumption (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>–<xref rid=\"B62\" ref-type=\"bibr\">62</xref>). The effects of fructose on fatty acid synthesis is likely to be dose-dependent, due to the lipogenic effects of fructose. Long-term increases in circulating FFAs have also been shown in offspring of mothers fed diets high in sucrose and fructose (HFC-55). Toop et al. showed increased hepatic lipid composition and content in fructose offspring (<xref rid=\"B63\" ref-type=\"bibr\">63</xref>). They went on to hypothesize that increased hepatic <italic>de novo</italic> lipogenesis was caused, in part, by increased circulating FFAs. In the current study, increases in FFAs were observed in pentadecanoic acid (15:0), dma16:0, margaric acid (17:0) palmitoleic acid, total omega-7 and total saturates. More importantly, fructose offspring serum displayed these specific changes in serum FFAs at day 0, when samples were taken prior to their first feed and before receiving any milk from the mother. These increases in circulating FFAs were consistently significantly higher at all other time points (days 7, 14, and 21). This clearly indicates an <italic>in utero</italic> metabolic programming of offspring FFA metabolism. Our findings are of particular importance because of the consistency in the increase of specific FFAs observed. As prolonged FFAs may also be present as a consequence of dysfunctional adipose lipolysis and altered beta-oxidation function (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>). Additionally, the specific type of FFA increases also has important metabolic implications (<xref rid=\"B64\" ref-type=\"bibr\">64</xref>). The majority of the significantly increased FFAs observed in fructose offspring and maternal milk were long-chain fatty acids which are primary components of storage triglycerides.</p><p>The majority of offspring plasma saturated fats are associated with hyperglycemia, hyperinsulinemia and insulin resistance. Chronic elevations of circulating FFAs augment fat storage, insulin resistance and development of metabolic disease (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>). Our results provide evidence of maternal fructose increasing <italic>de novo</italic> lipogenesis, shown by increased circulating triglycerides, uric acid, LDL and HDL prior to pregnancy, changes in milk lipid composition at birth, perturbed offspring metabolic status and potential predisposition to metabolic disease in later-life. A limitation of the current study is a lack of molecular and lipogenic enzymic analysis in offspring. Few studies have investigated similar models to our own. However, a previous study by Clayton et al., showed that genes related to hepatic lipogenesis (SPBP1c) were increased, showing a phenotype with differential expression in lipogenic gene expression. It would be interesting to determine hepatic lipogenic enzyme activity and activation of molecular gene expression to investigate a “true” programming or causal effects of maternal fructose intake and preturbed offspring hepatic metabolism. Nevertheless, this study focused on the primary outcomes of maternal and offspring phenotype where we show, in principle, at multiple time points, a perinatally acquired predisposition to metabolic dysfunction in offspring. While the precise mechanism driving the offspring hyperlipidemia and deleterious FFA profile is not fully understood, the current study clearly demonstrates how maternal fructose can influence the developmental programming of offspring lipid metabolism during critical windows of plasticity prior to suckling, which may alter an individual's later-life susceptibility to metabolic disease. However, more studies are needed to clarify the potential gene and functional enzymatic pathways which are preturbed in models of maternal fructose intake.</p><p>In conclusion, we have shown for the first time, highly significant, consistent changes in young offspring FFA serum profiles, despite offspring consuming no fructose themselves. A specific pattern of increased FFAs were present prior to suckling d0 and to d21 of age, as well as increased uric acid and triglycerides in offspring of fructose-fed dams. Taken together, we provide evidence that indicates unregulated <italic>de novo</italic> fatty acid synthesis in offspring from mothers fed a moderate 10% (w/v) fructose water prior to and during pregnancy. We would suggest that suboptimal maternal diets, in particular, those high in fructose and refined sugars contribute to the rise in metabolic diseases observed over the last 40–50 years. Our study, along with other current research, emphasizes the importance of limiting added refined fructose such as sugar-sweetened beverage intake and striving for a more nutritionally-balanced diet in women prior to/during pregnancy and lactation.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by University of Otago Animal Ethics Committee.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>CG designed research and funding acquisition. ES, RD, and CG conducted research. ES wrote the first draft manuscript. RD, MB, and CG critically evaluated the paper. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>The authors wish to express their gratitude for the help and support provided by the staff at the University of Otago Biomedical Research Unit including Maureen Prowse, Heather Barnes, Taylor Wilson and Katherine Wright, University of Adelaide Waite Lipid Lab Analysis Service.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> The authors also gratefully acknowledge funding support from the Lottery Grants Board, Nutricia Research Foundation, Preterm Birth Research Grant and Otago Foundation Trust.</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s9\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fendo.2020.00550/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fendo.2020.00550/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><label>Supplementary Table 1</label><caption><p>Effects of fructose on dam 60-days post-dietary intervention and midgestation OGTT Gluose, Insulin and Matsuda-ISI CD (<italic>n</italic> = 6); FD (<italic>n</italic> = 6). All data was analyzed using an independent <italic>T</italic>-test using IBM SPSS statistics 25. Data presented as group mean ± SEM.</p></caption><media xlink:href=\"Table_1.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bidwell</surname><given-names>AJ</given-names></name></person-group>. <article-title>Chronic fructose ingestion as a major health concern: is a sedentary lifestyle making it worse?</article-title> A review. <source>Nutrients</source>. 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] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"iso-abbrev\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"doi\">10.1111/(ISSN)1750-2659</journal-id><journal-id journal-id-type=\"publisher-id\">IRV</journal-id><journal-title-group><journal-title>Influenza and Other Respiratory Viruses</journal-title></journal-title-group><issn pub-type=\"ppub\">1750-2640</issn><issn pub-type=\"epub\">1750-2659</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32157809</article-id><article-id pub-id-type=\"pmc\">PMC7431636</article-id><article-id pub-id-type=\"doi\">10.1111/irv.12736</article-id><article-id pub-id-type=\"publisher-id\">IRV12736</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Formal Systematic Review (Commissioned or Non‐commissioned)</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Formal Systematic Review (Commissioned or Non‐commissioned)</subject></subj-group></article-categories><title-group><article-title>Seroprevalence of H7N9 infection among humans: A systematic review and meta‐analysis</article-title><alt-title alt-title-type=\"left-running-head\">WANG et al.</alt-title></title-group><contrib-group><contrib id=\"irv12736-cr-0001\" contrib-type=\"author\"><name><surname>Wang</surname><given-names>Qiang</given-names></name><xref ref-type=\"aff\" rid=\"irv12736-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12736-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12736-cr-0002\" contrib-type=\"author\"><name><surname>Xu</surname><given-names>Ke</given-names></name><xref ref-type=\"aff\" rid=\"irv12736-aff-0003\">\n<sup>3</sup>\n</xref></contrib><contrib id=\"irv12736-cr-0003\" contrib-type=\"author\"><name><surname>Xie</surname><given-names>Weihua</given-names></name><xref ref-type=\"aff\" rid=\"irv12736-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12736-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12736-cr-0004\" contrib-type=\"author\"><name><surname>Yang</surname><given-names>Liuqing</given-names></name><xref ref-type=\"aff\" rid=\"irv12736-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12736-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12736-cr-0005\" contrib-type=\"author\"><name><surname>Chen</surname><given-names>Haiyan</given-names></name><xref ref-type=\"aff\" rid=\"irv12736-aff-0004\">\n<sup>4</sup>\n</xref></contrib><contrib id=\"irv12736-cr-0006\" contrib-type=\"author\"><name><surname>Shi</surname><given-names>Naiyang</given-names></name><xref ref-type=\"aff\" rid=\"irv12736-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12736-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12736-cr-0007\" contrib-type=\"author\"><name><surname>Bao</surname><given-names>Changjun</given-names></name><xref ref-type=\"aff\" rid=\"irv12736-aff-0003\">\n<sup>3</sup>\n</xref></contrib><contrib id=\"irv12736-cr-0008\" contrib-type=\"author\"><name><surname>Huang</surname><given-names>Haodi</given-names></name><xref ref-type=\"aff\" rid=\"irv12736-aff-0003\">\n<sup>3</sup>\n</xref></contrib><contrib id=\"irv12736-cr-0009\" contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Xuefeng</given-names></name><xref ref-type=\"aff\" rid=\"irv12736-aff-0003\">\n<sup>3</sup>\n</xref></contrib><contrib id=\"irv12736-cr-0010\" contrib-type=\"author\"><name><surname>Liao</surname><given-names>Yilan</given-names></name><xref ref-type=\"aff\" rid=\"irv12736-aff-0005\">\n<sup>5</sup>\n</xref></contrib><contrib id=\"irv12736-cr-0011\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Jin</surname><given-names>Hui</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0003-0071-9755</contrib-id><xref ref-type=\"aff\" rid=\"irv12736-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12736-aff-0002\">\n<sup>2</sup>\n</xref><address><email>jinhui_hld@163.com</email></address></contrib></contrib-group><aff id=\"irv12736-aff-0001\">\n<label><sup>1</sup></label>\n<named-content content-type=\"organisation-division\">Department of Epidemiology and Health Statistics</named-content>\n<named-content content-type=\"organisation-division\">School of Public Health</named-content>\n<institution>Southeast University</institution>\n<city>Nanjing</city>\n<country country=\"CN\">China</country>\n</aff><aff id=\"irv12736-aff-0002\">\n<label><sup>2</sup></label>\n<named-content content-type=\"organisation-division\">Key Laboratory of Environmental Medicine Engineering</named-content>\n<named-content content-type=\"organisation-division\">Ministry of Education</named-content>\n<named-content content-type=\"organisation-division\">School of Public Health</named-content>\n<institution>Southeast University</institution>\n<city>Nanjing</city>\n<country country=\"CN\">China</country>\n</aff><aff id=\"irv12736-aff-0003\">\n<label><sup>3</sup></label>\n<institution>Jiangsu Provincial Center for Disease Control and Prevention</institution>\n<city>Nanjing</city>\n<country country=\"CN\">China</country>\n</aff><aff id=\"irv12736-aff-0004\">\n<label><sup>4</sup></label>\n<named-content content-type=\"organisation-division\">Department of Laboratory Medicine</named-content>\n<named-content content-type=\"organisation-division\">The First Affiliated Hospital</named-content>\n<institution>Nanjing Medical University</institution>\n<city>Nanjing</city>\n<country country=\"CN\">China</country>\n</aff><aff id=\"irv12736-aff-0005\">\n<label><sup>5</sup></label>\n<named-content content-type=\"organisation-division\">The State Key Laboratory of Resources and Environmental Information System</named-content>\n<named-content content-type=\"organisation-division\">Institute of Geographic Sciences and Natural Resources Research</named-content>\n<institution>Chinese Academy of Sciences</institution>\n<city>Beijing</city>\n<country country=\"CN\">China</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label><bold>Correspondence</bold><break/>\nHui Jin, Department of Epidemiology and Health Statistics, School of Public Health, Southeast University, 87# Dingjiaqiao, Nanjing 210009, China.<break/>\nEmail: <email>jinhui_hld@163.com</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>10</day><month>3</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>9</month><year>2020</year></pub-date><volume>14</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1111/irv.v14.5</issue-id><fpage>587</fpage><lpage>595</lpage><history><date date-type=\"received\"><day>21</day><month>8</month><year>2019</year></date><date date-type=\"rev-recd\"><day>27</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>19</day><month>2</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 John Wiley & Sons Ltd <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Influenza and Other Respiratory Viruses</italic> Published by John Wiley & Sons Ltd.</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:IRV-14-587.pdf\"/><abstract id=\"irv12736-abs-0001\"><title>Abstract</title><p>In spring 2013, a novel avian‐origin influenza A (H7N9) virus emerged in mainland China. The burden of H7N9 infection was estimated based on systematic review and meta‐analysis. The systematic search for available literature was conducted using Chinese and English databases. We calculated the pooled seroprevalence of H7N9 infection and its 95% confidence interval by using Freeman‐Tukey double arcsine transformation. Out of 16 890 records found using Chinese and English databases, 54 articles were included in the meta‐analysis. These included studies of a total of 64 107 individuals. The pooled seroprevalence of H7N9 infection among humans was 0.122% (95% CI: 0.023, 0.275). In high‐risk populations, the highest pooled seroprevalence was observed among close contacts (1.075%, 95% CI: 0.000, 4.357). The seroprevalence among general population was (0.077%, 95% CI: 0.011, 0.180). Our study discovered that asymptomatic infection of H7N9 virus did occur, even if the seroprevalence among humans was low.</p></abstract><kwd-group><kwd id=\"irv12736-kwd-0001\">H7N9</kwd><kwd id=\"irv12736-kwd-0002\">influenza A</kwd><kwd id=\"irv12736-kwd-0003\">meta‐analysis</kwd><kwd id=\"irv12736-kwd-0004\">seroprevalence</kwd><kwd id=\"irv12736-kwd-0005\">systematic review</kwd></kwd-group><funding-group><award-group id=\"funding-0001\"><funding-source>Jiangsu Provincial Major Science & Technology Demostation Project</funding-source><award-id>BE2015714</award-id><award-id>BE2017749</award-id></award-group><award-group id=\"funding-0002\"><funding-source>Chinese National Natural Fund</funding-source><award-id>81573258</award-id></award-group><award-group id=\"funding-0003\"><funding-source>Jiangsu Provincial Key Medical Discipline</funding-source><award-id>ZDXKA2016008</award-id></award-group><award-group id=\"funding-0004\"><funding-source>Jiangsu Provincial Six Talent Peak</funding-source><award-id>WSN‐002</award-id></award-group></funding-group><counts><fig-count count=\"2\"/><table-count count=\"2\"/><page-count count=\"9\"/><word-count count=\"5898\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>September 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.6 mode:remove_FC converted:18.08.2020</meta-value></custom-meta></custom-meta-group></article-meta><notes><p content-type=\"self-citation\">\n<mixed-citation publication-type=\"journal\" id=\"irv12736-cit-1001\">\n<string-name>\n<surname>Wang</surname>\n<given-names>Q</given-names>\n</string-name>, <string-name>\n<surname>Xu</surname>\n<given-names>K</given-names>\n</string-name>, <string-name>\n<surname>Xie</surname>\n<given-names>W</given-names>\n</string-name>, et al. <article-title>Seroprevalence of H7N9 infection among humans: A systematic review and meta‐analysis</article-title>. <source xml:lang=\"en\">Influenza Other Respi Viruses</source>. <year>2020</year>;<volume>14</volume>:<fpage>587</fpage>–<lpage>595</lpage>. <pub-id pub-id-type=\"doi\">10.1111/irv.12736</pub-id>\n</mixed-citation>\n</p><fn-group id=\"irv12736-ntgp-0001\"><fn id=\"irv12736-note-0001\"><p>The peer review history for this article is available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://publons.com/publon/10.1111/irv.12736\">https://publons.com/publon/10.1111/irv.12736</ext-link>\n</p></fn><fn fn-type=\"funding\" id=\"irv12736-note-0002\"><p>\n<bold>Funding information</bold>\n</p><p>This work was supported by Chinese National Natural Fund (Grant number: 81573258); Jiangsu Provincial Major Science & Technology Demostation Project (Grant numbers: BE2015714, and BE2017749); Jiangsu Provincial Six Talent Peak (Grant number: WSN‐002); Jiangsu Provincial Key Medical Discipline (Grant number: ZDXKA2016008).</p></fn></fn-group></notes></front><body id=\"irv12736-body-0001\"><sec id=\"irv12736-sec-0001\"><label>1</label><title>INTRODUCTION</title><p>In February and March 2013, a novel avian‐origin influenza A (H7N9) virus was identified, which caused more than 100 human cases in mainland China.<xref rid=\"irv12736-bib-0001\" ref-type=\"ref\">1</xref>, <xref rid=\"irv12736-bib-0002\" ref-type=\"ref\">2</xref> Up to September 5, 2018, a total of 1567 H7N9 human cases were reported, including more than 615 mortalities.<xref rid=\"irv12736-bib-0003\" ref-type=\"ref\">3</xref> The case fatality rate of H7N9 patients was close to 40%.<xref rid=\"irv12736-bib-0003\" ref-type=\"ref\">3</xref>, <xref rid=\"irv12736-bib-0004\" ref-type=\"ref\">4</xref> In March 2019, the cases of H7N9 infection re‐emerged in China after a period of 14 months.<xref rid=\"irv12736-bib-0005\" ref-type=\"ref\">5</xref> The majority of the H7N9 patients lived in mainland China. Hong Kong, Taiwan, Malaysia, and Canada also reported human cases of sporadic H7N9 infection, which were imported from mainland China.<xref rid=\"irv12736-bib-0004\" ref-type=\"ref\">4</xref>\n</p><p>The patients with H7N9 infection who presented with severe clinical symptoms and showed a high case fatality rate have attracted global attention. The proportion of the population with mild or asymptomatic infection, that is, the iceberg below the sea level, was also worthy of attention. A number of serologic studies have been conducted on the seroprevalence of H7N9 infection among humans. However, it is difficult to estimate the extent of infection owing to different variables, such as study populations, test methods, and positive cutoff values in various studies. The purpose of our meta‐analysis was to examine the seroprevalence of influenza A (H7N9) infection among humans and to estimate the overall burden of H7N9 infection.</p></sec><sec sec-type=\"methods\" id=\"irv12736-sec-0002\"><label>2</label><title>METHOD</title><sec id=\"irv12736-sec-0003\"><label>2.1</label><title>Search strategy</title><p>A systematic search of the relevant literature was conducted for relevant articles published before October 22, 2018. The search terms were as follows: [“H7N9” OR “influenza A” OR “influenza A virus”] AND [“seroprevalence” OR “seropositive” OR “seronegative” OR “serologic” OR “serological” OR “seroepidemiology”]. Both Chinese and English databases were searched. Chinese databases consisted of the China National Knowledge Infrastructure, Chinese Science and Technology Periodical Database (VIP), and WanFang Database. English databases consisted of PubMed, Web of Science, and the Cochrane Library. Additionally, we searched the World Health Organization (WHO)'s website, regional health department's website, and reference lists of selected studies.</p></sec><sec id=\"irv12736-sec-0004\"><label>2.2</label><title>Inclusion and exclusion criteria</title><p>The inclusion criteria were as follows: (a) studies reporting the seroprevalence of H7N9 infection among humans and (b) cross‐sectional, retrospective, and cohort studies or routine surveillance.</p><p>The exclusion criteria were as follows: (a) study only examined the H7 subtype, (b) non‐H7N9 virus strains used in experiments, (c) study subjects were H7N9 patients or influenza patients, (d) sample size was too small (N < 10), (e) duplicated data, (f) study did not provide key data, that is, one‐third of the data (total number, the number of seropositive cases, and seroprevalence) were missing, and (g) conference papers.</p><p>Our aim was to summarize the antibody level against the H7N9 subtype. We excluded the serological studies that only examined H7 sole subtype or used non‐H7N9 virus strains in the test for the following reasons. First, the results of the tests only for the H7 subtype could only indicate previous infection with the H7 subtype, such as H7N1. Second, the hemagglutinin gene fragment of the influenza virus is constantly mutating. The H7 antigen of other subtypes is different from the H7N9 antigen reported in China because the H7 antigen can change.<xref rid=\"irv12736-bib-0006\" ref-type=\"ref\">6</xref>, <xref rid=\"irv12736-bib-0007\" ref-type=\"ref\">7</xref> Therefore, the results of the detection of other subtypes of H7 were not convincing. Although previous studies have shown cross‐reactivities between H7N9 and divergent H7 subtypic viruses,<xref rid=\"irv12736-bib-0008\" ref-type=\"ref\">8</xref>, <xref rid=\"irv12736-bib-0009\" ref-type=\"ref\">9</xref> the specific avian influenza A(H7N9) virus should be used in hemagglutinin inhibition (HI) or microneutralization (MN) assays.<xref rid=\"irv12736-bib-0010\" ref-type=\"ref\">10</xref>\n</p></sec><sec id=\"irv12736-sec-0005\"><label>2.3</label><title>Quality assessment and data abstraction</title><p>Two researchers (QW and KX) independently reviewed and assessed each included article according to the following 10 criteria<xref rid=\"irv12736-bib-0011\" ref-type=\"ref\">11</xref>: (a) whether it was a population‐based study, (b) whether the study time and location were provided, (c) whether the study population was ≥100 subjects, (d) whether the study population had avian exposure, (e) whether the characteristics of the study population were mentioned, (f) whether HI was carried out, (g) whether MN was carried out, (h) whether horse red blood cells were used in the HI assay, (i) whether the seropositive cutoff value was mentioned in the study, and (j) whether the seropositive cutoff value provided in the study referred to the WHO criteria. If the two researchers were in disagreement about the quality of a study, a third researcher (HJ) would make the final decision. “Yes” indicated a score of one, and “No” or “Not provided” indicated a score of zero; finally, we calculated the total score of the 10 items.</p><p>Similarly, data abstraction was carried out by two researchers (QW and KX). After extraction, data were checked by a third researcher (HJ). If there was a difference, the original literature would be reviewed for re‐extraction. The following data were extracted: first author, publication year, study type, population sample, study region, fieldwork dates, sample size, number of seropositive cases, seroprevalence, test method, seropositive cutoff value, HI test cell, and number of humans in each dilution titer (1:10‐1:640). For cohort studies, the number of people who showed seroconversion, the criteria of seroconversion, and follow‐up time was also extracted.</p></sec><sec id=\"irv12736-sec-0006\"><label>2.4</label><title>Data analysis</title><p>Excel and Stata software were used in this study. The data were subjected to Freeman‐Tukey double arcsine transformation, and we reported the pooled seroprevalence and its 95% confidence interval (CI) using the DerSimonian‐Laird random effects.<xref rid=\"irv12736-bib-0012\" ref-type=\"ref\">12</xref>, <xref rid=\"irv12736-bib-0013\" ref-type=\"ref\">13</xref> Analyses were conducted using the metaprop package in Stata software.<xref rid=\"irv12736-bib-0014\" ref-type=\"ref\">14</xref> We assessed the heterogeneity between the studies with the <italic>I</italic>\n<sup>2</sup> statistic. If the heterogeneity test result was <italic>I</italic>\n<sup>2</sup> < 50%, a fixed effect model was used; otherwise, a random effect model was used.</p><p>The WHO has suggested the criteria for confirming whether the results of H7N9 serologic tests are positive: for single‐serum samples, HI ≥ 1:160; for paired serum samples (acute and convalescent sera), a 4‐fold rise in HI titer.<xref rid=\"irv12736-bib-0015\" ref-type=\"ref\">15</xref> In single‐serum samples, sera with HI titer of 20‐80 should be confirmed by MN or WB assay.<xref rid=\"irv12736-bib-0015\" ref-type=\"ref\">15</xref> In addition to pooling seroprevalence according to the original study criteria, we re‐judged the seropositive results according to the WHO criteria to explore the influence of different thresholds on pooled seroprevalence.<xref rid=\"irv12736-bib-0011\" ref-type=\"ref\">11</xref> Based on the studies that reported the number of humans in each titer, we re‐judged the seropositive results in the included studies. The statistical significance of H7N9 seroprevalences that were calculated by the WHO criteria and original study criteria was assessed using the Wilcoxon rank sum test. We performed statistical tests for the included studies that provided number of humans in each titer to ensure comparability. For cohort studies, the incidence of seroconversion was analyzed after calculating data using the same standard unit (per person‐months), defined as follows: number of seroconverted humans in the cohorts divided by the number of person‐months of follow‐up. We further performed stratified subgroup and meta‐regression analyses.</p></sec></sec><sec sec-type=\"results\" id=\"irv12736-sec-0007\"><label>3</label><title>RESULTS</title><sec id=\"irv12736-sec-0008\"><label>3.1</label><title>Search results</title><p>A total of 16 890 records were obtained from Chinese and English databases according to the search terms mentioned before, of which 71 articles were reviewed in full‐text (Figure <xref rid=\"irv12736-fig-0001\" ref-type=\"fig\">1</xref>). Further, 17 articles were excluded on the basis of the exclusion criteria: 5 studies only examined H7 sole subtype, 1 study used other H7 subtype in the test, 1 study involved H7N9 patients who survived, 6 studies did not provide the data, 3 studies provided replicated data, and 1 study was a conference study. Finally, 54 studies were included in the analysis, consisting of a total of 64 107 individuals.</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"irv12736-fig-0001\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Flowchart of the literature search and study selection</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"IRV-14-587-g001\"/></fig></sec><sec id=\"irv12736-sec-0009\"><label>3.2</label><title>Study characteristics and quality assessment</title><p>The 54 included studies were conducted in different regions and with different populations (Table <xref rid=\"irv12736-sup-0001\" ref-type=\"supplementary-material\">S1</xref>). One study was from Taiwan, one from Hong Kong, and the rest were from mainland China. One study conducted in India was excluded because a non‐H7N9 virus strain was used in the experiments,<xref rid=\"irv12736-bib-0016\" ref-type=\"ref\">16</xref> and one study in Vietnam was excluded because it only examined the H7 subtype.<xref rid=\"irv12736-bib-0017\" ref-type=\"ref\">17</xref> There were 43 articles that involved poultry workers, 5 articles that involved swine workers, 8 articles that involved close contacts, and 14 articles that involved the general population. Some articles provided data on various study populations.</p><p>Of the 54 studies, 3 studies reported the seroprevalence before 2013, 13 studies reported it during the first epidemic wave (1/2013‐9/2013), 14 studies during the second epidemic wave (10/2013‐9/2014), and 4 studies during the third epidemic wave (10/2014‐9/2015). The other studies did not provide the study period or provided ambiguous dates that were difficult to classify; 23 studies followed the WHO criteria, 21 studies did not, and others did not specify the criteria. The scores of quality assessment ranged from 3 to 10 points, with an average of 7.2 ± 1.8 (Table <xref rid=\"irv12736-sup-0002\" ref-type=\"supplementary-material\">S2</xref>).</p></sec><sec id=\"irv12736-sec-0010\"><label>3.3</label><title>Seroprevalence of influenza A (H7N9)</title><p>The pooled seroprevalence of H7N9 infection among humans was 0.122% (95% CI: 0.023, 0.275). The seroprevalence reported in the included studies ranged from 0.000% to 17.143%. In the 37 included studies that reported the number of people in each titer, the pooled seroprevalence was 0.046% (95% CI: 0.000, 0.193) according to the original study criteria, but was 0.003% (95% CI: 0.000, 0.081) according to the WHO criteria. However, the difference was not statistically significant (<italic>Z</italic> = −1.334, <italic>P</italic> = .182). Of the 37 articles, 14 were not in accordance with the WHO criteria. The pooled incidence of seroconversion in our study was 0.087% (95% CI: 0.007, 0.223) per person‐months.</p><p>The seroprevalence of the H7N9 virus varied widely in different regions (Table <xref rid=\"irv12736-tbl-0001\" ref-type=\"table\">1</xref>, Figure <xref rid=\"irv12736-fig-0002\" ref-type=\"fig\">2</xref>). We reported the seroprevalence in different regions of mainland China, including the eastern, central, and western regions.<xref rid=\"irv12736-bib-0018\" ref-type=\"ref\">18</xref> The seroprevalence in the eastern region was higher than that in the other two regions.</p><table-wrap id=\"irv12736-tbl-0001\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Seroprevalence in different groups<xref ref-type=\"fn\" rid=\"irv12736-note-0003\">\n<sup>a</sup>\n</xref>\n</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Variable</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"> </th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Ref (n)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Event</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Seroprevalence (%)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">95% CI (%)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic>I</italic>\n<sup>2</sup> (%)</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">54</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">64 107</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">410</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.122</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.023, 0.275</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">88.6</td></tr><tr><td align=\"left\" rowspan=\"4\" colspan=\"1\">Different populations</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Poultry workers</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">27 383</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">317</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.254</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.041, 0.584</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">90.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Swine workers</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">8596</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.005</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.000, 0.064</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">19.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Close contacts</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">793</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.075</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.000, 4.357</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">74.7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">General population</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">25 620</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.077</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.011, 0.180</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">71.1</td></tr><tr><td align=\"left\" rowspan=\"4\" colspan=\"1\">Time</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Before 2013</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3089</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.000</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.000, 0.053</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">First epidemic wave</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10 166</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">95</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.109</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.000, 0.670</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">90.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Second epidemic wave</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">22 550</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">166</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.441</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.101, 0.942</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">93.1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Third epidemic wave</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6005</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.000</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.000, 0.000</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">53.6</td></tr><tr><td align=\"left\" rowspan=\"3\" colspan=\"1\">Region in mainland China</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Eastern</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">34</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">52 458</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">389</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.129</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.009, 0.346</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">92.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Central</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6819</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.000</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.000, 0.000</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">8.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Western</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4514</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.000</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.000, 0.006</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.0</td></tr><tr><td align=\"left\" rowspan=\"5\" colspan=\"1\">Seropositive value</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">HI titer ≥ 1:20</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4942</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.007</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.000, 0.226</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">66.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HI titer ≥ 1:40</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9200</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">93</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.056</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.247, 2.348</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">95.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HI titer ≥ 1:80</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">18 698</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">94</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.147</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.001, 0.440</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">90.1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HI titer ≥ 1:160</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">7971</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">146</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.500</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.003, 1.501</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">92.8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HI titer ≥ 1:20 and MN titer ≥ 1:20</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6975</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.000</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.000, 0.033</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">40.1</td></tr><tr><td align=\"left\" rowspan=\"3\" colspan=\"1\">Test HI cell</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Turkey red blood cell</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4910</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.013</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.000, 0.506</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">82.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Horse red blood cell</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">47 961</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">303</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.158</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.026, 0.364</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">90.1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Chicken red blood cell</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3781</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.000</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.000, 0.229</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">66.5</td></tr><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">Test method</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">HI</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">40</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">39 263</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">329</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.203</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.026, 0.490</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">91.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HI an MN</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">23 114</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">81</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.078</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.000, 0.245</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">79.3</td></tr><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">Sample size</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">＜500</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">46</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">18 659</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">212</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.169</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.005, 0.484</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">86.1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">≥500</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">19</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">45 317</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">198</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.213</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.078, 0.398</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">91.2</td></tr></tbody></table><table-wrap-foot id=\"irv12736-ntgp-0002\"><fn id=\"irv12736-note-0003\"><label><sup>a</sup></label><p>HI: Hemagglutination inhibition test; MN: Microneutralization test.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"irv12736-fig-0002\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Seroprevalence of H7N9 virus among humans</p></caption><graphic id=\"nlm-graphic-3\" xlink:href=\"IRV-14-587-g002\"/></fig><p>The seroprevalence among close contacts was 1.075% (95% CI: 0.000, 4.357), ranging from 0.000% to 14.286%. The seroprevalence among close contacts was the highest in Jiangsu province (Table <xref rid=\"irv12736-sup-0003\" ref-type=\"supplementary-material\">S3</xref>). The seroprevalence rates among poultry and swine workers were 0.254% (95% CI: 0.041, 0.584) and 0.005% (95% CI: 0.000, 0.064), respectively. The seroprevalence among poultry workers was the highest in Hong Kong and the lowest in some central and western provinces in mainland China. The seroprevalence among humans before 2013 was 0.000%. The seroprevalence was higher in the first two epidemic waves than in the third one.</p></sec><sec id=\"irv12736-sec-0011\"><label>3.4</label><title>Meta‐analysis regression</title><p>The results of univariate analysis showed that time and population significantly affected the heterogeneity of the meta‐analysis results (Table <xref rid=\"irv12736-tbl-0002\" ref-type=\"table\">2</xref>). We added region to the multivariate analysis because its adjusted <italic>R</italic>\n<sup>2</sup> was 2.55% in the univariate analysis. The results showed that the variables included in the regression were time and population, and the adjusted <italic>R</italic>\n<sup>2</sup> was 15.89%, which suggested that time and population can explain part of the heterogeneity.</p><table-wrap id=\"irv12736-tbl-0002\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>The results of meta‐regression<xref ref-type=\"fn\" rid=\"irv12736-note-0004\">\n<sup>a</sup>\n</xref>\n</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Covariate</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Coefficient</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">95% CI</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic>t</italic>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Adjusted <italic>R</italic>\n<sup>2</sup> (%)</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"6\" rowspan=\"1\">Univariate analysis</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Time</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Other time</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4.25</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">First and second epidemic wave</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.068</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.009, 1.126</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2.27</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.025</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Region in mainland China</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Western</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2.55</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Eastern</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.084</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.017, 0.185</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.65</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.102</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Central</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.017</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.095, 0.129</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.30</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.763</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Population</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Swine worker</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4.24</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Close contacts</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.220</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.054, 0.385</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2.63</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.010</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Poultry workers</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.103</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.006, 0.200</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2.09</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.038</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">General population</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.024</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.082, 0.130</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.44</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.657</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Test method</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">HI and MN</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">−0.15</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">HI</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.039</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.026, 0.103</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.19</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.237</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Test HI cell</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Chicken red blood cell</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">−2.24</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Turkey red blood cell</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.011</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.148, 0.169</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.13</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.895</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Horse red blood cell</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.016</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.112, 0.144</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.25</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.805</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Sample size</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">n ≥ 500</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">n < 500</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.039</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.020, 0.098</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.30</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.197</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Multivariate analysis</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"left\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">15.89</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Time</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Other time</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">First and second epidemic wave</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.062</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.005, 0.119</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2.17</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.032</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Region in mainland China</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Western</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Eastern</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.099</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.002, 0.201</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.94</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.055</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Central</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.022</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.088, 0.133</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.40</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.693</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Population</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Swine worker</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Close contacts</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.152</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.027, 0.332</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.68</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.095</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Poultry workers</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.151</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.057, 0.245</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3.18</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.002</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">General population</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.050</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−0.050, 0.150</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.98</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.327</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr></tbody></table><table-wrap-foot id=\"irv12736-ntgp-0003\"><fn id=\"irv12736-note-0004\"><label><sup>a</sup></label><p>“‐”: the first line of every covariate represented reference; adjusted <italic>R</italic>\n<sup>2</sup> was used to indicate the degree of heterogeneity explained by study characteristics.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap></sec></sec><sec sec-type=\"discussion\" id=\"irv12736-sec-0012\"><label>4</label><title>DISCUSSION</title><p>We performed this systematic review and meta‐analysis of the serological studies on influenza A (H7N9) to estimate the burden of this virus among humans. Strikingly, mild or asymptomatic human infection with H7N9 did exist, even if the proportion was small. Similar to influenza A (H5N1 and H9N2), the reported human cases were only a tip of the iceberg of a large number of infections.<xref rid=\"irv12736-bib-0011\" ref-type=\"ref\">11</xref>, <xref rid=\"irv12736-bib-0019\" ref-type=\"ref\">19</xref>\n</p><p>High seroprevalence among poultry workers suggests that avian exposure is a risk factor for infection. A previous study on H5N1 and H9N2 infections suggested that age and chronic lung problems were consistent with elevated titers.<xref rid=\"irv12736-bib-0020\" ref-type=\"ref\">20</xref> Some case‐control studies on clinical H7N9 patients also found that patients with chronic obstructive lung disease (COPD) and those receiving immunosuppressive medications were highly susceptible to be infected with influenza virus infection.<xref rid=\"irv12736-bib-0021\" ref-type=\"ref\">21</xref>, <xref rid=\"irv12736-bib-0022\" ref-type=\"ref\">22</xref> Hence, avian exposure, chronic lung problems, and poor immune status may be associated with influenza A (H7N9) infection, in both clinical H7N9 cases and silent infections.</p><p>Poultry workers with intense avian exposure tend to have high risk of being infected with the influenza virus.<xref rid=\"irv12736-bib-0020\" ref-type=\"ref\">20</xref> However, the data were consistent with the same phenomenon seen with H5N1, where the virus was so poorly adapted to humans that most hosts could not be productively infected, leading to high exposure to the virus but low seroprevalence.<xref rid=\"irv12736-bib-0023\" ref-type=\"ref\">23</xref> Previous studies have provided evidence that the host gene plays an important role in susceptibility to infection and clinical outcomes.<xref rid=\"irv12736-bib-0024\" ref-type=\"ref\">24</xref>, <xref rid=\"irv12736-bib-0025\" ref-type=\"ref\">25</xref> The severe influenza cases are the result of rare genetic susceptibilities, attributable to the interferon‐induced transmembrane protein 3 (IFITM3) or other risk factors.<xref rid=\"irv12736-bib-0023\" ref-type=\"ref\">23</xref>, <xref rid=\"irv12736-bib-0026\" ref-type=\"ref\">26</xref>, <xref rid=\"irv12736-bib-0027\" ref-type=\"ref\">27</xref>, <xref rid=\"irv12736-bib-0028\" ref-type=\"ref\">28</xref> The possible association between glycine decarboxylase (GLDC), IFITM3, and toll‐like receptors 3 (TLR3) and the outcomes of influenza A (H7N9) infection was also examined by a few researchers.<xref rid=\"irv12736-bib-0029\" ref-type=\"ref\">29</xref>, <xref rid=\"irv12736-bib-0030\" ref-type=\"ref\">30</xref> Controversially, an epidemiological study analyzing clusters of H7N9 patients found that genetic susceptibility to H7N9 virus infection was limited.<xref rid=\"irv12736-bib-0031\" ref-type=\"ref\">31</xref> The association between susceptibility‐conferring genes and influenza A (H7N9) virus infection warrants further in‐depth studies.</p><p>The risk of infection among humans was high as per the pooled seroprevalence of close contacts. Close contacts were defined as healthcare workers who had not taken effective protective measures and family members who took care of patients during the treatment of suspected or confirmed infection; the staff who lived with the patients or had experienced other close contact situations from one day before the suspicion or confirmation of infection were placed in isolation.<xref rid=\"irv12736-bib-0032\" ref-type=\"ref\">32</xref> Articles providing information about close contacts were reviewed for further evidence. Of the 8 studies that reported the data, the seroprevalence in 5 studies was 0.000%; in the remaining 3 studies, it was 3.200%, 6.667%, and 14.286%. In the three studies that reported seropositivity, only one study reported that none were exposed to poultry, swine, or other animals.<xref rid=\"irv12736-bib-0033\" ref-type=\"ref\">33</xref> The seropositive close contacts in the study included healthcare workers and family members.<xref rid=\"irv12736-bib-0033\" ref-type=\"ref\">33</xref> Previous studies suggested that the transmissibility of H7N9 virus among persons cannot be ignored, even if it was limited.<xref rid=\"irv12736-bib-0034\" ref-type=\"ref\">34</xref>, <xref rid=\"irv12736-bib-0035\" ref-type=\"ref\">35</xref>, <xref rid=\"irv12736-bib-0036\" ref-type=\"ref\">36</xref> The close contacts of H7N9 patients require protective measures and more close attention.</p><p>Swine, the intermediate hosts that facilitate the reassortment of influenza viruses, were likely to cause infection in humans.<xref rid=\"irv12736-bib-0037\" ref-type=\"ref\">37</xref>, <xref rid=\"irv12736-bib-0038\" ref-type=\"ref\">38</xref> The animal could provide a suitable vessel for the influenza A (H7N9) virus to survive and evolve.<xref rid=\"irv12736-bib-0039\" ref-type=\"ref\">39</xref> Controversially, the seroprevalence of H7N9 among swine workers was low in our pooled results. On one hand, this might be associated with swine infected with H7N9. The previous serological reports did not provide evidence of swine involvement in H7N9 virus ecology.<xref rid=\"irv12736-bib-0040\" ref-type=\"ref\">40</xref>, <xref rid=\"irv12736-bib-0041\" ref-type=\"ref\">41</xref>, <xref rid=\"irv12736-bib-0042\" ref-type=\"ref\">42</xref> One study had shown that the H7N9 virus might not efficiently infect swine.<xref rid=\"irv12736-bib-0043\" ref-type=\"ref\">43</xref> On the other hand, the virus transmission ability from pigs to other mammals might be limited.<xref rid=\"irv12736-bib-0044\" ref-type=\"ref\">44</xref> The reasons mentioned above may explain the low seroprevalence among swine workers. More studies need to explore the difference between the adaption of H7N9 and other influenza A subtypes to swine.</p><p>With regard to time, cases of subclinical H7N9 infection were not reported before 2013. Compared with the first epidemic wave, mild or asymptomatic infection showed a downward trend during the third epidemic wave. The small number of studies on seroprevalence during this wave might have led to this finding. Besides, studies showed that antibody titers in people waned over time.<xref rid=\"irv12736-bib-0045\" ref-type=\"ref\">45</xref>, <xref rid=\"irv12736-bib-0046\" ref-type=\"ref\">46</xref> China experienced six waves of H7N9 epidemics.<xref rid=\"irv12736-bib-0003\" ref-type=\"ref\">3</xref> The highest number of humans cases was reported in the fifth epidemic wave.<xref rid=\"irv12736-bib-0047\" ref-type=\"ref\">47</xref> However, only three laboratory‐confirmed human cases were reported in the recent wave.<xref rid=\"irv12736-bib-0003\" ref-type=\"ref\">3</xref> The situation of asymptomatic infection with the H7N9 virus is unclear. Studies on the seroprevalence of the H7N9 virus among humans need to be performed, especially considering the sixth wave when cases of H7N9 infection were rarely reported.</p><p>Of note, there were some limitations to our study. First, cross‐sectional studies accounted for most of the included studies. The proportion rather than incidence was provided in these studies. The serologic results of a cross‐sectional study can be misleading because antibodies may wane over time. The risk factors for infection could not be explored exhaustively because the included studies provided inadequate information about the demographic, health, and exposure variables. Second, the different seropositive thresholds might have led to the overestimation of the seroprevalence among humans. Third, cross‐reactive immunity cannot be ignored despite excluding those articles. Fourth, the result of the meta‐regression did not explain the source of heterogeneity. Probably, the proportion was too small or even zero in most of the included studies.</p></sec><sec sec-type=\"conclusions\" id=\"irv12736-sec-0013\"><label>5</label><title>CONCLUSION</title><p>Our study found that subclinical infection with the H7N9 virus did occur, even if the seroprevalence among humans was low. Be it the high‐risk group or general population, a certain degree of infection did exist. Stringent seropositive standards should be developed and observed to ensure that serology assays are reliable and convincing. Sensitive detection tests for the influenza A (H7N9) virus are need to be carried out to provide warnings before the evolvement and adaptation of the virus to the human body.</p></sec><sec sec-type=\"COI-statement\" id=\"irv12736-sec-0015\"><title>CONFLICT OF INTEREST</title><p>The authors declare that they have no competing interests.</p></sec><sec id=\"irv12736-sec-0016\"><title>AUTHOR CONTRIBUTIONS</title><p>QW and HJ designed the study. QW, KX, LQY, and WHX conducted the literature search and review. QW and KX reviewed citations and extracted data. QW, NYS, HYC, HDH, CJB, XFZ, and YLL analyzed the data. HJ and QW interpreted the results. All authors critically revised for important intellectual content. All authors approved the final version. <bold>Qiang Wang</bold>: Conceptualization (equal); data curation (lead); formal analysis (lead); funding acquisition (equal); methodology (equal); writing‐review & editing (lead). <bold>Ke Xu</bold>: Data curation (equal); formal analysis (equal); methodology (equal). <bold>Weihua Xie</bold>: Data curation (equal); methodology (equal). <bold>Liuqing Yang</bold>: Data curation (equal); methodology (equal). <bold>Haiyan Chen</bold>: Methodology‐Supporting. <bold>Naiyang Shi</bold>: Data curation (equal); methodology (equal). <bold>Changjun Bao</bold>: Formal analysis (equal); supervision (equal). <bold>Haodi Huang</bold>: Formal analysis (equal). <bold>Xuefeng Zhang</bold>: Formal analysis (equal); supervision (equal). <bold>Yilan Liao</bold>: Formal analysis (equal). <bold>Hui Jin</bold>: Conceptualization (equal); methodology (equal); supervision (lead); writing‐review & editing (equal).</p></sec><sec sec-type=\"supplementary-material\"><title>Supporting information</title><supplementary-material content-type=\"local-data\" id=\"irv12736-sup-0001\"><caption><p>Table S1</p></caption><media xlink:href=\"IRV-14-587-s001.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"irv12736-sup-0002\"><caption><p>Table S2</p></caption><media xlink:href=\"IRV-14-587-s002.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"irv12736-sup-0003\"><caption><p>Table S3</p></caption><media xlink:href=\"IRV-14-587-s003.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"irv12736-sec-0014\"><title>ACKNOWLEDGEMENTS</title><p>We thank all the editors and reviewers for their work and suggestions.</p></ack><ref-list content-type=\"cited-references\" id=\"irv12736-bibl-0001\"><title>REFERENCES</title><ref id=\"irv12736-bib-0001\"><label>1</label><mixed-citation publication-type=\"journal\" id=\"irv12736-cit-0001\">\n<string-name>\n<surname>Gao</surname>\n<given-names>RB</given-names>\n</string-name>, <string-name>\n<surname>Cao</surname>\n<given-names>B</given-names>\n</string-name>, <string-name>\n<surname>Hu</surname>\n<given-names>YW</given-names>\n</string-name>, et al. <article-title>Human infection with a novel avian‐origin influenza A (H7N9) virus</article-title>. <source xml:lang=\"en\">N Engl J Med</source>. <year>2013</year>;<volume>368</volume>(<issue>20</issue>):<fpage>1888</fpage>‐<lpage>1897</lpage>.<pub-id pub-id-type=\"pmid\">23577628</pub-id></mixed-citation></ref><ref id=\"irv12736-bib-0002\"><label>2</label><mixed-citation publication-type=\"journal\" id=\"irv12736-cit-0002\">\n<string-name>\n<surname>Li</surname>\n<given-names>Q</given-names>\n</string-name>, <string-name>\n<surname>Zhou</surname>\n<given-names>L</given-names>\n</string-name>, <string-name>\n<surname>Zhou</surname>\n<given-names>MH</given-names>\n</string-name>, et al. <article-title>Epidemiology of human infections with avian influenza A(H7N9) virus in China</article-title>. <source xml:lang=\"en\">N Engl J Med</source>. <year>2014</year>;<volume>370</volume>:<fpage>520</fpage>‐<lpage>532</lpage>.<pub-id pub-id-type=\"pmid\">23614499</pub-id></mixed-citation></ref><ref id=\"irv12736-bib-0003\"><label>3</label><mixed-citation publication-type=\"book\" id=\"irv12736-cit-0003\">\n<collab collab-type=\"authors\">World Health Organization</collab>\n. <source xml:lang=\"en\">Human Infection with Avian Influenza A (H7N9) Virus – China: Update</source>. <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.who.int/csr/don/05-september-2018-ah7n9-china/en/\">https://www.who.int/csr/don/05‐september‐2018‐ah7n9‐china/en/</ext-link>. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"iso-abbrev\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"doi\">10.1111/(ISSN)1750-2659</journal-id><journal-id journal-id-type=\"publisher-id\">IRV</journal-id><journal-title-group><journal-title>Influenza and Other Respiratory Viruses</journal-title></journal-title-group><issn pub-type=\"ppub\">1750-2640</issn><issn pub-type=\"epub\">1750-2659</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32390298</article-id><article-id pub-id-type=\"pmc\">PMC7431638</article-id><article-id pub-id-type=\"doi\">10.1111/irv.12751</article-id><article-id pub-id-type=\"publisher-id\">IRV12751</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Short Article</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Short Article</subject></subj-group></article-categories><title-group><article-title>Prediction of serious RSV‐related outcomes in older adults with outpatient RSV respiratory illness during 12 consecutive seasons</article-title><alt-title alt-title-type=\"left-running-head\">KIEKE et al.</alt-title></title-group><contrib-group><contrib id=\"irv12751-cr-0001\" contrib-type=\"author\"><name><surname>Kieke</surname><given-names>Burney A.</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-7430-0570</contrib-id><xref ref-type=\"aff\" rid=\"irv12751-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12751-cr-0002\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Belongia</surname><given-names>Edward A.</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-7478-0415</contrib-id><xref ref-type=\"aff\" rid=\"irv12751-aff-0001\">\n<sup>1</sup>\n</xref><address><email>belongia.edward@marshfieldclinic.org</email></address></contrib><contrib id=\"irv12751-cr-0003\" contrib-type=\"author\"><name><surname>McClure</surname><given-names>David L.</given-names></name><xref ref-type=\"aff\" rid=\"irv12751-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12751-cr-0004\" contrib-type=\"author\"><name><surname>Shinde</surname><given-names>Vivek</given-names></name><xref ref-type=\"aff\" rid=\"irv12751-aff-0002\">\n<sup>2</sup>\n</xref></contrib></contrib-group><aff id=\"irv12751-aff-0001\">\n<label><sup>1</sup></label>\n<institution>Marshfield Clinic Research Institute</institution>\n<city>Marshfield</city>\n<named-content content-type=\"country-part\">Wisconsin</named-content>\n<country country=\"US\">USA</country>\n</aff><aff id=\"irv12751-aff-0002\">\n<label><sup>2</sup></label>\n<institution>Novavax, Inc</institution>\n<city>Gaithersburg</city>\n<named-content content-type=\"country-part\">Maryland</named-content>\n<country country=\"US\">USA</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label><bold>Correspondence</bold><break/>\nEdward A. Belongia, Marshfield Clinic Research Institute, 1000 N. Oak Ave. (ML2), Marshfield, WI, 54449.<break/>\nEmail: <email>belongia.edward@marshfieldclinic.org</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>10</day><month>5</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>9</month><year>2020</year></pub-date><volume>14</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1111/irv.v14.5</issue-id><fpage>479</fpage><lpage>482</lpage><history><date date-type=\"received\"><day>23</day><month>8</month><year>2019</year></date><date date-type=\"rev-recd\"><day>28</day><month>1</month><year>2020</year></date><date date-type=\"accepted\"><day>16</day><month>4</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 John Wiley & Sons Ltd <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Influenza and Other Respiratory Viruses</italic> Published by John Wiley & Sons Ltd.</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:IRV-14-479.pdf\"/><abstract id=\"irv12751-abs-0001\"><title>Abstract</title><p>We developed and evaluated a model to predict serious outcomes among 243 adults ≥60 years old with medically attended respiratory illness and laboratory‐confirmed respiratory syncytial virus (RSV); 47 patients had a serious outcome defined as hospital admission, emergency department (ED) visit, or pneumonia diagnosis. The model used logistic regression with penalized maximum likelihood estimation. The reduced penalized model included age ≥ 75 years, ≥1 ED visit in prior year, crackles/rales, tachypnea, wheezing, new/increased sputum, and new/increased dyspnea. The optimal score cutoff yielded sensitivity and specificity of 66.0% and 81.6%. This prediction model provided moderate utility for identifying older adults with elevated risk of complicated RSV illness.</p></abstract><kwd-group><kwd id=\"irv12751-kwd-0001\">adult</kwd><kwd id=\"irv12751-kwd-0002\">penalized maximum likelihood estimation</kwd><kwd id=\"irv12751-kwd-0003\">prediction model</kwd><kwd id=\"irv12751-kwd-0004\">respiratory syncytial viruses</kwd><kwd id=\"irv12751-kwd-0005\">severity</kwd></kwd-group><funding-group><award-group id=\"funding-0001\"><funding-source>Novavax</funding-source></award-group></funding-group><counts><fig-count count=\"1\"/><table-count count=\"1\"/><page-count count=\"4\"/><word-count count=\"2211\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>September 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.6 mode:remove_FC converted:18.08.2020</meta-value></custom-meta></custom-meta-group></article-meta><notes><p content-type=\"self-citation\">\n<mixed-citation publication-type=\"journal\" id=\"irv12751-cit-1001\">\n<string-name>\n<surname>Kieke</surname>\n<given-names>BA</given-names>\n</string-name>, <string-name>\n<surname>Belongia</surname>\n<given-names>EA</given-names>\n</string-name>, <string-name>\n<surname>McClure</surname>\n<given-names>DL</given-names>\n</string-name>, <string-name>\n<surname>Shinde</surname>\n<given-names>V</given-names>\n</string-name>. <article-title>Prediction of serious RSV‐related outcomes in older adults with outpatient RSV respiratory illness during 12 consecutive seasons</article-title>. <source xml:lang=\"en\">Influenza Other Respi Viruses</source>. <year>2020</year>;<volume>14</volume>:<fpage>479</fpage>–<lpage>482</lpage>. <pub-id pub-id-type=\"doi\">10.1111/irv.12751</pub-id>\n</mixed-citation>\n</p><fn-group id=\"irv12751-ntgp-0001\"><fn fn-type=\"funding\" id=\"irv12751-note-0002\"><p>\n<bold>Funding information</bold>\n</p><p>Support for this research was provided by Novavax, Inc</p></fn></fn-group></notes></front><body id=\"irv12751-body-0001\"><sec id=\"irv12751-sec-0001\"><label>1</label><title>INTRODUCTION</title><p>The epidemiology and burden of respiratory syncytial virus (RSV)‐related respiratory illness is not well‐defined in adults.<xref rid=\"irv12751-bib-0001\" ref-type=\"ref\">\n<sup>1</sup>\n</xref> The symptoms of RSV mimic those of other viral respiratory pathogens, and specific diagnostic testing is rarely performed in the outpatient setting.<xref rid=\"irv12751-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref> Multiple studies have documented that RSV is an important cause of respiratory illness in adults, particularly those who are immunocompromised or have cardiopulmonary disease.<xref rid=\"irv12751-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref>, <xref rid=\"irv12751-bib-0004\" ref-type=\"ref\">\n<sup>4</sup>\n</xref> In 2018, we published a report describing the epidemiology and outcomes of RSV infection among adults with outpatient respiratory illness who were systematically recruited and tested for respiratory viruses during 12 influenza seasons.<xref rid=\"irv12751-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref> Among 243 patients ≥ 60 years of age with RT‐PCR‐confirmed RSV, 47 had a serious outcome defined as hospital admission, emergency department visit, or pneumonia. For this study, we analyzed typical clinical and demographic characteristics among patients with RSV in the prior study to develop a predictive model that identifies patients with an elevated risk for a serious outcome as defined above.</p></sec><sec sec-type=\"methods\" id=\"irv12751-sec-0002\"><label>2</label><title>METHODS</title><sec id=\"irv12751-sec-0003\"><label>2.1</label><title>Patient population and study design</title><p>This was a secondary analysis of a previously published study reporting clinical features, severity, and incidence of RSV illness among older adults living in and around Marshfield, Wisconsin.<xref rid=\"irv12751-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref> Adults ≥60 years old with medically attended acute respiratory illness (primarily outpatient) were systematically recruited by research staff for an influenza vaccine effectiveness study from the 2004‐2005 through 2015‐2016 influenza seasons. Symptom eligibility criteria varied by season, but fever/feverishness or cough were required during most seasons. Multiplex reverse‐transcription polymerase chain reaction (RT‐PCR) was performed on archived samples to detect RSV and other viruses. The multiplex panel included RSV A and B, human rhinovirus, human metapneumovirus, parainfluenza viruses 1‐3, influenza A (H3, H1, and H1N1pdm09), influenza B, and adenoviruses B/E and C. Symptoms were assessed during the enrollment interview, and medical records were abstracted for all RSV cases. Serious RSV outcomes were defined as acute care hospital admission, emergency department (ED) visit for acute illness, or pneumonia occurring within 28 days after enrollment. A physician independently validated all serious outcomes. All participants provided informed consent for influenza study participation, and subsequent multiplex testing was approved by the institutional review board with a waiver of informed consent.</p></sec><sec id=\"irv12751-sec-0004\"><label>2.2</label><title>Statistical modeling</title><p>The modeling dataset contained information from 243 adults with RSV illness, including 47 who had a serious outcome and 196 with a non‐serious outcome. A model to predict presence of a serious RSV outcome was built via logistic regression using penalized maximum likelihood estimation (PMLE).<xref rid=\"irv12751-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref>, <xref rid=\"irv12751-bib-0007\" ref-type=\"ref\">\n<sup>7</sup>\n</xref>, <xref rid=\"irv12751-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> Models employing PMLE, a form of shrinkage estimation, effectively use fewer degrees of freedom (<italic>df</italic>) than standard MLE models and hence have a higher threshold for what would constitute overfitting. In this context, overfitting translates to the number of candidate predictors evaluated being high relative to the number of patients with serious RSV outcome events. PMLE directly adjusts for “over‐optimism,” the fact that predictive performance measures derived from the dataset on which the model was built will generally be more favorable than would be expected when the model is applied to a new data source.</p><p>Penalized maximum likelihood estimation is well suited for situations with a high potential for overfitting as described above. However, since the modeling dataset included only 47 serious outcome events, a univariate screening of candidate predictors was performed prior to multivariable modeling. The set of candidate variables included signs and symptoms (both self‐reported and physical examination findings) as well as other clinical and demographic factors.</p><p>A goal of the PMLE analyses was to impose shrinkage such that the <italic>df</italic> from the standard MLE‐based logistic regression model (one <italic>df</italic> for each parameter required to represent the terms in the model) were reduced to the point where overfitting was not present. We implemented the shrinkage by selecting a penalty factor and applying this factor in the process of estimating regression coefficients for the full penalized model. Optimization of the corrected Akaike information criterion, a goodness of fit measure, was the basis for selecting the penalty factor. To further reduce the effective <italic>df</italic>, a reduced penalized model containing a parsimonious set of important predictors was established by applying a backward stepdown procedure to the full penalized model.<xref rid=\"irv12751-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref>\n</p><p>We assigned a score to each patient that represents the likelihood of a serious RSV outcome using a system based on the estimated regression coefficients. A score of 100 was assigned to the predictor with the largest regression coefficient and the remaining scores reflected the importance of other predictors relative to the predictor with a score of 100. For each patient, scores across predictors were summed to generate a total score.</p><p>The final step in the predictive modeling process was to establish an “optimal” cutoff for dichotomizing the score to represent either presence or absence of a serious RSV outcome. We evaluated two well‐known criteria for selecting cutoffs: the “closest to ideal” criterion and the Youden index.<xref rid=\"irv12751-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref>, <xref rid=\"irv12751-bib-0010\" ref-type=\"ref\">\n<sup>10</sup>\n</xref> In this context, the term “optimal” corresponds to selecting a cutoff with a desirable tradeoff between sensitivity and specificity. We generated an empirical receiver operating characteristic (ROC) curve for the reduced penalized model that depicts the combinations of 1‐specificity (false‐positive rate) and sensitivity (true‐positive rate) across the observed range of possible cutoff values for the patient score. To evaluate the robustness of the selected cutoff, we computed its sensitivity and specificity in 500 bootstrap samples.</p><p>Analyses were carried out in TIBCO Spotfire S+® 8.2 for Windows (TIBCO Software Inc), and SAS 9.4 (SAS Institute Inc).</p></sec></sec><sec sec-type=\"results\" id=\"irv12751-sec-0005\"><label>3</label><title>RESULTS</title><p>The univariate screening process yielded 11 variables for inclusion in the initial multivariable model: age ≥75 years, ≥1 ED visit in the prior year, ≥1 hospital admission in the prior year, congestive heart failure (CHF), chronic obstructive pulmonary disease (COPD), examination findings of crackles/rales, fever ≥100°F, tachypnea and wheezing, and patient self‐reported new/increased sputum and new/increased dyspnea. Any coinfection and influenza coinfection status were included in the screening process and neither satisfied the criteria for inclusion in the multivariable modeling. Serious RSV outcomes were more common in patients with coinfections (relative risk = 2.0 overall and 3.2 for influenza coinfections). However, these patients comprised a small percentage of the study population. Only 2.1% (5/243) of patients with RSV had influenza coinfection, and 9.5% (23/243) had coinfection with any virus. The full penalized model yielded 6.5 effective <italic>df</italic>, representing a 41% reduction relative to the corresponding conventional logistic regression model. Area under the ROC curve (AUC) expected in similar patient samples in the future (ie, the AUC corrected for “over‐optimism”) was 0.763. The reduced penalized model accounted for 97.1% of the variation in the full penalized model, used 4.3 effective <italic>df</italic> and contained the following 7 terms: age ≥75, ≥1 ED visit in the prior year, crackles/rales, tachypnea, wheezing, new/increased sputum, and new/increased dyspnea.</p><p>Observed patient scores ranged from 0 to 426. Crackles/rales had the highest score followed closely by tachypnea (Table <xref rid=\"irv12751-tbl-0001\" ref-type=\"table\">1</xref>). The “closest to ideal” criterion and Youden index both identified an optimal score cutoff value of 143 with sensitivity and specificity of 66.0% and 81.6%, respectively. The empirical ROC curve for the reduced penalized model, with the selected cutoff annotated, is shown in the Figure <xref rid=\"irv12751-fig-0001\" ref-type=\"fig\">1</xref>. In the bootstrap samples, the median sensitivity and specificity were within 0.7% of the observed values. The lower and upper quartiles in the bootstrap samples were within 5.2% of the observed value for sensitivity and 1.8% for specificity<bold>.</bold>\n</p><table-wrap id=\"irv12751-tbl-0001\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 1</label><caption><p>Individual predictor regression coefficients and scores from the reduced penalized model for predicting serious RSV outcomes (hospital admission, ED visit, or pneumonia) among patients ≥ 60 y old with PCR‐confirmed RSV</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Predictor</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Regression coefficient (RC)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Score<xref ref-type=\"fn\" rid=\"irv12751-note-0003\">\n<sup>a</sup>\n</xref>\n</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Crackles/rales<xref ref-type=\"fn\" rid=\"irv12751-note-0004\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.9651533526</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">100</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Tachypnea<xref ref-type=\"fn\" rid=\"irv12751-note-0004\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.9175530877</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">95</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Wheezing<xref ref-type=\"fn\" rid=\"irv12751-note-0004\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.6302838213</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">65</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">New/increased dyspnea<xref ref-type=\"fn\" rid=\"irv12751-note-0005\">\n<sup>c</sup>\n</xref>\n</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.6110782569</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">63</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">≥1 ED visit in the prior year</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.5821523979</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">60</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">New/increased sputum<xref ref-type=\"fn\" rid=\"irv12751-note-0005\">\n<sup>c</sup>\n</xref>\n</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.4156787828</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">43</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age ≥75 y</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.4133028716</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">43</td></tr></tbody></table><table-wrap-foot id=\"irv12751-ntgp-0002\"><fn id=\"irv12751-note-0003\"><label><sup>a</sup></label><p>100 × RC/maximum(RC) rounded to an integer, where the maximum RC is 0.9651533526 from the first row in Table <xref rid=\"irv12751-tbl-0001\" ref-type=\"table\">1</xref>.</p></fn><fn id=\"irv12751-note-0004\"><label><sup>b</sup></label><p>Based on examination by a healthcare provider.</p></fn><fn id=\"irv12751-note-0005\"><label><sup>c</sup></label><p>Based on patient self‐report.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><fig fig-type=\"FIGURE\" xml:lang=\"en\" id=\"irv12751-fig-0001\" orientation=\"portrait\" position=\"float\"><label>FIGURE 1</label><caption><p>Empirical receiver operating characteristic (ROC) curve for the reduced penalized model</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"IRV-14-479-g001\"/></fig></sec><sec sec-type=\"discussion\" id=\"irv12751-sec-0006\"><label>4</label><title>DISCUSSION</title><p>This predictive model was moderately successful in predicting serious RSV outcomes in older adults with RSV infection. It was based on patient characteristics readily available in clinical practice and may be useful for identifying and risk stratifying patients with an increased likelihood of serious complications of RSV infection. It could also be useful for constructing clinically meaningful efficacy endpoints for prelicensure clinical trials of RSV vaccines. While the aim of such vaccines would be to prevent serious outcomes such as hospitalizations, ED visits or pneumonia caused by RSV infection, capturing these outcomes typically requires prohibitively large clinical trials. Use of clinical endpoints based on prediction scores derived from readily captured data could be a more efficient approach to measuring vaccine efficacy against serious outcomes of interest.</p><p>The strengths of this analysis include systematic screening and testing of patients in the original study, recruitment from a stable community cohort, and inclusion of 12 consecutive seasons with access to outpatient and inpatient electronic medical records. Weaknesses included the small number of serious outcomes captured among adults seeking outpatient care, lack of racial/ethnic diversity, and inability to examine predictors of hospitalization or more severe hospitalized illness. The number of coinfections was small, and it is possible that a larger study would have identified influenza coinfection as a significant predictor of serious outcomes. Validation of this prediction model in larger populations is needed to determine if data collected during an acute respiratory illness visit is useful to predict the likelihood of a subsequent hospital admission or other serious outcome.</p><p>In conclusion, a prediction model using clinical information available during the initial evaluation of RSV illness was moderately useful for assessing the risk of a serious outcome.</p></sec><sec sec-type=\"COI-statement\" id=\"irv12751-sec-0007\"><title>CONFLICTS OF INTEREST</title><p>EAB, BAK, and DLM have received research support from Novavax, Inc VS is employed by Novavax, Inc</p></sec></body><back><ref-list content-type=\"cited-references\" id=\"irv12751-bibl-0001\"><title>REFERENCES</title><ref id=\"irv12751-bib-0001\"><label>1</label><mixed-citation publication-type=\"journal\" id=\"irv12751-cit-0001\">\n<string-name>\n<surname>Kim</surname>\n<given-names>L</given-names>\n</string-name>, <string-name>\n<surname>Rha</surname>\n<given-names>B</given-names>\n</string-name>, <string-name>\n<surname>Abramson</surname>\n<given-names>JS</given-names>\n</string-name>, et al. <article-title>Identifying gaps in respiratory syncytial virus disease epidemiology in the United States prior to the introduction of vaccines</article-title>. <source xml:lang=\"en\">Clin Infect Dis</source>. <year>2017</year>;<volume>65</volume>(<issue>6</issue>):<fpage>1020</fpage>‐<lpage>1025</lpage>.<pub-id pub-id-type=\"pmid\">28903503</pub-id></mixed-citation></ref><ref id=\"irv12751-bib-0002\"><label>2</label><mixed-citation publication-type=\"journal\" id=\"irv12751-cit-0002\">\n<string-name>\n<surname>Branche</surname>\n<given-names>AR</given-names>\n</string-name>, <string-name>\n<surname>Falsey</surname>\n<given-names>AR</given-names>\n</string-name>. <article-title>Respiratory syncytial virus infection in older adults: an under‐recognized problem</article-title>. <source xml:lang=\"en\">Drugs Aging</source>. <year>2015</year>;<volume>32</volume>(<issue>4</issue>):<fpage>261</fpage>‐<lpage>269</lpage>.<pub-id pub-id-type=\"pmid\">25851217</pub-id></mixed-citation></ref><ref id=\"irv12751-bib-0003\"><label>3</label><mixed-citation publication-type=\"journal\" id=\"irv12751-cit-0003\">\n<string-name>\n<surname>Walsh</surname>\n<given-names>EE</given-names>\n</string-name>, <string-name>\n<surname>Falsey</surname>\n<given-names>AR</given-names>\n</string-name>. <article-title>Respiratory syncytial virus infection in adult populations</article-title>. <source xml:lang=\"en\">Infect Disord Drug Targets</source>. <year>2012</year>;<volume>12</volume>(<issue>2</issue>):<fpage>98</fpage>‐<lpage>102</lpage>.<pub-id pub-id-type=\"pmid\">22335500</pub-id></mixed-citation></ref><ref id=\"irv12751-bib-0004\"><label>4</label><mixed-citation publication-type=\"journal\" id=\"irv12751-cit-0004\">\n<string-name>\n<surname>Falsey</surname>\n<given-names>AR</given-names>\n</string-name>, <string-name>\n<surname>Walsh</surname>\n<given-names>EE</given-names>\n</string-name>, <string-name>\n<surname>Esser</surname>\n<given-names>MT</given-names>\n</string-name>, <string-name>\n<surname>Shoemaker</surname>\n<given-names>K</given-names>\n</string-name>, <string-name>\n<surname>Yu</surname>\n<given-names>L</given-names>\n</string-name>, <string-name>\n<surname>Griffin</surname>\n<given-names>MP</given-names>\n</string-name>. <article-title>Respiratory syncytial virus‐associated illness in adults with advanced chronic obstructive pulmonary disease and/or congestive heart failure</article-title>. <source xml:lang=\"en\">J Med Virol</source>. <year>2019</year>;<volume>91</volume>(<issue>1</issue>):<fpage>65</fpage>‐<lpage>71</lpage>.<pub-id pub-id-type=\"pmid\">30132922</pub-id></mixed-citation></ref><ref id=\"irv12751-bib-0005\"><label>5</label><mixed-citation publication-type=\"journal\" id=\"irv12751-cit-0005\">\n<string-name>\n<surname>Belongia</surname>\n<given-names>EA</given-names>\n</string-name>, <string-name>\n<surname>King</surname>\n<given-names>JP</given-names>\n</string-name>, <string-name>\n<surname>Kieke</surname>\n<given-names>BA</given-names>\n</string-name>, et al. <article-title>Clinical features, severity, and incidence of RSV illness during 12 consecutive seasons in a community cohort of adults >/=60 years old</article-title>. <source xml:lang=\"en\">Open Forum Infect Dis</source>. <year>2018</year>;<volume>5</volume>(<issue>12</issue>):<elocation-id>ofy316</elocation-id>.<pub-id pub-id-type=\"pmid\">30619907</pub-id></mixed-citation></ref><ref id=\"irv12751-bib-0006\"><label>6</label><mixed-citation publication-type=\"book\" id=\"irv12751-cit-0006\">\n<string-name>\n<surname>Harrell</surname>\n<given-names>FE</given-names>\n</string-name>. <source xml:lang=\"en\">Regression Modeling Strategies</source>. <publisher-loc>New York, NY</publisher-loc>: <publisher-name>Springer‐Verlag</publisher-name>; 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"iso-abbrev\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"doi\">10.1111/(ISSN)1750-2659</journal-id><journal-id journal-id-type=\"publisher-id\">IRV</journal-id><journal-title-group><journal-title>Influenza and Other Respiratory Viruses</journal-title></journal-title-group><issn pub-type=\"ppub\">1750-2640</issn><issn pub-type=\"epub\">1750-2659</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32614504</article-id><article-id pub-id-type=\"pmc\">PMC7431640</article-id><article-id pub-id-type=\"doi\">10.1111/irv.12744</article-id><article-id pub-id-type=\"publisher-id\">IRV12744</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Original Article</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Original Articles</subject></subj-group></article-categories><title-group><article-title>Recent influenza activity in tropical Puerto Rico has become synchronized with mainland US</article-title><alt-title alt-title-type=\"left-running-head\">PAZ–BAILEY et al.</alt-title></title-group><contrib-group><contrib id=\"irv12744-cr-0001\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Paz–Bailey</surname><given-names>Gabriela</given-names></name><xref ref-type=\"aff\" rid=\"irv12744-aff-0001\">\n<sup>1</sup>\n</xref><address><email>gmb5@cdc.gov</email></address></contrib><contrib id=\"irv12744-cr-0002\" contrib-type=\"author\"><name><surname>Quandelacy</surname><given-names>Talia M.</given-names></name><xref ref-type=\"aff\" rid=\"irv12744-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12744-cr-0003\" contrib-type=\"author\"><name><surname>Adams</surname><given-names>Laura E.</given-names></name><xref ref-type=\"aff\" rid=\"irv12744-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12744-cr-0004\" contrib-type=\"author\"><name><surname>Olsen</surname><given-names>Sonja J.</given-names></name><xref ref-type=\"aff\" rid=\"irv12744-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12744-cr-0005\" contrib-type=\"author\"><name><surname>Blanton</surname><given-names>Lenee</given-names></name><xref ref-type=\"aff\" rid=\"irv12744-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12744-cr-0006\" contrib-type=\"author\"><name><surname>Munoz-Jordan</surname><given-names>Jorge L.</given-names></name><xref ref-type=\"aff\" rid=\"irv12744-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12744-cr-0007\" contrib-type=\"author\"><name><surname>Lozier</surname><given-names>Matthew</given-names></name><xref ref-type=\"aff\" rid=\"irv12744-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12744-cr-0008\" contrib-type=\"author\"><name><surname>Alvarado</surname><given-names>Luisa I.</given-names></name><xref ref-type=\"aff\" rid=\"irv12744-aff-0003\">\n<sup>3</sup>\n</xref></contrib><contrib id=\"irv12744-cr-0009\" contrib-type=\"author\"><name><surname>Johansson</surname><given-names>Michael A.</given-names></name><xref ref-type=\"aff\" rid=\"irv12744-aff-0001\">\n<sup>1</sup>\n</xref></contrib></contrib-group><aff id=\"irv12744-aff-0001\">\n<label><sup>1</sup></label>\n<institution>Centers for Disease Control and Prevention</institution>\n<city>San Juan</city>\n<country country=\"PR\">Puerto Rico</country>\n</aff><aff id=\"irv12744-aff-0002\">\n<label><sup>2</sup></label>\n<institution>Centers for Disease Control and Prevention</institution>\n<city>Atlanta</city>\n<named-content content-type=\"country-part\">Georgia</named-content>\n</aff><aff id=\"irv12744-aff-0003\">\n<label><sup>3</sup></label>\n<institution>Saint Luke’s Episcopal Hospital and Ponce Health Sciences University</institution>\n<city>Ponce</city>\n<country country=\"PR\">Puerto Rico</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label><bold>Correspondence</bold><break/>\nGabriela Paz–Bailey, Centers for Disease Control and Prevention, 1324 Calle Canada, San Juan 00920, Puerto Rico.<break/>\nEmail: <email>gmb5@cdc.gov</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>02</day><month>7</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>9</month><year>2020</year></pub-date><volume>14</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1111/irv.v14.5</issue-id><fpage>515</fpage><lpage>523</lpage><history><date date-type=\"received\"><day>10</day><month>12</month><year>2019</year></date><date date-type=\"rev-recd\"><day>31</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>01</day><month>4</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 John Wiley & Sons Ltd <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Influenza and Other Respiratory Viruses</italic> Published by John Wiley & Sons Ltd.</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:IRV-14-515.pdf\"/><abstract id=\"irv12744-abs-0001\"><title>Abstract</title><sec id=\"irv12744-sec-0001\"><title>Background</title><p>We used data from the Sentinel Enhanced Dengue Surveillance System (SEDSS) to describe influenza trends in southern Puerto Rico during 2012‐2018 and compare them to trends in the United States.</p></sec><sec id=\"irv12744-sec-0002\"><title>Methods</title><p>Patients with fever onset ≤ 7 days presenting were enrolled. Nasal/oropharyngeal swabs were tested for influenza A and B viruses by PCR. Virologic data were obtained from the US World Health Organization (WHO) Collaborating Laboratories System and the National Respiratory and Enteric Virus Surveillance System (NREVSS). We compared influenza A and B infections identified from SEDSS and WHO/NREVSS laboratories reported by US Department of Health and Human Services (HHS) region using time series decomposition methods, and analysed coherence of climate and influenza trends by region.</p></sec><sec id=\"irv12744-sec-0003\"><title>Results</title><p>Among 23,124 participants, 9% were positive for influenza A and 5% for influenza B. Influenza A and B viruses were identified year‐round, with no clear seasonal patterns from 2012 to 2015 and peaks in December‐January in 2016‐2017 and 2017‐2018 seasons. Influenza seasons in HHS regions were relatively synchronized in recent years with the seasons in Puerto Rico. We observed high coherence between absolute humidity and influenza A and B virus in HHS regions. In Puerto Rico, coherence was much lower in the early years but increased to similar levels to HHS regions by 2017‐2018.</p></sec><sec id=\"irv12744-sec-0004\"><title>Conclusions</title><p>Influenza seasons in Puerto Rico have recently become synchronized with seasons in US HHS regions. Current US recommendations are for everyone 6 months and older to receive influenza vaccination by the end of October seem appropriate for Puerto Rico.</p></sec></abstract><kwd-group><kwd id=\"irv12744-kwd-0001\">COVID-19</kwd><kwd id=\"irv12744-kwd-0002\">influenza</kwd><kwd id=\"irv12744-kwd-0003\">Puerto Rico</kwd><kwd id=\"irv12744-kwd-0006\">synchrony</kwd><kwd id=\"irv12744-kwd-0007\">trends</kwd><kwd id=\"irv12744-kwd-0008\">United States</kwd></kwd-group><funding-group><award-group id=\"funding-0001\"><funding-source><institution-wrap><institution>National Center for Emerging and Zoonotic Infectious Diseases </institution><institution-id institution-id-type=\"open-funder-registry\">10.13039/100006088</institution-id></institution-wrap></funding-source></award-group></funding-group><counts><fig-count count=\"2\"/><table-count count=\"2\"/><page-count count=\"9\"/><word-count count=\"6747\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>September 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.6 mode:remove_FC converted:18.08.2020</meta-value></custom-meta></custom-meta-group></article-meta><notes><p content-type=\"self-citation\">\n<mixed-citation publication-type=\"journal\" id=\"irv12744-cit-1001\">\n<string-name>\n<surname>Paz–Bailey</surname>\n<given-names>G</given-names>\n</string-name>, <string-name>\n<surname>Quandelacy</surname>\n<given-names>TM</given-names>\n</string-name>, <string-name>\n<surname>Adams</surname>\n<given-names>LE</given-names>\n</string-name>, et al. <article-title>Recent influenza activity in tropical Puerto Rico has become synchronized with mainland US</article-title>. <source xml:lang=\"en\">Influenza Other Respi Viruses</source>. <year>2020</year>;<volume>14</volume>:<fpage>515</fpage>–<lpage>523</lpage>. <pub-id pub-id-type=\"doi\">10.1111/irv.12744</pub-id>\n</mixed-citation>\n</p><fn-group id=\"irv12744-ntgp-0001\"><fn id=\"irv12744-note-0001\"><p>The peer review history for this article is available at https://publons.com/publon/10.1111/irv.12744</p></fn></fn-group></notes></front><body id=\"irv12744-body-0001\"><sec sec-type=\"background\" id=\"irv12744-sec-0005\"><label>1</label><title>BACKGROUND</title><p>Influenza is a significant public health problem globally. Seasonal influenza has a high disease burden, reducing school attendance, increasing worker absenteeism and impacting daily productivity.<xref rid=\"irv12744-bib-0001\" ref-type=\"ref\">\n<sup>1</sup>\n</xref> Since 2010, CDC estimates that influenza is associated with between 9.3‐49 million illnesses, 140 000‐960 000 hospitalizations and 12 000‐79 000 deaths each year in the United States (US).<xref rid=\"irv12744-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref> Though antivirals treat infection, vaccination remains the primary tool to prevent influenza‐associated morbidity and mortality.<xref rid=\"irv12744-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref> The US Advisory Committee on Immunization Practices (ACIP) recommends routine annual influenza vaccination for individuals, aged ≥6‐months who do not have contraindications.<xref rid=\"irv12744-bib-0004\" ref-type=\"ref\">\n<sup>4</sup>\n</xref> Influenza immunization programmes must consider disease seasonality to most effectively use immunization.<xref rid=\"irv12744-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref> As a US Territory, Puerto Rico follows the same seasonal influenza vaccination recommendations as the continental US. However, given its tropical climate and distant geographic location, Puerto Rico's seasonality may differ compared with the continental United States. This difference may impact public health measures, for example when to recommend the annual influenza vaccine in Puerto Rico.</p><p>In high‐income temperate countries, influenza has been well described. Most seasonal epidemics in temperate regions occur during the winter months, between November and March in the Northern Hemisphere and between April and September in the Southern Hemisphere.<xref rid=\"irv12744-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref> These seasonal patterns are thought to be driven by annual changes in climate, contact rates, human immunity and other factors.<xref rid=\"irv12744-bib-0001\" ref-type=\"ref\">\n<sup>1</sup>\n</xref>, <xref rid=\"irv12744-bib-0007\" ref-type=\"ref\">\n<sup>7</sup>\n</xref>, <xref rid=\"irv12744-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> Some tropical and subtropical regions experience annual epidemics coinciding with local rainy seasons,<xref rid=\"irv12744-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref> whereas others have semi‐annual epidemics or year‐round influenza activity without well‐defined influenza seasons.<xref rid=\"irv12744-bib-0010\" ref-type=\"ref\">\n<sup>10</sup>\n</xref>, <xref rid=\"irv12744-bib-0011\" ref-type=\"ref\">\n<sup>11</sup>\n</xref> Influenza seasonality in tropical and subtropical regions is less understood.<xref rid=\"irv12744-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref>\n</p><p>Few studies examined influenza in Puerto Rico, and none examine Puerto Rico influenza trends in relation to patterns in the continental United States. Comparing seasonal patterns of influenza in Puerto Rico to seasonality in the continental United States would help inform immunization programmes, surveillance and mitigation measures on the island. We analysed data from enhanced acute febrile illness (AFI) surveillance sites in southern Puerto Rico during 2012 to 2018 to define typical periods of influenza activity. To better understand the temporal dynamics of influenza epidemics and possible drivers, we used time series decomposition methods to isolate seasonal oscillations of influenza epidemics in Puerto Rico and to compare them to influenza epidemics in US Health and Human Service [HHS] regions (Figure <xref rid=\"irv12744-sup-0001\" ref-type=\"supplementary-material\">S1</xref>). We also explored coherence between climate and influenza trends for Puerto Rico and HHS regions, to determine whether climatic differences explained differences in influenza trends.</p></sec><sec sec-type=\"methods\" id=\"irv12744-sec-0006\"><label>2</label><title>METHODS</title><sec id=\"irv12744-sec-0007\"><label>2.1</label><title>Sentinel enhanced dengue surveillance system</title><p>The Sentinel Enhanced Dengue Surveillance System (SEDSS)<xref rid=\"irv12744-bib-0012\" ref-type=\"ref\">\n<sup>12</sup>\n</xref> recruits febrile patients presenting at emergency departments (ED) and one outpatient clinic in Ponce, Puerto Rico. SEDSS started in May 2012 at Saint Luke's Episcopal Hospital, a 427 inpatient bed tertiary care hospital that receives ~60 000 patients/year, and is the regional paediatric referral hospital for the Ponce and Mayaguez (~500 000 residents) Health Districts. The Outpatient Acute Care Clinic (2016‐present) services ~20 000 patients/year. Guayama Menonita Hospital (2013‐2015) is a 161‐bed secondary care hospital site that receives ~35 000 patients per year from southeastern Puerto Rico (~50 000 residents), and participated in SEDSS from 2013‐2015. The municipality of Ponce has a population of 133 191 persons for 2018. The age distribution is 5% are ≤5 years, 20% 5‐18 years, 22% ≥65 years (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.census.gov/\">https://www.census.gov/</ext-link>).</p><p>Patients presenting to the SEDSS sites were queried for current fever (body temperature of ≥38.0°C [oral] or ≥38.5°C [axillary]), or having reported a history of fever within the last 7 days. Those responding positively for fever were enrolled. Blood, urine, and combined nasopharyngeal and oropharyngeal swabs were collected from enrolled patients, and diagnostic testing was performed. Upper respiratory tract specimens were tested for influenza A and B viruses, adenovirus, respiratory syncytial virus (RSV), coronavirus, parainfluenza viruses and human metapneumovirus by real‐time reverse transcription polymerase chain reaction (RT–PCR) as previously described.<xref rid=\"irv12744-bib-0013\" ref-type=\"ref\">\n<sup>13</sup>\n</xref>, <xref rid=\"irv12744-bib-0014\" ref-type=\"ref\">\n<sup>14</sup>\n</xref>, <xref rid=\"irv12744-bib-0015\" ref-type=\"ref\">\n<sup>15</sup>\n</xref> For each respiratory pathogen, a positive result was defined as nucleic acid detected by PCR (cycle threshold cut–off 37.0). SEDSS participants provided demographic, history of exposure and clinical data during enrolment interview.</p><p>All enrolled patients provided informed consent. The Ponce Medical School Foundation and the US Centers for Disease Control and Prevention (CDC) ethics committees reviewed and approved the SEDSS study protocol.</p></sec><sec id=\"irv12744-sec-0008\"><label>2.2</label><title>US influenza viral surveillance</title><p>Virologic surveillance data from the 40th week of 2012 to the end of 2018 were obtained from the US World Health Organization (WHO) Collaborating Laboratories and the National Respiratory and Enteric Virus Surveillance System (NREVSS). The WHO Collaborating Laboratories System is maintained by CDC’s Influenza Division and the NREVSS is maintained by CDC’s Division of Viral Diseases. The data included the total number of respiratory specimens tested, and the number positive for influenza types A and B viruses each week. Data were collected, analysed and aggregated nationally and by HHS regions (Figure <xref rid=\"irv12744-sup-0001\" ref-type=\"supplementary-material\">S1</xref>).<fn id=\"irv12744-note-1001\"><label><sup>1</sup></label><p>Region 1 includes: Connecticut, Maine, Massachusetts, New Hampshire, Rhode Island, and Vermont. Region 2 includes: New Jersey, New York, Puerto Rico, and the Virgin Islands. Region 3 includes: Delaware, District of Columbia, Maryland, Pennsylvania, Virginia, and West Virginia. Region 4 includes: Alabama, Florida, Georgia, Kentucky, Mississippi, North Carolina, South Carolina, and Tennessee. Region 5 includes: Illinois, Indiana, Michigan, Minnesota, Ohio, and Wisconsin. Region 6 includes: Arkansas, Louisiana, New Mexico, Oklahoma, and Texas. Region 7 includes: Iowa, Kansas, Missouri, and Nebraska. Region 8 includes: Colorado, Montana, North Dakota, South Dakota, Utah, and Wyoming. Region 9 includes: Arizona, California, Hawaii, Nevada, American Samoa, Common wealth of the Northern Mariana Islands, Federated States of Micronesia, Guam, Marshall Islands, and Republic of Palau. Region 10 includes: Alaska, Idaho, Oregon, and Washington.</p></fn>\n</p></sec><sec id=\"irv12744-sec-0009\"><label>2.3</label><title>Data analyses</title><p>We compared the number of confirmed influenza A and B cases in SEDSS to virologic surveillance data collected by US WHO and NREVSS laboratories by HHS region. Weekly numbers of influenza cases combined influenza A and B virus types. The reporting period for each influenza seasons begin during week 40 and ends week 39 of the following year. For weekly case time series in Puerto Rico and HHS regions, the peak week was the week with the maximum number of positive cases. To compare the difference in timing (in weeks) of influenza epidemics between SEDSS and HHS regions, we used continuous wavelet transforms to isolate the seasonal component of the time series data. First, case count time series were normalized by logging the weekly case counts and standardizing by the mean and standard deviation of the time series. The seasonal time series for each region was reconstructed using Morlet wavelet decomposition to extract the major seasonal component (period: 40‐56 weeks) from the normalized case count time series. This range allowed us to capture fluctuations around the 52‐week yearly cycle. Then, we calculated coherence and phase difference analyses between the seasonal influenza A and B component from SEDSS to the seasonal influenza component of each HHS region.<xref rid=\"irv12744-bib-0016\" ref-type=\"ref\">\n<sup>16</sup>\n</xref> Coherence estimated the relatedness of the time series, with a coherence of 1 indicating the time series are exactly identical though possibly lagged, while a coherence of 0 indicates no association between the two time series. Seasonal epidemics in two regions were considered to be in phase (ie, occurring at the same time), if there was no difference between their phases (ie relative timing in weeks). Relative to the reference location, Puerto Rico, a positive phase difference would indicate that seasonal epidemics occurred earlier in Puerto Rico than the matched HHS region. In contrast, a negative phase difference indicated that seasonal epidemics were later in Puerto Rico than in the HHS Region. Results based on peak timing were confirmed by the wavelet phase analysis, which considers the entire epidemic cycle.<xref rid=\"irv12744-bib-0017\" ref-type=\"ref\">\n<sup>17</sup>\n</xref>\n</p><p>To explore if differences in seasonality between Puerto Rico and HHS regions were due to differences in climate, we obtained regional climate data (absolute humidity, temperature and precipitation) from 2012 to 2018 from the North American Regional Reanalysis data set from the National Oceanic and Atmospheric Administration (NOAA).<xref rid=\"irv12744-bib-0018\" ref-type=\"ref\">\n<sup>18</sup>\n</xref> Daily climate data were temporally and spatially aggregated to weekly means for each HHS region and Puerto Rico. Weekly mean time series data were normalized. In order to analyse the possible relationship to climate, we extended the timeframe considered for the seasonal component to 40‐80 weeks to include potential climatic trends that might occur outside the typical seasonal periodicity. For each region, we used the Morlet wavelet decomposition to extract the major seasonal component (period: 40‐80 weeks) for weekly influenza cases and the weekly mean absolute humidity, temperature and precipitation.</p></sec><sec id=\"irv12744-sec-0010\"><label>2.4</label><title>Role of funding source</title><p>The US CDC funded the study, and participated in the study design, data analysis, data interpretation and preparation of the manuscript. All authors had full access to study data, and all authors had final responsibility for the decision to submit for publication.</p></sec></sec><sec sec-type=\"results\" id=\"irv12744-sec-0011\"><label>3</label><title>RESULTS</title><p>Of 78 822 febrile patients presenting to SEDSS sites during 2012‐2018, 28 280 were offered participation and 23 124 (82%) participants enrolled in SEDSS. Among participants, 52% were female, and 60% were aged ≤17 years. Half of the participants were from the Ponce municipality, and most (72%) presented within 3 days of illness onset. While 82% were sent home after evaluation and treatment, 16% had hospital admittance. From 2012‐2013, 20% of the total enrollees were recruited, 20% in 2014‐2015, 43% in 2016‐2017 and 17% in 2018. Participants’ clinical and demographic characteristics did not change between years (Table <xref rid=\"irv12744-tbl-0001\" ref-type=\"table\">1</xref>). Overall, 14% of participants were positive for influenza; 9% for influenza A and 5% for influenza B viruses. Although there were no differences by gender, the percentage of participants with positive results for influenza A or B viruses differed by age (<italic>P</italic> < .001) and days after symptom onset (<italic>P</italic> < .001; Table <xref rid=\"irv12744-tbl-0002\" ref-type=\"table\">2</xref>).</p><table-wrap id=\"irv12744-tbl-0001\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Characteristics of participants by year of recruitment, Sentinel Enhanced Dengue Surveillance System, Ponce, Puerto Rico, 2012‐2018</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"bottom\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"2\" valign=\"bottom\" colspan=\"1\">Participant Characteristics</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Overall</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">2012‐2013</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">\n<italic>2014‐2015</italic>\n</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">\n<italic>2016‐2017</italic>\n</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">\n<italic>2018</italic>\n</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">N</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">%</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">N</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">%</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">N</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">%</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">N</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">%</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">N</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">%</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"11\" rowspan=\"1\">Gender</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Female</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">11 924</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">51.6</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2282</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">49.6</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2383</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">5264</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">53.5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1995</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">51.0</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Male</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">11 200</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">48.4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2320</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50.4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2388</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50.1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4578</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">46.5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1914</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">49.0</td></tr><tr><td align=\"left\" colspan=\"11\" rowspan=\"1\">Age</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\"><1 y</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1997</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.6</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">354</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.7</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">535</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.2</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">790</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">318</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.1</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">1‐5 y</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6395</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27.7</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1128</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">24.5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1582</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.2</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2483</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25.2</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1202</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">30.8</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">6‐17 y</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">5430</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23.5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1350</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29.3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1141</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23.9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2000</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20.3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">939</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">24.0</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">18‐64 y</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">7874</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34.1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1513</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32.9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1234</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25.9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3923</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39.9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1204</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">30.8</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">65 + y</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1428</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.2</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">257</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.6</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">279</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">646</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.6</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">246</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.3</td></tr><tr><td align=\"left\" colspan=\"11\" rowspan=\"1\">Municipality of residence</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Ponce</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">11 747</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">50.8</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2021</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">43.9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2075</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">43.5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">5608</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">57.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2043</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">52.3</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Juana Diaz</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2215</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.6</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">407</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.8</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">413</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.7</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1020</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10.4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">375</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.6</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Peñuelas</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2076</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">161</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">219</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.6</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1150</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.7</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">546</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.0</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Guayama</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1289</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.6</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">590</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.8</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">676</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.2</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.2</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.2</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Villalba</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1088</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.7</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">236</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">211</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">484</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">157</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.0</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Other municipality</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4709</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20.4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1187</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25.8</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1177</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">24.7</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1565</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15.9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">780</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20.0</td></tr><tr><td align=\"left\" colspan=\"11\" rowspan=\"1\">Days post‐onset of symptoms</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\"><3 d</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">16 632</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">71.9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2911</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">63.3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3784</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">79.3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6987</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">71.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2950</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">75.5</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">3‐7 d</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6416</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27.8</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1655</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">36.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">986</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20.7</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2852</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">923</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23.6</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">8 + d</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">76</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.8</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.9</td></tr><tr><td align=\"left\" colspan=\"11\" rowspan=\"1\">Disposition outcome</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Sent home</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">19 051</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">82.4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3192</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">69.4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3724</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">78.1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">8665</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">88.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3470</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">88.8</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Admitted</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3707</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1378</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29.9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1024</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21.5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">964</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.8</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">341</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.7</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Transferred</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">171</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.7</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">109</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">34</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.9</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Died</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Other</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">167</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.7</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">94</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">62</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">23 124</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4602</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4771</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9842</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3909</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100</td></tr></tbody></table><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><table-wrap id=\"irv12744-tbl-0002\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Characteristics of participants with Influenza A and Influenza B, Sentinel Enhanced Dengue Surveillance System, Ponce, Puerto Rico, 2012‐2018</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"bottom\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"2\" valign=\"bottom\" colspan=\"1\">Participant Characteristics</th><th align=\"left\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Total</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">All Influenza</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Influenza A</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Influenza B</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">N</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">%</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">%</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">%</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"8\" rowspan=\"1\">Gender</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Female</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">11 924</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1619</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1051</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">568</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.8</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Male</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">11 200</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1595</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">985</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">610</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.5</td></tr><tr><td align=\"left\" colspan=\"8\" rowspan=\"1\">Age</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\"><1 y</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1997</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">128</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">91</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.9</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">1‐5 y</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6395</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">680</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">468</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">212</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.3</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">6‐17 y</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">5430</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1031</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">19.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">517</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">514</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.5</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">18‐64 y</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">7874</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1194</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">846</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">348</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.4</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">65 + y</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1428</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">181</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">114</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">67</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.7</td></tr><tr><td align=\"left\" colspan=\"8\" rowspan=\"1\">Days post‐onset of symptoms</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\"><3 d</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">16 632</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2427</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1635</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">792</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.8</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">3‐7 d</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6416</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">778</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">394</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">384</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.0</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">8 + d</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">76</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.6</td></tr><tr><td align=\"left\" colspan=\"8\" rowspan=\"1\">Disposition outcome</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Sent home</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">19 051</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2864</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1800</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1064</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.6</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Admitted</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3707</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">320</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">217</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">103</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.8</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Transferred</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">171</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.8</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Died</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">.</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">.</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Other</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">167</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">23 124</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3214</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2036</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1178</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.1</td></tr></tbody></table><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>We compared the time series and peak timing of overall influenza and type‐specific patterns in Puerto Rico to the HHS regions. In Puerto Rico (SEDSS), influenza peaks varied substantially between 2013 and 2018 (Figure <xref rid=\"irv12744-fig-0001\" ref-type=\"fig\">1A</xref>). Specifically, influenza A virus had an atypical summer peak in 2013, but had subsequent peaks in the winter months (December, January and February) with similar timing to those in the HHS regions (Figure <xref rid=\"irv12744-fig-0001\" ref-type=\"fig\">1B</xref>). In the HHS regions, influenza peaked consistently during the winter months (December and January) throughout the study period. For influenza B virus, long‐term patterns in SEDSS were less clear with some peaks in the summer (2016) and others in the winter months (2017‐2018). Only 2017‐2018 appeared to be closely aligned with HHS regions, where influenza B virus peaked in the winter and early spring (January to March; Figure <xref rid=\"irv12744-fig-0001\" ref-type=\"fig\">1C</xref>). The seasonal components from the time series confirmed these trends (Figure <xref rid=\"irv12744-sup-0001\" ref-type=\"supplementary-material\">S2</xref>A‐C).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"irv12744-fig-0001\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>A, Number of influenza cases overall (1A‐C) and by type in the Sentinel Enhanced Dengue Surveillance System (SEDSS), Southern Puerto Rico (PR) and US HHS regions (US WHO/NRVESS), (B). Coherence (1D‐F) comparing the similarity between the influenza patterns in the US HHS regions with SEDSS (PR), and (C). Phase differences (1G‐I) comparing the timing of SEDSS (PR) influenza seasonal epidemics to those in each of the US HHS regions. Seasonal decomposition was defined as a 40‐ to 56‐wk period. Colour lines refer to the comparisons of the weekly seasonal time series of SEDSS (PR) to each US HHS region (PR‐HHS). For coherence, the dashed line at 1 refers to identical seasonal patterns of the US HHS region and the SEDSS (PR), whereas 0 refers to no association of the two time series. For phase differences, negative values indicate the seasonal pattern of SEDSS (PR) influenza cases is behind US HHS regions, and positive values indicate the seasonal pattern of SEDSS (PR) cases is ahead of US HHS regions</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"IRV-14-515-g001\"/></fig><p>We performed coherence and phase analyses to estimate the relatedness and relative timing (in weeks) of the influenza time series in SEDSS, Puerto Rico to those in each of the US HHS regions. Comparing SEDSS to the HHS regions for overall influenza, there was low coherence during 2013‐2015, and high coherence in 2016 (Figure <xref rid=\"irv12744-fig-0001\" ref-type=\"fig\">1D</xref>). Similarly, influenza A virus in SEDSS had high coherence with influenza A virus in each HHS time series in later years (Figure <xref rid=\"irv12744-fig-0001\" ref-type=\"fig\">1E</xref>). Influenza B virus coherence differed from A. Influenza B virus coherence was higher in the early years, decreasing to roughly 0.40‐0.45 in 2015‐2016, then increased in 2016‐2017 (Figure <xref rid=\"irv12744-fig-0001\" ref-type=\"fig\">1F</xref>). The average annual coherence of influenza was highest when comparing US HHS Region 2 (0.66) and US HHS Region 9 (0.67). It should be noted that US HHS region 2 includes Puerto Rico (along with the US Virgin Island, New York, and New Jersey). The southern states are represented in US HHS region 4 (Alabama, Florida, Georgia, Kentucky, Mississippi, North Carolina, South Carolina, and Tennessee), but this region had lower coherence with Puerto Rico (0.52), suggesting they do not have more similar influenza patterns (Table <xref rid=\"irv12744-sup-0002\" ref-type=\"supplementary-material\">S1</xref>). Similar coherence patterns are also observed when looking at influenza A and B.</p><p>From the phase analyses, the seasonal components of the overall time series showed a synchronization between SEDSS and the HHS regions across the study period (Figure <xref rid=\"irv12744-fig-0001\" ref-type=\"fig\">1G‐I</xref>). From 2013 to 2015, the SEDSS seasonal influenza patterns lagged behind the HHS regions by as much as 12‐18 weeks. However, this pattern inverted in 2016, after which the seasonal patterns in SEDSS led the patterns in the HHS regions by 0‐5 weeks. Influenza A and B viruses showed distinct patterns when comparing SEDSS to HHS regions. The sporadic and off‐season dynamics of influenza A virus in SEDSS in 2013‐2014 led to a high degree of asynchrony with the HHS regions. However, those dynamics quickly changed beginning in 2014, with seasonal influenza A having similar timing with HHS regions in early 2016 and onward. Seasonal influenza B virus patterns in Puerto Rico lagged behind the HHS regions by 2‐10 weeks until the 2016‐2017 season at which point they preceded HHS regions by 0‐4 weeks, showing better synchrony with the HHS regions.</p><p>To investigate possible climatic drivers of influenza A and B epidemics patterns within each geographic region, we assessed the coherence and phase differences between location‐specific climate variables and type‐specific influenza incidence (Figure <xref rid=\"irv12744-fig-0002\" ref-type=\"fig\">2A‐I</xref>). In terms of the overall time series climatic trends, Puerto Rico was consistently warmer and more humid over the time series compared with weekly average absolute humidity and temperature in HHS regions (Figure <xref rid=\"irv12744-fig-0002\" ref-type=\"fig\">2A‐B</xref>). In contrast, precipitation varied greatly across all regions. Some HHS regions had peak rainfall in distinctly different seasons compared with Puerto Rico (Figure <xref rid=\"irv12744-fig-0002\" ref-type=\"fig\">2C</xref>). Over the 40‐ to 80‐week component of the time series, we observed high coherence (range: 0.61‐0.99) between absolute humidity and influenza A and B viruses within the HHS regions (Figure <xref rid=\"irv12744-fig-0002\" ref-type=\"fig\">2D, G</xref>). However, this pattern was markedly different in Puerto Rico. Influenza A virus coherence was much lower in the early years, but by the 2017‐2018 season, coherence increased to similar levels as the HHS regions (range: 0.18‐0.94). Meanwhile, influenza B virus coherence was higher early in the time series, decreasing in 2015‐2016, and increasing in late 2016, but never having the higher coherence estimates observed in the HHS regions (range: 0.27‐0.72). Similar patterns were observed for coherence between mean weekly temperature and influenza A and B viruses (Figure <xref rid=\"irv12744-fig-0002\" ref-type=\"fig\">2E, H</xref>). Coherence between precipitation and influenza varied substantially for the HHS regions and Puerto Rico, but did not have consistently high coherence for the SEDSS Puerto Rico data (Figure <xref rid=\"irv12744-fig-0002\" ref-type=\"fig\">2F‐I</xref>). The phase difference analyses showed a high degree of asynchrony of influenza patterns in Puerto Rico and patterns for climate variables (Figure <xref rid=\"irv12744-sup-0001\" ref-type=\"supplementary-material\">S3</xref>C‐D). The seasonal precipitation patterns lagged behind influenza A virus trends in SEDSS by 20 weeks, while absolute humidity and temperature patterns led by about 25‐30 weeks (Figure <xref rid=\"irv12744-sup-0001\" ref-type=\"supplementary-material\">S3</xref>C). For influenza B virus, precipitation, absolute humidity and temperature seasonal components were ahead of the influenza B patterns by 15‐30 weeks (Figure <xref rid=\"irv12744-sup-0001\" ref-type=\"supplementary-material\">S3</xref>D). The seasonal wavelet decomposition for each HHS region showed that influenza peaked when temperature and absolute humidity were low (Figure <xref rid=\"irv12744-sup-0001\" ref-type=\"supplementary-material\">S4</xref>A‐E and K‐O for influenza A and Figure <xref rid=\"irv12744-sup-0001\" ref-type=\"supplementary-material\">S5</xref>A‐E, K‐O for influenza B). For each HHS region, influenza patterns consistently lagged approximately 30 weeks for influenza A and 20 weeks influenza B compared with temperature and absolute humidity patterns (Figure <xref rid=\"irv12744-sup-0001\" ref-type=\"supplementary-material\">S4</xref>F‐J, P‐T, <xref rid=\"irv12744-sup-0001\" ref-type=\"supplementary-material\">S5</xref>F‐J, P‐T).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"irv12744-fig-0002\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Trends in absolute humidity (A), temperature (B) and precipitation (C) in Puerto Rico and US HHS regions (US WHO/NRVESS) and annual coherence for influenza A (D, E, F) and B (G, H, I). Seasonal decomposition was defined as a 40‐ to 80‐wk period. Colour lines refer to the comparisons of the weekly seasonal time series of SEDSS, Puerto Rico to the weekly seasonal pattern for each climate variable. For coherence, the dashed line at 1 refers to identical seasonal patterns of SEDSS and the climate variables, whereas 0 refers to no association of the two time series</p></caption><graphic id=\"nlm-graphic-3\" xlink:href=\"IRV-14-515-g002\"/></fig></sec><sec sec-type=\"discussion\" id=\"irv12744-sec-0012\"><label>4</label><title>DISCUSSION</title><p>This is the first study comparing seasonal influenza trends in Puerto Rico to trends in the US HHS regions, and we found influenza trends between HHS regions and SEDSS, Puerto Rico varied over time but increased in synchrony in recent years. We observed distinct synchronization patterns by influenza virus types, showing different dynamics for each type over the time series. For influenza A, the predominant type in Puerto Rico and the HHS regions, the seasonal incidence pattern in Puerto Rico greatly differed from the HHS regions in 2012‐2014, but synchronized beginning in 2015‐2016. Influenza B cases in Puerto Rico had more consistent seasonality and also exhibited increased synchrony starting in the 2016‐2017 season. Relative to influenza in the HHS regions, both virus types in Puerto Rico switched from irregular, asynchronous seasonal epidemics, generally following those in the rest of the United States, to synchronous epidemics, preceding the seasonal epidemics in the continental United States.</p><p>One explanation of the seasonal differences between Puerto Rico and the continental United States may be the geographic differences in climate. Consistent with other studies, we found that influenza incidence in HHS regions had high coherence with humidity and temperature. In contrast, Puerto Rico lacked the high coherence with climate observed among the HHS regions, and only had higher coherence during the most recent influenza seasons. The absence of high coherence between influenza peaks and temperature and humidity in Puerto Rico may be because higher year‐round humidity and temperatures have less variation than in temperate regions. In vitro and animal model studies of influenza transmission showed low absolute humidity favours virus survival and aerosolized transmission<xref rid=\"irv12744-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref>, <xref rid=\"irv12744-bib-0020\" ref-type=\"ref\">\n<sup>20</sup>\n</xref> and epidemiological studies showed lower humidity is associated with the onset of influenza epidemics in the United States.<xref rid=\"irv12744-bib-0021\" ref-type=\"ref\">\n<sup>21</sup>\n</xref> Increased proximity between susceptible and infected hosts associated with cold weather is also frequently suggested as an important driver of influenza seasonality.<xref rid=\"irv12744-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> Seasonal variation in host immunity may also explain the seasonality of influenza in temperate countries. Studies also suggest an association between increased influenza activity and the rainy season in several tropical populations,<xref rid=\"irv12744-bib-0022\" ref-type=\"ref\">\n<sup>22</sup>\n</xref> but seasonal precipitation also had low coherence with influenza activity in Puerto Rico. While climatic conditions may likely affect influenza transmission in some locations, humidity, temperature and precipitation are not the main drivers of influenza in Puerto Rico. Other climatic factors not examined here, or other socio‐demographic factors may be more important drivers, though this needs further examination.</p><p>Perhaps, the most striking result of our study was the increased synchronization with HHS regions during recent years for both influenza virus types. As described above, the climate analysis suggests this synchrony was not driven by climate alone and likely influenced by other factors<xref rid=\"irv12744-bib-0023\" ref-type=\"ref\">\n<sup>23</sup>\n</xref> including travel and global and US influenza viral introductions into Puerto Rico. For instance, a previous study suggested that human mobility has synchronized epidemics among highly connected populations in the United States, with influenza viruses spreading from populous to less populous locations.<xref rid=\"irv12744-bib-0017\" ref-type=\"ref\">\n<sup>17</sup>\n</xref> It is possible that contact rates at these larger scales may be particularly important to the seasonal patterns of influenza in smaller populations, or for locations that are characterized by less climatic variability, like Puerto Rico. In such cases, seasonal variability in the volume of infected individuals entering a population (like migration from the United States to Puerto Rico during the winter) may exceed any climate‐mediated seasonality.<xref rid=\"irv12744-bib-0024\" ref-type=\"ref\">\n<sup>24</sup>\n</xref> Analysis of air passenger data and seasonal patterns may help investigate this hypothesis. The time lag of several weeks relative to HHS regions suggests that recent epidemics in Puerto Rico may lead rather than follow those in HHS regions; however, the different geographical scale may complicate this relationship. The SEDSS catchment area is just one region of Puerto Rico, and much smaller than any HHS region. Recent work found that the shape of epidemics varies across different geographies, such that even introductions from another location may result in epidemics with earlier sharper peaks than the source location.<xref rid=\"irv12744-bib-0025\" ref-type=\"ref\">\n<sup>25</sup>\n</xref> Other studies found highly synchronized epidemics in Australia coincided with years of emergent antigenically distinct subtypes, and synchrony across the continent was a function of global introductions of influenza viruses paired with domestic connectivity.<xref rid=\"irv12744-bib-0026\" ref-type=\"ref\">\n<sup>26</sup>\n</xref> Our study found that coherence was higher for certain HHS regions compared with others. The high coherence associated with US HHS Region 9 may be due to the fact that Region 9 is comprised of southwestern states (Arizona, California, Nevada), but also Pacific Island states (Hawaii) and territories (American Samoa, Commonwealth of the Northern Mariana Islands, Federated States of Micronesia, Guam, Marshall Islands, and Republic of Palau), and these Pacific Island areas may have similar patterns to Puerto Rico. Similar coherence patterns are also observed when looking at influenza A and B. This may be because even though southern states have warmer and more humid temperatures, they still experience seasonal fluctuations in temperature and humidity that are closer to temperate states compared with tropical locations such as Puerto Rico, and other tropical areas, such as Hawaii or American Samoa.</p><p>The annual coherence of influenza B increased from 2015 to the end of 2018. This increase was observed across all comparisons of US HHS Regions and SEDSS in Puerto Rico. Though we did not have data on influenza B lineages, there is evidence from other genomic analyses that the influenza B Victoria lineage had experienced high genetic reassortment in 2016,<xref rid=\"irv12744-bib-0027\" ref-type=\"ref\">\n<sup>27</sup>\n</xref> which may in part explain the increase in coherence and phase of the influenza B strains across the region. Increases in B Victoria (as opposed to B Yamagata) can also be seen over time in CDC’s virological surveillance from the 2016 to 2017 seasons onward.<xref rid=\"irv12744-bib-0028\" ref-type=\"ref\">\n<sup>28</sup>\n</xref>\n</p><p>This investigation leveraged a large study with consistent recruiting and diagnostic testing over multiple influenza seasons, yet still is subject to several limitations. First, the patient population was limited to two sentinel hospitals and one outpatient clinic in southern Puerto Rico. Because healthcare‐seeking behaviours may differ between populations served by different hospitals, the observed percentages of patients with a respiratory virus may not be representative of all Puerto Rican health facilities. Second, the 6.5 seasons captured through SEDSS are only a snapshot of a long history of influenza in Puerto Rico. For example, it is unclear if the seasons without a regular peak seen, such as those in the earlier years of the study, were common prior to 2012 or if earlier epidemics were more consistent with HHS regions. It is possible that there are cycles working on larger time scales that contribute to sporadic asynchrony or sporadic synchrony. We also could not account for vaccine effects. While Puerto Rico generally has lower influenza vaccination rates compared with US states,<xref rid=\"irv12744-bib-0029\" ref-type=\"ref\">\n<sup>29</sup>\n</xref> we did not capture vaccination information as part of the surveillance systems and therefore, could not assess its impact the local dynamics. Data on influenza A virus subtypes or B virus lineages were not available for this analysis. We did not examine travel between Puerto Rico and the mainland United States or other locations, but are components that may contribute substantially to influenza dynamics in Puerto Rico. Future studies could include phylogenetic analysis to confirm the timing of influenza strains circulating in the continental United States compared with Puerto Rico. HHS region two includes data from Puerto Rico, which may result in over‐estimating associations with that region; however, the patterns observed for this region were similar to others. We did not have data on influenza trends for all of Puerto Rico and were unable determine if trends in Ponce are similar to the island wide trends. Finally, few participants were 65 years of age and older, and possibly under‐estimating the number of infections in Puerto Rico.<xref rid=\"irv12744-bib-0029\" ref-type=\"ref\">\n<sup>29</sup>\n</xref>\n</p><p>Our study highlights the poorly understood complexity of influenza in tropical regions where climate is not a strong driver of seasonal dynamics. It also broadens our understanding of the relationship between influenza trends in the continental United States and Puerto Rico, and the role of climate. Currently in the United States, influenza vaccines are recommended to be received before the end of October. These recommendations would not have been ideal for Puerto Rico in the early study years. Influenza trends in Puerto Rico will need to be continuously monitored to better understand if these dynamics change again, and to ensure that interventions, like vaccinations, are implemented appropriately for influenza in Puerto Rico.</p></sec><sec sec-type=\"COI-statement\" id=\"irv12744-sec-0014\"><title>CONFLICT OF INTEREST</title><p>The authors report no conflict of interest.</p></sec><sec id=\"irv12744-sec-0015\"><title>AUTHOR CONTRIBUTIONS</title><p>GPB designed the study, contributed to the analyses, participated in the interpretation of results and wrote the first draft of the paper. TQ conducted the wavelet decomposition and phase difference analyses and co‐wrote the paper. LA conducted the descriptive analyses and contributed to the final draft of the paper. SO, LB and ML helped interpret results and edited the final draft of the paper. JM supervised the laboratory testing and reviewed the final draft of the paper. LIA is the principal investigator for the enhanced surveillance system, helped interpret study results and reviewed the final draft of the paper. MJ oversaw the analyses strategy, helped interpret study results and edited the final draft of the paper. All authors revised the draft paper critically for important intellectual content, provided final approval of the version to be published and are accountable for all aspects of the work with regards to accuracy and integrity.</p></sec><sec id=\"irv12744-sec-0016\"><title>DISCLAIMER</title><p>The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.</p></sec><sec sec-type=\"supplementary-material\"><title>Supporting information</title><supplementary-material content-type=\"local-data\" id=\"irv12744-sup-0001\"><caption><p>FigS1‐S5</p></caption><media xlink:href=\"IRV-14-515-s001.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"irv12744-sup-0002\"><caption><p>Table S1</p></caption><media xlink:href=\"IRV-14-515-s002.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"irv12744-sec-0013\"><title>ACKNOWLEDGEMENTS</title><p>We acknowledge funding provided by the Centers for Disease Control and Prevention. We would like to thank the contribution of all study participants. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"iso-abbrev\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"doi\">10.1111/(ISSN)1750-2659</journal-id><journal-id journal-id-type=\"publisher-id\">IRV</journal-id><journal-title-group><journal-title>Influenza and Other Respiratory Viruses</journal-title></journal-title-group><issn pub-type=\"ppub\">1750-2640</issn><issn pub-type=\"epub\">1750-2659</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32410402</article-id><article-id pub-id-type=\"pmc\">PMC7431641</article-id><article-id pub-id-type=\"doi\">10.1111/irv.12752</article-id><article-id pub-id-type=\"publisher-id\">IRV12752</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Original Article</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Original Articles</subject></subj-group></article-categories><title-group><article-title>Surveillance of medically‐attended influenza in elderly patients from Romania—data from three consecutive influenza seasons (2015/16, 2016/17, and 2017/18)</article-title><alt-title alt-title-type=\"left-running-head\">PIȚIGOI et al.</alt-title></title-group><contrib-group><contrib id=\"irv12752-cr-0001\" contrib-type=\"author\"><name><surname>Pițigoi</surname><given-names>Daniela</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0003-4118-8268</contrib-id><xref ref-type=\"aff\" rid=\"irv12752-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12752-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12752-cr-0002\" contrib-type=\"author\"><name><surname>Streinu‐Cercel</surname><given-names>Anca</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0003-2794-6720</contrib-id><xref ref-type=\"aff\" rid=\"irv12752-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12752-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12752-cr-0003\" contrib-type=\"author\"><name><surname>Ivanciuc</surname><given-names>Alina Elena</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-2947-3800</contrib-id><xref ref-type=\"aff\" rid=\"irv12752-aff-0003\">\n<sup>3</sup>\n</xref></contrib><contrib id=\"irv12752-cr-0004\" contrib-type=\"author\"><name><surname>Lazãr</surname><given-names>Mihaela</given-names></name><xref ref-type=\"aff\" rid=\"irv12752-aff-0003\">\n<sup>3</sup>\n</xref></contrib><contrib id=\"irv12752-cr-0005\" contrib-type=\"author\"><name><surname>Cherciu</surname><given-names>Carmen Maria</given-names></name><xref ref-type=\"aff\" rid=\"irv12752-aff-0003\">\n<sup>3</sup>\n</xref></contrib><contrib id=\"irv12752-cr-0006\" contrib-type=\"author\"><name><surname>Mihai</surname><given-names>Maria Elena</given-names></name><xref ref-type=\"aff\" rid=\"irv12752-aff-0003\">\n<sup>3</sup>\n</xref></contrib><contrib id=\"irv12752-cr-0007\" contrib-type=\"author\"><name><surname>Nițescu</surname><given-names>Maria</given-names></name><xref ref-type=\"aff\" rid=\"irv12752-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12752-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12752-cr-0008\" contrib-type=\"author\"><name><surname>Aramă</surname><given-names>Victoria</given-names></name><xref ref-type=\"aff\" rid=\"irv12752-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12752-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12752-cr-0009\" contrib-type=\"author\"><name><surname>Crăciun</surname><given-names>Maria Dorina</given-names></name><xref ref-type=\"aff\" rid=\"irv12752-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12752-aff-0004\">\n<sup>4</sup>\n</xref></contrib><contrib id=\"irv12752-cr-0010\" contrib-type=\"author\"><name><surname>Streinu‐Cercel</surname><given-names>Adrian</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-6382-5067</contrib-id><xref ref-type=\"aff\" rid=\"irv12752-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12752-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12752-cr-0011\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Săndulescu</surname><given-names>Oana</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-2586-4070</contrib-id><xref ref-type=\"aff\" rid=\"irv12752-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12752-aff-0002\">\n<sup>2</sup>\n</xref><address><email>oana.sandulescu@umfcd.ro</email></address></contrib></contrib-group><aff id=\"irv12752-aff-0001\">\n<label><sup>1</sup></label>\n<institution>Carol Davila University of Medicine and Pharmacy</institution>\n<city>Bucharest</city>\n<country country=\"RO\">Romania</country>\n</aff><aff id=\"irv12752-aff-0002\">\n<label><sup>2</sup></label>\n<institution>National Institute for Infectious Diseases “Prof. Dr. Matei Balș”</institution>\n<city>Bucharest</city>\n<country country=\"RO\">Romania</country>\n</aff><aff id=\"irv12752-aff-0003\">\n<label><sup>3</sup></label>\n<institution>“Cantacuzino” National Medico‐Military Institute for Research and Development</institution>\n<city>Bucharest</city>\n<country country=\"RO\">Romania</country>\n</aff><aff id=\"irv12752-aff-0004\">\n<label><sup>4</sup></label>\n<institution>Grigore Alexandrescu Clinical Children's Emergency Hospital</institution>\n<city>Bucharest</city>\n<country country=\"RO\">Romania</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label><bold>Correspondence</bold><break/>\nOana Săndulescu, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania, National Institute for Infectious Diseases “Prof. Dr. Matei Balș”, No. 1 Dr. Calistrat Grozovici street, Bucharest 021105, Romania.<break/>\nEmail: <email>oana.sandulescu@umfcd.ro</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>5</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>9</month><year>2020</year></pub-date><volume>14</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1111/irv.v14.5</issue-id><fpage>530</fpage><lpage>540</lpage><history><date date-type=\"received\"><day>09</day><month>5</month><year>2019</year></date><date date-type=\"rev-recd\"><day>14</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>16</day><month>4</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 John Wiley & Sons Ltd <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Influenza and Other Respiratory Viruses</italic> Published by John Wiley & Sons Ltd.</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:IRV-14-530.pdf\"/><abstract id=\"irv12752-abs-0001\"><title>Abstract</title><sec id=\"irv12752-sec-0001\"><title>Background</title><p>Influenza is an acute infection affecting all age groups; however, elderly patients are at an increased risk. We aim to describe the clinical characteristics and the circulation of influenza virus types in elderly patients admitted for severe acute respiratory infection (SARI) to a tertiary care hospital in Bucharest, Romania, part of the I‐MOVE+ hospital network.</p></sec><sec id=\"irv12752-sec-0002\"><title>Methods</title><p>We conducted an active surveillance study at the National Institute for Infectious Diseases “Prof. Dr Matei Balș,” Bucharest, Romania, during three consecutive influenza seasons: 2015/16, 2016/17, and 2017/18. All patients aged 65 and older admitted to our hospital for SARI were tested for influenza by PCR.</p></sec><sec id=\"irv12752-sec-0003\"><title>Results</title><p>A total of 349 eligible patients were tested during the study period, and 149 (42.7%) were confirmed with influenza. Most patients, 321 (92.5%) presented at least one underlying condition at the time of hospital admission, the most frequent being cardiovascular disease, 270 (78.3%). The main influenza viral subtype circulating in 2015/16 was A(H1N1)pdm09, followed by A(H3N2) in 2016/17 and B influenza in 2017/18. Case fatality was highest in the 2015/16 season (3.7%), 0% in 2016/17, and 1.0% in 2017/18. Vaccination coverage in elderly patients with SARI from our study population was 22 (6.3%) over the three seasons.</p></sec><sec id=\"irv12752-sec-0004\"><title>Conclusions</title><p>Our study has highlighted a high burden of comorbidities in elderly patients presenting with SARI during winter season in Romania. The influenza vaccine coverage rate needs to be substantially increased in the elderly population, through targeted interventions.</p></sec></abstract><kwd-group><kwd id=\"irv12752-kwd-0001\">case fatality</kwd><kwd id=\"irv12752-kwd-0002\">comorbidities</kwd><kwd id=\"irv12752-kwd-0003\">elderly</kwd><kwd id=\"irv12752-kwd-0004\">influenza</kwd><kwd id=\"irv12752-kwd-0005\">subtype</kwd></kwd-group><funding-group><award-group id=\"funding-0001\"><funding-source>Horizon 2020 Framework Programme</funding-source><award-id>6334446</award-id></award-group></funding-group><counts><fig-count count=\"2\"/><table-count count=\"3\"/><page-count count=\"11\"/><word-count count=\"8039\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>September 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.6 mode:remove_FC converted:18.08.2020</meta-value></custom-meta></custom-meta-group></article-meta><notes><p content-type=\"self-citation\">\n<mixed-citation publication-type=\"journal\" id=\"irv12752-cit-1001\">\n<string-name>\n<surname>Pițigoi</surname>\n<given-names>D</given-names>\n</string-name>, <string-name>\n<surname>Streinu‐Cercel</surname>\n<given-names>A</given-names>\n</string-name>, <string-name>\n<surname>Ivanciuc</surname>\n<given-names>AE</given-names>\n</string-name>, et al. <article-title>Surveillance of medically‐attended influenza in elderly patients from Romania—data from three consecutive influenza seasons (2015/16, 2016/17, and 2017/18)</article-title>. <source xml:lang=\"en\">Influenza Other Respi Viruses</source>. <year>2020</year>;<volume>14</volume>:<fpage>530</fpage>–<lpage>540</lpage>. <pub-id pub-id-type=\"doi\">10.1111/irv.12752</pub-id>\n</mixed-citation>\n</p><fn-group id=\"irv12752-ntgp-0001\"><fn fn-type=\"funding\" id=\"irv12752-note-0001\"><p>\n<bold>Funding information</bold>\n</p><p>This work was supported by the project “I‐MOVE+ ‐ Hospital‐based test negative case control studies to measure seasonal influenza vaccine effectiveness against influenza laboratory confirmed SARI hospitalization among the elderly across the European Union and European Economic Area Member States,” European Union's HORIZON 2020 Research and Innovation Programme Grant Agreement No 6334446.</p></fn></fn-group></notes></front><body id=\"irv12752-body-0001\"><sec sec-type=\"background\" id=\"irv12752-sec-0005\"><label>1</label><title>BACKGROUND</title><p>Influenza is an acute infection affecting all age groups; however, elderly patients are at an increased risk, particularly due to a clustering of comorbidities which puts them at risk of severe influenza, complicated influenza, or influenza‐related decompensation of underlying conditions.<xref rid=\"irv12752-bib-0001\" ref-type=\"ref\">\n<sup>1</sup>\n</xref>\n</p><p>On a global level, in 2017, influenza has been estimated to be responsible for 54 481 000 episodes of medically‐diagnosed lower respiratory tract infections leading to 9 459 000 hospitalizations, and specifically in Romania, with a population in 2017 of 19.6 million people, 174 000 episodes of lower respiratory tract infections with 65 000 hospitalizations, as calculated by the Global Burden of Disease Study 2017.<xref rid=\"irv12752-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref> Continued surveillance of influenza is warranted in each country, in order to better inform public health policies and to fill the existing information gaps, particularly for specific influenza risk groups.</p><p>In Romania, influenza surveillance is performed at national level by two surveillance systems, one for severe acute respiratory infections (SARI: testing one patient hospitalized with SARI per week from 20 hospitals throughout six counties, during the influenza surveillance season), and one for influenza‐like illness (ILI: testing all patients with ILI attending 192 sentinel general practitioners from 16 counties every Tuesday).<xref rid=\"irv12752-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref>\n</p><p>The National Institute for Infectious Diseases “Prof. Dr Matei Balș,” a tertiary care hospital in Bucharest, Romania, was part of the I‐MOVE+ hospital network (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.i-moveplus.eu/\">http://www.i‐moveplus.eu/</ext-link>) from 2015 to 2018 and implemented a protocol for systematic screening for influenza in elderly patients admitted to the hospital for SARI.<xref rid=\"irv12752-bib-0004\" ref-type=\"ref\">\n<sup>4</sup>\n</xref>\n</p><p>Here, we aim to describe the clinical features of the elderly patients admitted with SARI in our hospital and the circulation of influenza virus types in these patients during three consecutive influenza seasons in Bucharest, Romania, in order to characterize the epidemiology of influenza in this particular patient population, at high risk for influenza‐related morbidity.</p></sec><sec sec-type=\"methods\" id=\"irv12752-sec-0006\"><label>2</label><title>METHODS</title><p>An active epidemiologic surveillance study was implemented at the National Institute for Infectious Diseases “Prof. Dr Matei Balș,” Bucharest, Romania, during three consecutive influenza seasons: 2015/16, 2016/17, and 2017/18. The study consisted of systematic daily screening of all consecutive admissions in patients aged 65 and older admitted to our hospital with an acute (onset <7 days) illness meeting the following SARI case definition: one or more general signs or symptoms, defined as: fever/feverishness, malaise, headache, myalgia, and altered clinical state (asthenia, anorexia, confusion, or weight loss) associated with one or more respiratory signs or symptoms defined as: cough, odynophagia, and dyspnea.<xref rid=\"irv12752-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref>\n</p><p>Patients were excluded from the study if they met any of the following exclusion criteria: had a contraindication for influenza vaccine, had SARI onset ≥48 hours after hospital admission, were unwilling to participate or unable to communicate and give consent (either by the patient or by her/his legal representative), were institutionalized, had a respiratory specimen taken ≥8 days after SARI onset, and had tested positive for any influenza virus in the current season before the onset of symptoms leading to the current hospitalization. No information was collected on the number of exclusions overall and by each criterion, and therefore, this information is not available and will not be reported in Results.</p><p>For each consenting patient, a standardized medical questionnaire was filled out by study investigators through review of medical (hospital or general practitioner) records and patient/relative interview, collecting demographic variables, a complete medical history, influenza vaccination status in the respective season, and data related to the current SARI episode's onset, characteristics and outcome, as previously described.<xref rid=\"irv12752-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref> Two respiratory specimens (a nasal swab and a pharyngeal swab) were collected according to the methodology for national surveillance of influenza, ARI, and SARI,<xref rid=\"irv12752-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref> placed at 4°C immediately, and transported within 24 hours at 2‐8°C to the National Reference Laboratory (NRL) at the “Cantacuzino” National Medico‐Military Institute for Research and Development, Bucharest, Romania.</p><p>At the NRL, detection, typing, subtyping, and genetic lineages differentiation of influenza viruses were done using real‐time reverse transcription‐polymerase chain reaction (RT‐PCR). All samples were tested for the presence of influenza A and B viruses. For influenza A viruses, a second real‐time RT‐PCR analysis was performed for determination of A/H1 pdm09 or A/H3 subtype. The sequences and protocols for type A, B and subtype A/H3 primers and probes were obtained from Erasmus Medical Center Rotterdam. For detection of subtype A/H1pdm09, we used the protocols from CDC Atlanta in the season 2015/2016 and starting with the 2016/2017 season the WHO protocol (Molecular diagnosis influenza virus humans update 2014).<xref rid=\"irv12752-bib-0007\" ref-type=\"ref\">\n<sup>7</sup>\n</xref> For a proportion of influenza B viruses, a second real‐time RT‐PCR analysis (single nucleotide polymorphism—SNP) was performed for lineage determination: Yamagata or Victoria‐like lineage. MGB probes were designed for both B virus lineages that can be detected and discriminated simultaneously, as only one of the two probes will give a fluorescent signal.<xref rid=\"irv12752-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> All real‐time RT‐PCR reactions were performed using commercial kits—SuperScript<sup>®</sup> One‐Step qRT‐PCR System (Invitrogen).</p><p>For sequencing, influenza‐positive samples were passaged in adherent MDCK‐SIAT1 cells. Genetic characterization was done by sequencing the hemagglutinin (HA) coding region by Sanger sequencing using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems). The HA sequences were analyzed using the commercial software Sequencher® version 5.4.6 DNA sequence analysis software (Gene Codes Corporation).</p><p>Phenotypic testing of inhibition of neuraminidase (NA) activity for viral susceptibility to oseltamivir was performed with the fluorescence kit (NA‐Fluor™ Assay, MUNANA‐Methylumbelliferyl‐N‐acetylneuraminic acid substrate; Thermo Fisher Scientific).</p><p>Screening for influenza in our study was performed each season during the duration of the declared influenza season, based on the results of national surveillance, as follows: from week 53/2015 to week 20/2016 in the first influenza season, from week 48/2016 to week 18/2017 in the second season, and from week 50/2017 to week 17/2018 in the third season.</p><p>The study protocol was approved by the Ethics Committee of the “Cantacuzino” National Medico‐Military Institute for Research and Development—approvals number 46/03.09.2015, 108/07.09.2016, and 251/14.09.2017. Written informed consent was obtained from all subjects prior to inclusion in the study.</p><p>We report descriptive data as number and percentage for categorical variables, and as median and interquartile range (IQR) or range for non‐parametric continuous variables. Statistical associations were tested using the chi‐squared test for categorical variables and Mann–Whitney's <italic>U</italic> test for continuous non‐parametric variables. Two‐tailed <italic>P</italic> values <.05 were interpreted as statistically significant. IBM SPSS Statistics for Windows, version 20 (IBM Corp.) was used for the statistical analysis.</p></sec><sec sec-type=\"results\" id=\"irv12752-sec-0007\"><label>3</label><title>RESULTS</title><p>A total of 349 eligible patients were tested in this study, ranging between 53 and 191 by season included in the study. The baseline characteristics overall and by each of the three influenza seasons are presented in Table <xref rid=\"irv12752-tbl-0001\" ref-type=\"table\">1</xref>. The median (IQR) age was 74 (68, 80) years, and 43.6% of patients were men.</p><table-wrap id=\"irv12752-tbl-0001\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 1</label><caption><p>Characteristics of patients included in the study in the three influenza seasons analyzed</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Studied variable</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2015/16</p>\n<p>(n = 191)</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2016/17</p>\n<p>(n = 53)</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2017/18</p>\n<p>(n = 105)</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Overall</p>\n<p>(n = 349)</p>\n</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Male gender</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">88 (46.1%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">26 (49.1%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">38 (36.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">152 (43.6%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age, median (IQR)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">74 (67, 80)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">77 (71, 85)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">73 (69, 78.5)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">74 (68, 80)</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Smoking status</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Never smoked</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">138 (72.6%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">42 (79.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">68 (64.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">248 (71.3%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Former smoker</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">39 (20.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">9 (17.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">26 (24.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">74 (21.3%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Current smoker</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">13 (6.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (3.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">11 (10.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">26 (7.5%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vaccinated against influenza in the current seasonal</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">15 (7.9%, 95% CI: 4.5%‐12.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (3.8%, 95% CI: 0.5%‐13.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5 (4.8%, 95% CI: 1.6%‐10.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">22 (6.3%, 95% CI: 4.0%‐9.4%)</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Comorbidities</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Patients with comorbidities<xref ref-type=\"fn\" rid=\"irv12752-note-0004\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">177 (93.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">49 (92.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">95 (91.3%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">321 (92.5%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Number of comorbidities, median (range)<xref ref-type=\"fn\" rid=\"irv12752-note-0004\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (0‐6)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (0‐4)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (0‐5)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (0‐6)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Frailty index, median (IQR)<xref ref-type=\"fn\" rid=\"irv12752-note-0004\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.2 (0.1, 0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.2 (0.1, 0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.2 (0.1, 0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.2 (0.1, 0.3)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Cardiovascular disease<xref ref-type=\"fn\" rid=\"irv12752-note-0005\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">149 (78.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">42 (79.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">79 (77.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">270 (78.3%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Diabetes<xref ref-type=\"fn\" rid=\"irv12752-note-0005\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">66 (34.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">15 (28.3%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">31 (30.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">112 (32.5%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Obesity<xref ref-type=\"fn\" rid=\"irv12752-note-0006\">\n<sup>c</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">55 (28.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">10 (19.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">33 (31.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">98 (29.2%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Chronic pulmonary disease<xref ref-type=\"fn\" rid=\"irv12752-note-0005\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">48 (25.3%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">10 (18.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">20 (19.6%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">78 (22.6%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Chronic liver disease<xref ref-type=\"fn\" rid=\"irv12752-note-0007\">\n<sup>d</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6 (3.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6 (11.3%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">9 (8.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">21 (6.1%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Chronic kidney disease<xref ref-type=\"fn\" rid=\"irv12752-note-0008\">\n<sup>e</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">30 (15.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7 (13.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">10 (9.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">47 (13.7%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Hematologic cancer<xref ref-type=\"fn\" rid=\"irv12752-note-0004\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6 (3.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (3.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (1.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">9 (2.6%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Non‐hematologic cancer<xref ref-type=\"fn\" rid=\"irv12752-note-0004\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">18 (9.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (1.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">12 (11.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">31 (8.9%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Rheumatologic disease<xref ref-type=\"fn\" rid=\"irv12752-note-0009\">\n<sup>f</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">30 (15.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7 (13.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">11 (11.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">48 (14.0%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Immune suppression<xref ref-type=\"fn\" rid=\"irv12752-note-0004\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4 (2.1%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5 (4.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">9 (2.6%)</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Laboratory results</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Laboratory‐confirmed influenza A<xref ref-type=\"fn\" rid=\"irv12752-note-0010\">\n<sup>g</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">66 (97.1%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">18 (90.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">9 (14.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">93 (62.4%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Subtype A(H1N1)pdm09<xref ref-type=\"fn\" rid=\"irv12752-note-0011\">\n<sup>h</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">59 (89.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (22.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">61 (65.6%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Subtype A(H3N2)<xref ref-type=\"fn\" rid=\"irv12752-note-0011\">\n<sup>h</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7 (10.6%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">18 (100%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7 (77.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">32 (34.4%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Laboratory‐confirmed influenza B<xref ref-type=\"fn\" rid=\"irv12752-note-0010\">\n<sup>g</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (2.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (10.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">53 (86.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">57 (38.3%)<xref ref-type=\"fn\" rid=\"irv12752-note-0012\">\n<sup>i</sup>\n</xref>\n</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Lineage B/Victoria<xref ref-type=\"fn\" rid=\"irv12752-note-0013\">\n<sup>j</sup>\n</xref>\n</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (100%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (3.5%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Lineage B/Yamagata<xref ref-type=\"fn\" rid=\"irv12752-note-0013\">\n<sup>j</sup>\n</xref>\n</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6 (11.3%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6 (10.5%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Lineage not determined<xref ref-type=\"fn\" rid=\"irv12752-note-0013\">\n<sup>j</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (100%)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">47 (88.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">49 (86.0%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Antiviral treatment</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">118 (61.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">30 (56.6%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">81 (77.1%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">229 (65.6%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Death during hospitalization</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7 (3.7%, 95% CI: 1.5%‐7.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0 (0.0%, 95% CI: 0.0%‐6.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (1.0%, 95% CI: 0.0%‐5.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">8 (2.3%, 95% CI: 1.0%‐4.5%)</td></tr></tbody></table><table-wrap-foot id=\"irv12752-ntgp-0002\"><title>Note</title><fn id=\"irv12752-note-0002\"><p>All data is presented as number (percentage), unless otherwise specified. Frailty index was calculated by dividing the number of active comorbidities by the total number of comorbidities assessed for each patient.</p></fn><fn id=\"irv12752-note-0003\"><p>Abbreviations: IQR, interquartile range; N/A, not applicable.</p></fn><fn id=\"irv12752-note-0004\"><label><sup>a</sup></label><p>Missing data for 2 patients (1 in 2015/16, 1 in 2017/18).</p></fn><fn id=\"irv12752-note-0005\"><label><sup>b</sup></label><p>Missing data for 4 patients (1 in 2015/16, 3 in 2017/18).</p></fn><fn id=\"irv12752-note-0006\"><label><sup>c</sup></label><p>Missing data for 2 patients (1 in 2015/16, 1 in 2016/17).</p></fn><fn id=\"irv12752-note-0007\"><label><sup>d</sup></label><p>Missing data for 3 patients (1 in 2015/16, 2 in 2017/18).</p></fn><fn id=\"irv12752-note-0008\"><label><sup>e</sup></label><p>Missing data for 5 patients (2 in 2015/16, 1 in 2016/17, 2 in 2017/18).</p></fn><fn id=\"irv12752-note-0009\"><label><sup>f</sup></label><p>Missing data for 7 patients (2 in 2015/16, 5 in 2017/18).</p></fn><fn id=\"irv12752-note-0010\"><label><sup>g</sup></label><p>Percentage calculated among cases positive for influenza.</p></fn><fn id=\"irv12752-note-0011\"><label><sup>h</sup></label><p>Percentage calculated among cases positive for influenza A.</p></fn><fn id=\"irv12752-note-0012\"><label><sup>i</sup></label><p>In the 2017/2018 season, one case of double infection was diagnosed, positive for both A/H1 and B.</p></fn><fn id=\"irv12752-note-0013\"><label><sup>j</sup></label><p>Percentage calculated among cases positive for influenza B.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>Most patients (92.5%) had at least one underlying condition at the time of hospital admission, the most frequent being cardiovascular disease, present in 78.3% of cases, followed by diabetes (32.5%), obesity (29.2%), and chronic lung disease (22.6%). Other types of comorbidities were less frequent, being reported in less than 15% of patients (Table <xref rid=\"irv12752-tbl-0001\" ref-type=\"table\">1</xref>). Patients had a median number of 2 comorbidities, with an overall range of 0‐6 underlying diseases. The median (IQR) number of visits to the general practitioner during the past 12 months was 1 (0, 3), with a range of 0‐10 visits. None of the patients reported an episode of laboratory‐confirmed influenza in the previous season.</p><p>In the season 2015/16, laboratory‐confirmed influenza cases were identified between weeks 04/2016 and 15/2016, in the following season between weeks 48/2016 and 08/2017, and in the third season between weeks 02/2018 and 13/2018. Influenza virus type and subtype distribution by season is presented in Figure <xref rid=\"irv12752-fig-0001\" ref-type=\"fig\">1</xref>.</p><fig fig-type=\"FIGURE\" xml:lang=\"en\" id=\"irv12752-fig-0001\" orientation=\"portrait\" position=\"float\"><label>FIGURE 1</label><caption><p>Distribution of influenza cases by virus type and subtype/lineage in elderly patients from Bucharest Romania in the three consecutive studied seasons: 2015/16, 2016/17, and 2017/18</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"IRV-14-530-g001\"/></fig><p>Overall, 149 (42.7%) of the tested patients were positive for influenza. During the first two influenza seasons investigated, we noticed a predominant circulation of influenza A viruses in the study population (97.1% of all influenza‐positive cases in 2015/16 and 90.0% in 2016/17). We saw an apparent switch between seasons from A(H1N1)pdm09 (89.4% of influenza A cases in 2015/16) to A(H3N2) (100% of influenza A cases in 2016/17), while in the third season, there was a distinct predominance of influenza B (86.9% of all influenza‐positive cases in 2017/18).</p><p>The full hemagglutinin gene was sequenced from 45 influenza viruses isolated from 2015 to 2018. All characterized A(H1N1)pdm09 viruses (n = 21) fell into clade A/California/7/2009 with the amino acid variations that define group 6B viruses (A/South Africa/3626/2013). The majority of the sequences (13 out of the 15) from the 2017/18 influenza season also possessed the amino acid substitutions of the new emerging subgroups 6B.1 (eg, A/Michigan/45/2015) and 6B.2 subgroup (eg, A/England/377/2015). The 18 HA sequences of A(H3N2) viruses analyzed for amino acid substitutions fell within genetic group 3C, subgroup 3C.2a like A/Hong Kong/5738/2014. The two viruses tested in 2016/17 (B/Victoria lineage) fell into the B/Brisbane/60/2008 genetic clade and all (n = 4) viruses tested in 2017/18 (B/Yamagata lineage) fell in clade 3 (B/Phuket/3073/2013). During the study period, 20 (13.4%) strains of influenza virus were tested for antiviral resistance; all strains showed susceptibility to oseltamivir.</p><p>Among the SARI criteria, fever (94.6%), malaise (90.6%), and myalgia (71.7%) were the most frequently encountered signs or symptoms in patients testing positive for influenza, and specifically fever (OR = 3.0) and odynophagia (OR = 1.8) were significantly predictive for testing positive for influenza (Table <xref rid=\"irv12752-tbl-0002\" ref-type=\"table\">2</xref>). The distribution of comorbidities was comparable between patients with and without influenza (Table <xref rid=\"irv12752-tbl-0002\" ref-type=\"table\">2</xref>). We observed no significant differences in clinical signs or symptoms, or in the distribution of comorbidities between patients with influenza A and influenza B (Table <xref rid=\"irv12752-tbl-0003\" ref-type=\"table\">3</xref>).</p><table-wrap id=\"irv12752-tbl-0002\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 2</label><caption><p>Distribution of SARI criteria and other patient characteristics in patients testing negative and positive for influenza</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Studied variable</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Patients testing negative for influenza</p>\n<p>(n = 200)</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Patients testing positive for influenza</p>\n<p>(n = 149)</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Overall</p>\n<p>(n = 349)</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Statistical analysis</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">SARI criteria</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Fever<xref ref-type=\"fn\" rid=\"irv12752-note-0016\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">169 (85.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">141 (94.6%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">310 (89.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">OR = 3.0, 95% CI: 1.3‐6.6, <italic>P</italic> = .008</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Feverishness<xref ref-type=\"fn\" rid=\"irv12752-note-0017\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">11 (5.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">14 (9.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">25 (7.2%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .209</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Malaise<xref ref-type=\"fn\" rid=\"irv12752-note-0017\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">186 (93.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">135 (90.6%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">321 (92.2%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .418</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Headache<xref ref-type=\"fn\" rid=\"irv12752-note-0018\">\n<sup>c</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">129 (65.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">97 (65.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">226 (65.3%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = 1.000</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Myalgia<xref ref-type=\"fn\" rid=\"irv12752-note-0016\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">135 (68.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">106 (71.1%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">241 (69.5%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .638</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Altered clinical state<xref ref-type=\"fn\" rid=\"irv12752-note-0016\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">138 (69.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">98 (65.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">236 (68.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .486</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Cough<xref ref-type=\"fn\" rid=\"irv12752-note-0017\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">190 (95.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">143 (96.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">333 (95.7%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = 1.000</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Odynophagia<xref ref-type=\"fn\" rid=\"irv12752-note-0016\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">64 (32.3%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">68 (45.6%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">132 (38.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">OR = 1.8, 95% CI: 1.1‐2.7, <italic>P</italic> = .014</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Dyspnea<xref ref-type=\"fn\" rid=\"irv12752-note-0018\">\n<sup>c</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">129 (65.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">103 (69.1%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">232 (67.1%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .491</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Smoking status<xref ref-type=\"fn\" rid=\"irv12752-note-0017\">\n<sup>b</sup>\n</xref>\n</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Never smoked</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">139 (69.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">109 (73.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">248 (71.3%)</td><td align=\"left\" rowspan=\"3\" colspan=\"1\">\n<italic>P</italic> = .767</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Former smoker</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">45 (22.6%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29 (19.5%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">74 (21.3%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Current smoker</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15 (7.5%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11 (7.4%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26 (7.5%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vaccinated against influenza in the current season</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15 (7.5%, 95% CI: 4.3%‐12.1%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7 (4.7%, 95% CI: 1.9%‐9.5%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22 (6.3%, 95% CI: 4.0%‐9.4%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .375</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Comorbidities</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Patients with comorbidities<xref ref-type=\"fn\" rid=\"irv12752-note-0016\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">184 (92.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">137 (91.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">321 (92.5%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .837</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Number of comorbidities, median (range)<xref ref-type=\"fn\" rid=\"irv12752-note-0016\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (0‐6)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (0‐4)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (0‐6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .133</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Frailty index, median (IQR)<xref ref-type=\"fn\" rid=\"irv12752-note-0016\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.2 (0.1, 0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.2 (0.1, 0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.2 (0.1, 0.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Cardiovascular disease<xref ref-type=\"fn\" rid=\"irv12752-note-0019\">\n<sup>d</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">155 (78.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">115 (77.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">270 (78.3%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .895</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Diabetes<xref ref-type=\"fn\" rid=\"irv12752-note-0019\">\n<sup>d</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">59 (29.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">53 (35.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">112 (32.5%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .296</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Obesity<xref ref-type=\"fn\" rid=\"irv12752-note-0016\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">57 (28.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">41 (27.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">98 (28.2%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .811</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Chronic pulmonary disease<xref ref-type=\"fn\" rid=\"irv12752-note-0019\">\n<sup>d</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">52 (26.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">26 (17.6%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">78 (22.6%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .068</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Chronic liver disease<xref ref-type=\"fn\" rid=\"irv12752-note-0018\">\n<sup>c</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">14 (7.1%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7 (4.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">21 (6.1%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .496</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Chronic kidney disease<xref ref-type=\"fn\" rid=\"irv12752-note-0020\">\n<sup>e</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">36 (18.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">11 (7.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">47 (13.7%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">OR = 0.4, 95% CI: 0.2‐0.7, <italic>P</italic> = .004</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Hematologic cancer<xref ref-type=\"fn\" rid=\"irv12752-note-0016\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4 (2.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5 (3.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">9 (2.6%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .506</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Non‐hematologic cancer<xref ref-type=\"fn\" rid=\"irv12752-note-0016\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">14 (7.1%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">17 (11.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">31 (8.9%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .185</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Rheumatologic disease<xref ref-type=\"fn\" rid=\"irv12752-note-0021\">\n<sup>f</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">37 (19.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">11 (7.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">48 (14.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">OR = 0.4, 95% CI: 0.2‐0.7, <italic>P</italic> = .003</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Immune suppression<xref ref-type=\"fn\" rid=\"irv12752-note-0016\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">8 (4.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (0.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">9 (2.6%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .084</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Antiviral treatment</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">108 (54.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">121 (81.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">229 (65.6%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> < .001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Death during hospitalization</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4 (2.0%, 95% CI: 0.5%‐5.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4 (2.7%, 95% CI: 0.7%‐6.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">8 (2.3%, 95% CI: 1.0%‐4.5%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .728</td></tr></tbody></table><table-wrap-foot id=\"irv12752-ntgp-0003\"><title>Note</title><fn id=\"irv12752-note-0014\"><p>All data are presented as number (percentage), unless otherwise specified. Statistical analysis was performed with the two‐tailed chi‐squared test or Mann‐Whitney's <italic>U</italic> test. Frailty index was calculated by dividing the number of active comorbidities by the total number of comorbidities assessed for each patient.</p></fn><fn id=\"irv12752-note-0015\"><p>Abbreviations: 95% CI, 95% confidence interval; N/A, not applicable; OR, odds ratio; SARI, severe acute respiratory infection.</p></fn><fn id=\"irv12752-note-0016\"><label><sup>a</sup></label><p>Missing data for 2 patients.</p></fn><fn id=\"irv12752-note-0017\"><label><sup>b</sup></label><p>Missing data for 1 patient.</p></fn><fn id=\"irv12752-note-0018\"><label><sup>c</sup></label><p>Missing data for 3 patients.</p></fn><fn id=\"irv12752-note-0019\"><label><sup>d</sup></label><p>Missing data for 4 patients.</p></fn><fn id=\"irv12752-note-0020\"><label><sup>e</sup></label><p>Missing data for 5 patients.</p></fn><fn id=\"irv12752-note-0021\"><label><sup>f</sup></label><p>Missing data for 7 patients.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><table-wrap id=\"irv12752-tbl-0003\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 3</label><caption><p>Distribution of SARI criteria and other patient characteristics in patients with laboratory‐confirmed influenza A and B</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Studied variable</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Patients positive for influenza A</p>\n<p>(n = 93)</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Patients positive for influenza B</p>\n<p>(n = 56)</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Overall</p>\n<p>(n = 149)</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Statistical analysis</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Fever</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">87 (93.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">54 (96.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">141 (94.6%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .711</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Feverishness</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3 (3.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">11 (19.6%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">14 (9.4%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">OR = 7.3, 95% CI: 2.0‐27.6, <italic>P</italic> = .002</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Malaise</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">84 (90.3%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">51 (91.1%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">135 (90.6%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = 1.000</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Headache<xref ref-type=\"fn\" rid=\"irv12752-note-0024\">\n<sup>a</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">64 (69.6%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">33 (58.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">97 (65.5%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .214</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Myalgia</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">68 (73.1%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">38 (67.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">106 (71.1%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .576</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Altered clinical state</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">63 (67.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">35 (62.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">98 (65.8%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .593</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cough</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">89 (95.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">54 (96.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">143 (96.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = 1.000</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Odynophagia</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">41 (44.1%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">27 (48.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">68 (45.6%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .734</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Dyspnea</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">65 (69.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">38 (67.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">103 (69.1%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .855</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Smoking status</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Never smoked</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">73 (78.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">36 (64.3%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">109 (73.2%)</td><td align=\"left\" rowspan=\"3\" colspan=\"1\">\n<italic>P</italic> = .091</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Former smoker</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">13 (14.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16 (28.6%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29 (19.5%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Current smoker</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7 (7.5%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (7.1%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11 (7.4%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vaccinated against influenza in the current season</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6 (6.5%, 95% CI: 2.4%‐13.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (1.8%, 95% CI: 0.0%‐9.6%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7 (4.7%, 95% CI: 1.9%‐9.5%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .254</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Comorbidities</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Patients with comorbidities<xref ref-type=\"fn\" rid=\"irv12752-note-0025\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">86 (92.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">51 (91.1%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">137 (91.9%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .764</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Number of comorbidities, median (range)<xref ref-type=\"fn\" rid=\"irv12752-note-0025\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (0‐4)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (0‐4)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (0‐4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Frailty index, median (IQR)<xref ref-type=\"fn\" rid=\"irv12752-note-0025\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.2 (0.1, 0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.2 (0.1, 0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.2 (0.1, 0.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Cardiovascular disease<xref ref-type=\"fn\" rid=\"irv12752-note-0026\">\n<sup>c</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">73 (78.5%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">42 (76.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">115 (77.7%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .839</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Diabetes<xref ref-type=\"fn\" rid=\"irv12752-note-0026\">\n<sup>c</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">33 (35.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">20 (35.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">53 (35.8%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .999</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Obesity<xref ref-type=\"fn\" rid=\"irv12752-note-0025\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">23 (24.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">18 (32.1%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">41 (27.5%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .348</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Chronic pulmonary disease<xref ref-type=\"fn\" rid=\"irv12752-note-0026\">\n<sup>c</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">15 (16.3%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">11 (19.6%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">26 (17.6%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .659</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Chronic liver disease<xref ref-type=\"fn\" rid=\"irv12752-note-0027\">\n<sup>d</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (2.2%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5 (8.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7 (4.7%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .105</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Chronic kidney disease<xref ref-type=\"fn\" rid=\"irv12752-note-0028\">\n<sup>e</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5 (5.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6 (10.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">11 (7.4%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .333</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Hematologic cancer<xref ref-type=\"fn\" rid=\"irv12752-note-0025\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4 (4.3%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (1.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5 (3.4%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .651</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Non‐hematologic cancer<xref ref-type=\"fn\" rid=\"irv12752-note-0025\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">11 (11.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6 (10.7%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">17 (11.4%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .999</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Rheumatologic disease<xref ref-type=\"fn\" rid=\"irv12752-note-0029\">\n<sup>f</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">9 (9.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (3.6%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">11 (7.5%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .211</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Immune suppression<xref ref-type=\"fn\" rid=\"irv12752-note-0025\">\n<sup>b</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (1.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (0.7%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Antiviral treatment</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">78 (83.9%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">43 (76.8%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">121 (81.2%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .289</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Death during hospitalization<xref ref-type=\"fn\" rid=\"irv12752-note-0030\">\n<sup>g</sup>\n</xref>\n</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4 (4.3%, 95% CI: 1.2%‐10.6%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0 (0.0%, 95% CI: 0.0%‐6.4%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4 (2.7%, 95% CI: 0.7%‐6.7%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = .297</td></tr></tbody></table><table-wrap-foot id=\"irv12752-ntgp-0004\"><title>Note</title><fn id=\"irv12752-note-0022\"><p>All data is presented as number (percentage), unless otherwise specified. Statistical analysis was performed with the two‐tailed chi‐squared test or Mann‐Whitney's <italic>U</italic> test. Frailty index was calculated by dividing the number of active comorbidities by the total number of comorbidities assessed for each patient.</p></fn><fn id=\"irv12752-note-0023\"><p>Abbreviations: 95% CI, 95% confidence interval; N/A, not applicable; OR, odds ratio; SARI, severe acute respiratory infection.</p></fn><fn id=\"irv12752-note-0024\"><label><sup>a</sup></label><p>Missing data for 1 patient.</p></fn><fn id=\"irv12752-note-0025\"><label><sup>b</sup></label><p>Missing data for 2 patients.</p></fn><fn id=\"irv12752-note-0026\"><label><sup>c</sup></label><p>Missing data for 4 patients.</p></fn><fn id=\"irv12752-note-0027\"><label><sup>d</sup></label><p>Missing data for 3 patients.</p></fn><fn id=\"irv12752-note-0028\"><label><sup>e</sup></label><p>Missing data for 5 patients.</p></fn><fn id=\"irv12752-note-0029\"><label><sup>f</sup></label><p>Missing data for 7 patients.</p></fn><fn id=\"irv12752-note-0030\"><label><sup>g</sup></label><p>Death occurred in 3 patients with influenza A/H1 and one with A/H3, all in the 2015/16 season. Four more deaths occurred in patients who tested negative for influenza in the 2017/18 season.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>Overall, 65.6% of the patients included in the study received antiviral treatment with oseltamivir during hospital admission, and the proportion was higher in patients with laboratory‐confirmed influenza (81.2%). The clinical course of disease was generally favorable. However, eight patients died during hospital admission, all having at least one underlying condition; of them, four had been confirmed with influenza, and four were negative for influenza. All four deaths that occurred in patients with laboratory‐confirmed influenza were caused by influenza A [three A(H1N1)pdm09 and one A(H3N2)]; none of these four patients had been vaccinated against influenza, three were females, all were aged 76 years old, and had a range of 1‐3 comorbidities, as follows: three had diabetes, two had chronic lung disease, one had cardiovascular disease and one had cancer. The overall calculated case fatality in this study was 2.3%: 2.7% in patients with laboratory‐confirmed influenza and 2.0% in the patients testing negative (Table <xref rid=\"irv12752-tbl-0002\" ref-type=\"table\">2</xref>). Case fatality was higher in the 2015/16 influenza season than in the subsequent seasons (3.7%, 0.0%, 1.0%) (Table <xref rid=\"irv12752-tbl-0001\" ref-type=\"table\">1</xref>).</p><p>A total of 22 (6.3%) patients had been vaccinated against influenza in the respective current season, all with standard‐dose inactivated trivalent vaccine, as follows: 20 with Influvac (Abbott Biologicals BV) and 2 with Vaxigrip (Sanofi Pasteur SA). Among these, 15 tested negative for influenza. Out of the remaining seven that tested positive, six were diagnosed in the first season (2015/16) with influenza A [4 A(H1N1)pdm09 and 2 A(H3N2)], and one in the third season (2017/18) with influenza B (no lineage data available). They had been vaccinated within a range of 6 to 24 weeks prior to symptom onset. Three were male and four were female, and their ages ranged from 66 to 81 years old. Only one of them had no comorbidities, the others presenting 0‐3 underlying conditions, as follows: five had cardiovascular disease, two had diabetes mellitus, two had cancer, one had chronic kidney disease, one had rheumatologic disease, and one was obese; no deaths were recorded among these seven vaccine failures during their hospitalization for influenza.</p></sec><sec sec-type=\"discussion\" id=\"irv12752-sec-0008\"><label>4</label><title>DISCUSSION</title><p>In this paper, we have reported the clinical and epidemiological characteristics of elderly patients admitted in our hospital for SARI in three consecutive influenza seasons from Bucharest, Romania.</p><p>Our study has highlighted a high burden of comorbidities in elderly patients presenting with SARI during winter season in Romania, over 90% with at least one comorbid disease. The synergy between old age, potential immune hyporeactivity, and the presence of multiple chronic diseases may put this group of patients at an increased risk of decompensating the underlying condition during influenza infection, particularly cardiovascular diseases,<xref rid=\"irv12752-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref> which were highly prevalent in our study population, or of developing bacterial pneumonia.<xref rid=\"irv12752-bib-0010\" ref-type=\"ref\">\n<sup>10</sup>\n</xref>, <xref rid=\"irv12752-bib-0011\" ref-type=\"ref\">\n<sup>11</sup>\n</xref>, <xref rid=\"irv12752-bib-0012\" ref-type=\"ref\">\n<sup>12</sup>\n</xref>, <xref rid=\"irv12752-bib-0013\" ref-type=\"ref\">\n<sup>13</sup>\n</xref>\n</p><p>Clinical characteristics of influenza did not differ significantly between elderly patients with influenza A and B in our study. This is in line with previous research from our institute in children<xref rid=\"irv12752-bib-0014\" ref-type=\"ref\">\n<sup>14</sup>\n</xref> and from France in all age groups, showing that clinical characteristics are mostly indistinguishable between viral types and subtypes.<xref rid=\"irv12752-bib-0015\" ref-type=\"ref\">\n<sup>15</sup>\n</xref> Among the SARI criteria, fever and odynophagia associated higher odds of testing positive for influenza in our study; this is consistent with the data reported by Falsey et al, which showed that the combination of fever with cough or odynophagia exhibited the best balance of sensitivity and specificity for diagnosing influenza.<xref rid=\"irv12752-bib-0016\" ref-type=\"ref\">\n<sup>16</sup>\n</xref> Falsey et al also suggested that in elderly patients, the threshold for defining fever could be as low as 37.3°C.<xref rid=\"irv12752-bib-0016\" ref-type=\"ref\">\n<sup>16</sup>\n</xref>\n</p><p>Overall, influenza A(H1N1)pdm09 was the main viral subtype circulating in 2015/16, followed by A(H3N2) in 2016/17 and B (supposedly mainly B/Yamagata) in 2017/18 in our study. These data are in line with that reported on a national level by the Romanian National Center for Surveillance and Control of Transmissible Diseases, with certain particularities, discussed below.</p><p>For the 2015/16 season, influenza A was the main circulating type in Romania in all age groups, accounting for 90.6% of all influenza cases reported and analyzed on a national level,<xref rid=\"irv12752-bib-0017\" ref-type=\"ref\">\n<sup>17</sup>\n</xref> compared to 97.1% in our study of elderly patients, and the main subtype was A(H1N1)pdm09, accounting for 93.5% of all subtyped A strains nationally,<xref rid=\"irv12752-bib-0017\" ref-type=\"ref\">\n<sup>17</sup>\n</xref> compared with 89.4% in our study. The A(H3N2) subtype was relatively less frequently identified at the national level (6.5%<xref rid=\"irv12752-bib-0017\" ref-type=\"ref\">\n<sup>17</sup>\n</xref> compared with 10.6% in our study), while the circulation of B strains, reported at 9.4% in the country,<xref rid=\"irv12752-bib-0017\" ref-type=\"ref\">\n<sup>17</sup>\n</xref> was almost negligible in our study (2.9%) for the 2015/16 season. These slight differences might arise from a different sampling frame (one tertiary care hospital analyzing hospitalized SARI in our study, compared to an ILI‐based national surveillance system<xref rid=\"irv12752-bib-0017\" ref-type=\"ref\">\n<sup>17</sup>\n</xref> in the country as a whole), but they might also reflect to a certain degree a particularity of the patients admitted in our hospital that is a referral hospital for the south of Romania.<xref rid=\"irv12752-bib-0018\" ref-type=\"ref\">\n<sup>18</sup>\n</xref>\n</p><p>For the 2016/17 season, the data from our current study confirmed to some extent our previous report regarding the exclusive co‐circulation of A(H3N2) and B/Victoria.<xref rid=\"irv12752-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref> However, in our study of elderly patients the season was dominated by the circulation of A viruses (90.0% compared with the national estimate of 65.7% in all age groups<xref rid=\"irv12752-bib-0020\" ref-type=\"ref\">\n<sup>20</sup>\n</xref> and compared to our previous report of 33.9% in all age groups with an emphasis on children<xref rid=\"irv12752-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref>), suggesting that A viruses predominated in elderly patients in 2016/17; A(H3N2) accounted for 100% of all circulating A viruses in 2016/17 in the current study and in our previous report.<xref rid=\"irv12752-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref> Data from Bulgaria and Poland also report the predominance of influenza A virus (97.5%<xref rid=\"irv12752-bib-0021\" ref-type=\"ref\">\n<sup>21</sup>\n</xref> and 95.5%<xref rid=\"irv12752-bib-0022\" ref-type=\"ref\">\n<sup>22</sup>\n</xref> of all influenza cases, respectively), and specifically the A(H3N2) subtype in the 2016/17 season, but A(H1N1)pdm09 was also present in these two countries, to a lower extent.<xref rid=\"irv12752-bib-0021\" ref-type=\"ref\">\n<sup>21</sup>\n</xref>, <xref rid=\"irv12752-bib-0022\" ref-type=\"ref\">\n<sup>22</sup>\n</xref> Our data are also in agreement with a recent meta‐regression analysis reporting that type B influenza viruses are somewhat less frequent in elderly patients compared to the other age groups.<xref rid=\"irv12752-bib-0023\" ref-type=\"ref\">\n<sup>23</sup>\n</xref>\n</p><p>For the 2017/18 season, the predominance of B viruses was evident in our SARI study, accounting for 86.9% of all influenza cases, but the rate was higher than the one reported for ILI‐based surveillance at the national level (66.5%).<xref rid=\"irv12752-bib-0024\" ref-type=\"ref\">\n<sup>24</sup>\n</xref> In our study, lineage determination was only performed for a small proportion of B viruses circulating during this season (11.3%, 6/53 cases), but based on the results available from a different study concomitantly conducted in our hospital in the 2017/18 season, B/Yamagata appeared to be the main circulating strain in patients of all ages.<xref rid=\"irv12752-bib-0025\" ref-type=\"ref\">\n<sup>25</sup>\n</xref>\n</p><p>Case fatality was overall low, being higher in 2015/16 (3.7%), and lower in the following seasons, 0% in 2016/17 and 1.0% in 2017/18, and there were no significant changes in the healthcare system to account for this difference. Out of the four deaths recorded in patients with laboratory‐confirmed influenza, all occurred during the 2015/16 season, 3 occurred in patients with A(H1N1)pdm09 and one with A(H3N2) infection. The Romanian National Center for Surveillance and Control of Transmissible Diseases has reported that for patients with SARI in the 2015/16 influenza season, testing positive for A(H1N1)pdm09 associated a fourfold increased risk of death, compared to infection due to other viral subtypes, and that the overall severity of influenza was high that season.<xref rid=\"irv12752-bib-0026\" ref-type=\"ref\">\n<sup>26</sup>\n</xref> Furthermore, based on the same national report, the general fatality rate for SARI in 2015/16 was reported at 20.7% and at 49.0% for SARI confirmed as influenza,<xref rid=\"irv12752-bib-0026\" ref-type=\"ref\">\n<sup>26</sup>\n</xref> and another study has reported a case fatality proportion of 39.8% (95% CI: 29.5%‐50.8%) for that same season for SARI confirmed as influenza.<xref rid=\"irv12752-bib-0027\" ref-type=\"ref\">\n<sup>27</sup>\n</xref> Differences in the lower case fatality reported in our study compared to the national estimate could potentially be due to selection testing bias of severe cases in the conventional surveillance system, leading to an artificial increase in the reported fatality ratio. These differences may also be partly explained by the specific profile of our institute, which is a major reference center for infectious diseases in Romania, and is well equipped to manage influenza cases in a timely manner, and to institute specific antiviral treatment promptly after hospital admission of SARI cases. In our study, 65.6% of patients admitted with SARI received antiviral treatment. In SARI cases confirmed as influenza, the percentage was higher, 81.2%, whereas in patients who tested negative for influenza, 54.0% received an antiviral, but treatment was stopped after influenza was ruled out and an alternative diagnosis was established.</p><p>In Romania, influenza vaccination is provided each year by health authorities, through the general practitioners, to priority risk groups as defined by the World Health Organization (WHO). However, vaccine coverage remains generally low for most risk groups, and for elderly patients in particular, with a marked decrease in the immediate post‐2009‐pandemic period (49.4% to 19.1% for elderly patients in 2008/09 vs 2010/11), as also reported for other countries,<xref rid=\"irv12752-bib-0028\" ref-type=\"ref\">\n<sup>28</sup>\n</xref> and a slowly increasing trend in the past 3 years (Figure <xref rid=\"irv12752-fig-0002\" ref-type=\"fig\">2</xref>).<xref rid=\"irv12752-bib-0028\" ref-type=\"ref\">\n<sup>28</sup>\n</xref>, <xref rid=\"irv12752-bib-0029\" ref-type=\"ref\">\n<sup>29</sup>\n</xref>, <xref rid=\"irv12752-bib-0030\" ref-type=\"ref\">\n<sup>30</sup>\n</xref>, <xref rid=\"irv12752-bib-0031\" ref-type=\"ref\">\n<sup>31</sup>\n</xref>, <xref rid=\"irv12752-bib-0032\" ref-type=\"ref\">\n<sup>32</sup>\n</xref> In our study population, the influenza vaccination uptake was low over the three seasons. In the 2015/16 season, the vaccination uptake was 7.9% (95% CI: 4.5%‐12.7%) in our study population, and the national reported rate for elderly patients was 10.3%.<xref rid=\"irv12752-bib-0017\" ref-type=\"ref\">\n<sup>17</sup>\n</xref> For the 2016/17 season, the influenza vaccine coverage in our study was 3.8% (95% CI: 0.5%‐13.0%), and the national reported coverage was 8.2% in elderly patients.<xref rid=\"irv12752-bib-0020\" ref-type=\"ref\">\n<sup>20</sup>\n</xref> For the 2017/18 season, the vaccine coverage in our study (4.8%, 95% CI: 1.6%‐10.8%) was lower that the coverage in elderly patients on a national level, reported at 16.3%.<xref rid=\"irv12752-bib-0024\" ref-type=\"ref\">\n<sup>24</sup>\n</xref> Given the overall low number of vaccinated patients included in our study, we cannot conclude whether there was a protective effect of vaccination against hospital admission for SARI. Stronger data from Australia have shown that an influenza vaccine coverage of 80.2% in elderly patients was able to prevent 49.5% of hospital admissions due to influenza in the 2015 influenza season.<xref rid=\"irv12752-bib-0033\" ref-type=\"ref\">\n<sup>33</sup>\n</xref>\n</p><fig fig-type=\"FIGURE\" xml:lang=\"en\" id=\"irv12752-fig-0002\" orientation=\"portrait\" position=\"float\"><label>FIGURE 2</label><caption><p>Influenza vaccine coverage in Romania in the general population and in elderly patients, 2007/08 to 2018/19</p></caption><graphic id=\"nlm-graphic-3\" xlink:href=\"IRV-14-530-g002\"/></fig><p>We recorded a total number of seven cases of influenza in patients who had been vaccinated in the respective current season; most of these vaccine failures occurred in the 2015/16 season. The adjusted influenza vaccine effectiveness (IVE) in elderly patients (defined as 60 years and older) for the 2015/16 season in Spain was low (20.2% for preventing hospital admission with A(H1N1)pdm09),<xref rid=\"irv12752-bib-0034\" ref-type=\"ref\">\n<sup>34</sup>\n</xref> similar to the data from the I‐MOVE+ study in Poland, reporting an overall vaccine effectiveness of 21.0%.<xref rid=\"irv12752-bib-0035\" ref-type=\"ref\">\n<sup>35</sup>\n</xref> For Romania, the low vaccine uptake for the previous influenza seasons did not allow an adequate calculation of the IVE, with very small numbers of cases and controls included in the formula, which led to an adjusted IVE against hospitalized A(H1N1)pdm09 infection reported at −22.6%, but with a very wide confidence interval (−490.3% to 74.6%).<xref rid=\"irv12752-bib-0036\" ref-type=\"ref\">\n<sup>36</sup>\n</xref>\n</p><p>Published studies have shown that standard‐dose influenza vaccines may induce lower hemagglutination‐inhibition HAI titers and seroprotection rates in elderly patients.<xref rid=\"irv12752-bib-0037\" ref-type=\"ref\">\n<sup>37</sup>\n</xref>, <xref rid=\"irv12752-bib-0038\" ref-type=\"ref\">\n<sup>38</sup>\n</xref> In Romania, high‐dose influenza vaccines have so far not been available and therefore, the data we are reporting here refer to elderly patients who had received standard‐dose inactivated trivalent vaccines.</p><p>Our study's main strength is that it employed systematic screening of all elderly patients admitted for SARI to one tertiary care hospital in Romania, applying the same standardized methodology throughout three consecutive influenza seasons, thus allowing an analysis of the differences from season to season in terms of clinical characteristics, viral type/subtype circulation, case fatality, and use of healthcare resources.</p><p>This study also had a number of limitations. The series is relatively small, 349 patients from one single tertiary care center over the course of three consecutive influenza seasons. The overall low vaccination uptake in our study population and in the country precluded us from performing an analysis of vaccine effectiveness for this study site. Also, lineage determination was only performed for a small proportion of B viruses circulating in the third influenza season investigated, which did not allow an exact quantification of the circulation of B/Victoria vs. B/Yamagata strains, but our data, corroborated with other information from field literature, showed that in the 2017/18 season in the Bucharest‐Ilfov region B/Yamagata accounted for 90.4% of all characterized B strains circulating in hospitalized influenza.<xref rid=\"irv12752-bib-0025\" ref-type=\"ref\">\n<sup>25</sup>\n</xref>\n</p><p>Continued surveillance of influenza is needed in order to inform the best local practices. In elderly patients from our setting, given the high burden of comorbidities that we have characterized, interdisciplinary management and good control of underlying diseases are important, and should be coupled with targeted interventions to substantially increase vaccine uptake.</p></sec><sec sec-type=\"conclusions\" id=\"irv12752-sec-0009\"><label>5</label><title>CONCLUSIONS</title><p>Among elderly patients admitted to the hospital with SARI in Bucharest Romania, influenza A(H1N1)pdm09 was the main viral subtype circulating in 2015/16, A(H3N2) in 2016/17, and B influenza in 2017/18, respectively. Case fatality was low in general, but it was highest during the 2015/16 season with predominant circulation of influenza A(H1N1)pdm09. Vaccination uptake in elderly patients from our study population was low, highlighting the importance of promoting vaccination in the elderly patients in general and in those with underlying conditions that present higher risk for severe disease.</p></sec><sec sec-type=\"COI-statement\" id=\"irv12752-sec-0010\"><title>CONFLICTS OF INTEREST</title><p>DP is the technical project manager for the GIHSN project funded by Sanofi Pasteur and Foundation for Influenza Epidemiology, principal investigator of the I‐MOVE+ study funded through the European Union's HORIZON 2020 research and innovation program, and technical project manager for the DRIVE study, that has received support from the EU/EFPIA Innovative Medicines Initiative 2 Joint Undertaking (DRIVE, grant n° 777363). No conflict of interest.</p><p>AnSC is the principal investigator of the I‐MOVE+ study 2017/18 funded through the European Union's HORIZON 2020 research and innovation program, member of the research team of the DRIVE study, that has received support from the EU/EFPIA Innovative Medicines Initiative 2 Joint Undertaking (DRIVE, grant n° 777363), and subinvestigator in influenza clinical trials by Shionogi and Roche; no conflict of interest.</p><p>AEI is the coordinator of the molecular detection of influenza type and subtype by real‐time reverse transcription PCR through the IMOVE+ project. No conflict of interest.</p><p>ML is the coordinator of the genetic characterization of influenza strains. No conflict of interest.</p><p>CMC is the coordinator of the isolation of influenza viruses in cell culture and antigenic characterization in the IMOVE+ study. No conflict of interest.</p><p>MEM is the coordinator of the antiviral resistance in IMOVE+ study. No conflict of interest.</p><p>MN is the principal investigator of the I‐MOVE+ study 2016/18 funded through the European Union's HORIZON 2020 research and innovation program.</p><p>VA is the member of the research team of the GIHSN project funded by Sanofi Pasteur and Foundation for Influenza Epidemiology, member of the research team of the DRIVE study, that has received support from the EU/EFPIA Innovative Medicines Initiative 2 Joint Undertaking (DRIVE, grant n° 777363), and reports lectures and advisory boards for Sanofi and Astra‐Zeneca, outside of the submitted work. No conflict of interest.</p><p>MDC No conflict of interest.</p><p>ASC is the member of the research team of the GIHSN project funded by Sanofi Pasteur and Foundation for Influenza Epidemiology, member of the research team of the DRIVE study, that has received support from the EU/EFPIA Innovative Medicines Initiative 2 Joint Undertaking (DRIVE, grant n° 777363), principal investigator in influenza clinical trials by Shionogi and Roche, and reports lectures for Sanofi, outside of the submitted work. No conflict of interest.</p><p>OS is the member of the research team of the GIHSN project funded by Sanofi Pasteur and Foundation for Influenza Epidemiology, principal investigator for adults in the DRIVE study, that has received support from the EU/EFPIA Innovative Medicines Initiative 2 Joint Undertaking (DRIVE, grant n° 777363), subinvestigator in influenza clinical trials by Shionogi and Roche, and reports lectures for Sanofi, outside of the submitted work. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"iso-abbrev\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"doi\">10.1111/(ISSN)1750-2659</journal-id><journal-id journal-id-type=\"publisher-id\">IRV</journal-id><journal-title-group><journal-title>Influenza and Other Respiratory Viruses</journal-title></journal-title-group><issn pub-type=\"ppub\">1750-2640</issn><issn pub-type=\"epub\">1750-2659</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32390333</article-id><article-id pub-id-type=\"pmc\">PMC7431642</article-id><article-id pub-id-type=\"doi\">10.1111/irv.12748</article-id><article-id pub-id-type=\"publisher-id\">IRV12748</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Original Article</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Original Articles</subject></subj-group></article-categories><title-group><article-title>Applying the moving epidemic method to determine influenza epidemic and intensity thresholds using influenza‐like illness surveillance data 2009‐2018 in Tunisia</article-title><alt-title alt-title-type=\"left-running-head\">BOUGUERRA et al.</alt-title></title-group><contrib-group><contrib id=\"irv12748-cr-0001\" contrib-type=\"author\"><name><surname>Bouguerra</surname><given-names>Hind</given-names></name><xref ref-type=\"aff\" rid=\"irv12748-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12748-cr-0002\" contrib-type=\"author\"><name><surname>Boutouria</surname><given-names>Elyes</given-names></name><xref ref-type=\"aff\" rid=\"irv12748-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12748-cr-0003\" contrib-type=\"author\"><name><surname>Zorraga</surname><given-names>Mokhtar</given-names></name><xref ref-type=\"aff\" rid=\"irv12748-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12748-cr-0004\" contrib-type=\"author\"><name><surname>Cherif</surname><given-names>Amal</given-names></name><xref ref-type=\"aff\" rid=\"irv12748-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12748-cr-0005\" contrib-type=\"author\"><name><surname>Yazidi</surname><given-names>Rihab</given-names></name><xref ref-type=\"aff\" rid=\"irv12748-aff-0003\">\n<sup>3</sup>\n</xref></contrib><contrib id=\"irv12748-cr-0006\" contrib-type=\"author\"><name><surname>Abdeddaiem</surname><given-names>Naima</given-names></name><xref ref-type=\"aff\" rid=\"irv12748-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12748-cr-0007\" contrib-type=\"author\"><name><surname>Maazaoui</surname><given-names>Latifa</given-names></name><xref ref-type=\"aff\" rid=\"irv12748-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12748-cr-0008\" contrib-type=\"author\"><name><surname>ElMoussi</surname><given-names>Awatef</given-names></name><xref ref-type=\"aff\" rid=\"irv12748-aff-0004\">\n<sup>4</sup>\n</xref></contrib><contrib id=\"irv12748-cr-0009\" contrib-type=\"author\"><name><surname>Abid</surname><given-names>Salma</given-names></name><xref ref-type=\"aff\" rid=\"irv12748-aff-0004\">\n<sup>4</sup>\n</xref></contrib><contrib id=\"irv12748-cr-0010\" contrib-type=\"author\"><name><surname>Amine</surname><given-names>Slim</given-names></name><xref ref-type=\"aff\" rid=\"irv12748-aff-0004\">\n<sup>4</sup>\n</xref></contrib><contrib id=\"irv12748-cr-0011\" contrib-type=\"author\"><name><surname>Bouabid</surname><given-names>Leila</given-names></name><xref ref-type=\"aff\" rid=\"irv12748-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12748-cr-0012\" contrib-type=\"author\"><name><surname>Bougatef</surname><given-names>Souha</given-names></name><xref ref-type=\"aff\" rid=\"irv12748-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12748-cr-0013\" contrib-type=\"author\"><name><surname>Kouni Chahed</surname><given-names>Mohamed</given-names></name><xref ref-type=\"aff\" rid=\"irv12748-aff-0005\">\n<sup>5</sup>\n</xref></contrib><contrib id=\"irv12748-cr-0014\" contrib-type=\"author\"><name><surname>Ben Salah</surname><given-names>Afif</given-names></name><xref ref-type=\"aff\" rid=\"irv12748-aff-0003\">\n<sup>3</sup>\n</xref></contrib><contrib id=\"irv12748-cr-0015\" contrib-type=\"author\"><name><surname>Bettaieb</surname><given-names>Jihene</given-names></name><xref ref-type=\"aff\" rid=\"irv12748-aff-0003\">\n<sup>3</sup>\n</xref></contrib><contrib id=\"irv12748-cr-0016\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Bouafif Ben Alaya</surname><given-names>Nissaf</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-3326-9861</contrib-id><xref ref-type=\"aff\" rid=\"irv12748-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12748-aff-0005\">\n<sup>5</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12748-aff-0006\">\n<sup>6</sup>\n</xref><address><email>nissafba@yahoo.fr</email></address></contrib></contrib-group><aff id=\"irv12748-aff-0001\">\n<label><sup>1</sup></label>\n<named-content content-type=\"organisation-division\">National Observatory of New and Emerging Diseases</named-content>\n<institution>Ministry of Health</institution>\n<city>Tunis</city>\n<country country=\"TN\">Tunisia</country>\n</aff><aff id=\"irv12748-aff-0002\">\n<label><sup>2</sup></label>\n<institution>Direction of Primary Health Care</institution>\n<city>Tunis</city>\n<country country=\"TN\">Tunisia</country>\n</aff><aff id=\"irv12748-aff-0003\">\n<label><sup>3</sup></label>\n<institution>Pasteur Institute of Tunis</institution>\n<city>Tunis</city>\n<country country=\"TN\">Tunisia</country>\n</aff><aff id=\"irv12748-aff-0004\">\n<label><sup>4</sup></label>\n<named-content content-type=\"organisation-division\">Microbiology Laboratory</named-content>\n<named-content content-type=\"organisation-division\">Virology Unit</named-content>\n<institution>Charles Nicolle Hospital</institution>\n<city>Tunis</city>\n<country country=\"TN\">Tunisia</country>\n</aff><aff id=\"irv12748-aff-0005\">\n<label><sup>5</sup></label>\n<named-content content-type=\"organisation-division\">Faculté de Médecine de Tunis</named-content>\n<institution>Université de Tunis El Manar</institution>\n<city>Tunis</city>\n<country country=\"TN\">Tunisia</country>\n</aff><aff id=\"irv12748-aff-0006\">\n<label><sup>6</sup></label>\n<named-content content-type=\"organisation-division\">Faculté de Médecine de Tunis</named-content>\n<named-content content-type=\"organisation-division\">LR01ES04 Epidémiologie et Prévention des Maladies Cardiovasculaires en Tunisie</named-content>\n<institution>Université de Tunis El Manar</institution>\n<city>Tunis</city>\n<country country=\"TN\">Tunisia</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label><bold>Correspondence</bold><break/>\nNissaf Bouafif Ben Alaya, 5‐7 Rue Elkhartoum, Immeuble Diplomate, le Belvédère, 1002 Tunis, Tunisia.<break/>\nEmail: <email>nissafba@yahoo.fr</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>10</day><month>5</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>9</month><year>2020</year></pub-date><volume>14</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1111/irv.v14.5</issue-id><fpage>507</fpage><lpage>514</lpage><history><date date-type=\"received\"><day>06</day><month>4</month><year>2019</year></date><date date-type=\"rev-recd\"><day>12</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>15</day><month>12</month><year>2019</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 John Wiley & Sons Ltd <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Influenza and Other Respiratory Viruses</italic> Published by John Wiley & Sons Ltd.</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:IRV-14-507.pdf\"/><abstract id=\"irv12748-abs-0001\"><title>Abstract</title><sec id=\"irv12748-sec-0001\"><title>Background</title><p>Defining the start and assessing the intensity of influenza seasons are essential to ensure timely preventive and control measures and to contribute to the pandemic preparedness. The present study aimed to determine the epidemic and intensity thresholds of influenza season in Tunisia using the moving epidemic method.</p></sec><sec id=\"irv12748-sec-0002\"><title>Methods</title><p>We applied the moving epidemic method (MEM) using the R Language implementation (package “mem”). We have calculated the epidemic and the different intensity thresholds from historical data of the past nine influenza seasons (2009‐2010 to 2017‐2018) and assessed the impact of the 2009‐2010 pandemic year. Data used were the weekly influenza‐like illness (ILI) proportions compared with all outpatient acute consultations. The goodness of the model was assessed using a cross validation procedure.</p></sec><sec id=\"irv12748-sec-0003\"><title>Results</title><p>The average duration of influenza epidemic during a typical season was 20 weeks and ranged from 11 weeks (2009‐2010 season) to 23 weeks (2015‐2016 season). The epidemic threshold with the exclusion of the pandemic season was 6.25%. It had a very high sensitivity of 85% and a high specificity of 69%. The different levels of intensity were established as follows: low, if ILI proportion is below 9.74%, medium below 12.05%; high below 13.27%; and very high above this last rate.</p></sec><sec id=\"irv12748-sec-0004\"><title>Conclusions</title><p>This is the first mathematically based study of seasonal threshold of influenza in Tunisia. As in other studies in different countries, the model has shown both good specificity and sensitivity, which allows timely and accurate detection of the start of influenza seasons. The findings will contribute to the development of more efficient measures for influenza prevention and control.</p></sec></abstract><kwd-group><kwd id=\"irv12748-kwd-0001\">epidemic threshold</kwd><kwd id=\"irv12748-kwd-0002\">ILI surveillance</kwd><kwd id=\"irv12748-kwd-0003\">influenza</kwd><kwd id=\"irv12748-kwd-0004\">moving epidemic method</kwd></kwd-group><counts><fig-count count=\"3\"/><table-count count=\"4\"/><page-count count=\"8\"/><word-count count=\"5245\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>September 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.6 mode:remove_FC converted:18.08.2020</meta-value></custom-meta></custom-meta-group></article-meta><notes><p content-type=\"self-citation\">\n<mixed-citation publication-type=\"journal\" id=\"irv12748-cit-1001\">\n<string-name>\n<surname>Bouguerra</surname>\n<given-names>H</given-names>\n</string-name>, <string-name>\n<surname>Boutouria</surname>\n<given-names>E</given-names>\n</string-name>, <string-name>\n<surname>Zorraga</surname>\n<given-names>M</given-names>\n</string-name>, et al. <article-title>Applying the moving epidemic method to determine influenza epidemic and intensity thresholds using influenza‐like illness surveillance data 2009‐2018 in Tunisia</article-title>. <source xml:lang=\"en\">Influenza Other Respi Viruses</source>. <year>2020</year>;<volume>14</volume>:<fpage>507</fpage>–<lpage>514</lpage>. <pub-id pub-id-type=\"doi\">10.1111/irv.12748</pub-id>\n</mixed-citation>\n</p><fn-group id=\"irv12748-ntgp-0001\"><fn fn-type=\"equal\" id=\"irv12748-note-0001\"><p>Hind Bouguerra and Elyes Boutouria equally contributed to this work.</p></fn></fn-group></notes></front><body id=\"irv12748-body-0001\"><sec id=\"irv12748-sec-0005\"><label>1</label><title>INTRODUCTION</title><p>Seasonal influenza continues to be a public health problem worldwide. Although in most cases, it leads to an increased number of consultations, it may cause severe illness and death especially among high‐risk groups. In fact, the World Health Organization (WHO) has recently updated global estimates to more than 3 million severe cases and from 290 000 to 650 000 respiratory deaths due to influenza each year.<xref rid=\"irv12748-bib-0001\" ref-type=\"ref\">\n<sup>1</sup>\n</xref> These annual epidemics mobilize considerable resources from health services and even a small‐scale epidemic can have a significant socio‐economic burden.</p><p>Ongoing monitoring and assessment of seasonal influenza are therefore essential to ensure early warning of epidemics and tailored preventive and control measures in real‐time. The last 2009 pandemic revealed many deficiencies in most countries' influenza surveillance systems, especially the capacity to estimate the severity of the season in a timely manner. For this reason, the WHO has progressively developed a framework on pandemic influenza severity assessment (PISA) and recommended member states to apply the proposed tools and measures.<xref rid=\"irv12748-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref> The framework is based on different steps, including setting thresholds for selected parameters and applying them in the routine surveillance of seasonal epidemics.</p><p>Various mathematical and statistical models have been developed to establish thresholds for influenza activity and study the dynamics of the disease.<xref rid=\"irv12748-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref>, <xref rid=\"irv12748-bib-0004\" ref-type=\"ref\">\n<sup>4</sup>\n</xref>, <xref rid=\"irv12748-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref> This mathematical modeling provides valuable information and a strong support to the preparedness and response plan. Of the popular methods currently in use, the moving epidemic method (MEM) is one of the most recommended and so far had provided a robust signal to detect influenza epidemics in many countries.<xref rid=\"irv12748-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref>, <xref rid=\"irv12748-bib-0007\" ref-type=\"ref\">\n<sup>7</sup>\n</xref> First developed in Spain in 2001, the MEM was adopted by the same authors to determine influenza thresholds in many European countries.<xref rid=\"irv12748-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> One of its strengths is its ability to also define different intensity levels in a given region or country and the possibility to compare them between countries and/or seasons.<xref rid=\"irv12748-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref>, <xref rid=\"irv12748-bib-0010\" ref-type=\"ref\">\n<sup>10</sup>\n</xref>\n</p><p>In Tunisia, influenza surveillance was first based on the virological surveillance ensured by the National Influenza Centre (NIC) recognized by the WHO since 1980 and supported by the Primary Health Care Direction of Ministry of Health. Starting from the late 1990s, the epidemiological surveillance was established through the network of Influenza‐like illness (ILI) sentinel sites at primary healthcare centers in the 24 governorates of the country. This network was progressively improved mainly by reducing the number from 268 in 1999 to 113 ILI sites in 2014, better representativeness and training of all staff involved in the surveillance.<xref rid=\"irv12748-bib-0011\" ref-type=\"ref\">\n<sup>11</sup>\n</xref>, <xref rid=\"irv12748-bib-0012\" ref-type=\"ref\">\n<sup>12</sup>\n</xref> Each year, the proportion of ILI among the total number of consultations at ILI sentinel sites determines the intensity of influenza season. The epidemic threshold of 10% adopted since then was based on combination of criteria and a national approach.<xref rid=\"irv12748-bib-0013\" ref-type=\"ref\">\n<sup>13</sup>\n</xref>, <xref rid=\"irv12748-bib-0014\" ref-type=\"ref\">\n<sup>14</sup>\n</xref>\n</p><p>Given the importance of seasonality and intensity levels in influenza severity assessment, our study aimed to determine the epidemic and intensity thresholds of influenza season in Tunisia by applying the moving epidemic method (MEM) based on ILI historical surveillance data of the last 9 years (2009‐2018).</p></sec><sec sec-type=\"methods\" id=\"irv12748-sec-0006\"><label>2</label><title>METHODS</title><sec id=\"irv12748-sec-0007\"><label>2.1</label><title>Available data</title><p>Influenza surveillance in Tunisia is carried out each year from 1st October (week 40) to 30th April (week 18) over a period of 30 weeks. Data collection is based on standardized forms of weekly aggregated data of ILI cases. These paper forms are sent from ILI sites at the local level to the regional directions in each governorate then to the Primary Health Care Direction at the national level. Aggregated data forms consist of general information including ILI site, governorate, the number of ILI cases and the total number of outpatients by gender and age groups (0‐5 years; 6‐16 years and ≥16 years). In Tunisia, case definition of ILI was an outpatient with fever (≥38°C) and cough or sore throat with onset less than 5 days prior to presentation in the absence of a specific diagnosis.<xref rid=\"irv12748-bib-0011\" ref-type=\"ref\">\n<sup>11</sup>\n</xref> Since 2014, the case definition recommended by WHO has been used instead: acute respiratory illness, and measured fever ≥38°C, and cough, and onset in previous 10 days.<xref rid=\"irv12748-bib-0012\" ref-type=\"ref\">\n<sup>12</sup>\n</xref> Collected data are analyzed to compute weekly ILI proportions compared with all outpatient acute consultations at both the national and regional levels. We analyzed data from up to nine influenza seasons (2009‐10 to 2017‐18).</p></sec><sec id=\"irv12748-sec-0008\"><label>2.2</label><title>Moving epidemic method</title><p>We applied the moving epidemic method (MEM) to establish epidemic and intensity thresholds, based on previous publications and the WHO's interim guidance for influenza severity assessment.<xref rid=\"irv12748-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref>, <xref rid=\"irv12748-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref>, <xref rid=\"irv12748-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref> For that, we used the R Language implementation of MEM (package “mem”) which is available online for free.<xref rid=\"irv12748-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref>, <xref rid=\"irv12748-bib-0015\" ref-type=\"ref\">\n<sup>15</sup>\n</xref> This method, based on a complex mathematical algorithm, can be summarized in three steps. First, determine the start, scope and end of the influenza epidemics by dividing the season in three periods (pre‐epidemic, epidemic and post‐epidemic periods). Then, epidemic thresholds are computed using the pre‐ and post‐epidemic values of historical seasons. Only a set of pre‐ and post‐epidemic values are used, of which we chose the highest n values for each season, with n = 30/number of seasons.<xref rid=\"irv12748-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> This step requires moving the epidemic seasons in order to match the epidemic periods, after which we compute the geometric mean of weekly rates as well as different levels of confidence intervals (50%, 90% and 95%). Last, thresholds for the different intensity levels are determined by the upper limits of these confidence intervals; each upper limit represents the threshold of one level of intensity of the epidemic. Five levels of intensity are thus defined:\n<list list-type=\"simple\" id=\"irv12748-list-0001\"><list-item><label>–</label><p>Baseline: below the epidemic threshold</p></list-item><list-item><label>–</label><p>Low level: between the epidemic and the medium thresholds</p></list-item><list-item><label>–</label><p>Medium level: between the medium and the high thresholds</p></list-item><list-item><label>–</label><p>High level: between the high and the very high thresholds</p></list-item><list-item><label>–</label><p>Very high level: above the very high threshold</p></list-item></list>\n</p><p>For the present work, we have described the epidemic and intensity thresholds from historical data of a period beginning in October 2009 and ending in April 2018.</p><p>The epidemic period is defined as the period of weeks with increased weekly values in a season. The periods of weeks before and after the epidemic period represent the pre‐epidemic and post‐epidemic periods, respectively. The epidemic threshold is the value which defines the start of the epidemic period while intensity thresholds represent the values marking the limit of the intensity levels. Besides, epidemic percentage is the sum of values in the epidemic period over the total sum of values of the whole influenza season, which reflects the coverage percentage of the epidemic period.</p></sec><sec id=\"irv12748-sec-0009\"><label>2.3</label><title>Cross‐validation procedure of the model</title><p>The goodness of the model was assessed using a cross validation procedure. This procedure is based on the extraction of each season from the historical series and using it as \"a target season\", for which we calculate the beginning and end of the epidemic period. Subsequently, the pre‐ and post‐epidemic thresholds are calculated on the basis of the remaining seasons and excluding the target season. These steps were repeated for all the available seasons.</p><p>Values of the target season inside and outside of the defined epidemic period were compared to the thresholds calculated using all historical information but the target season.</p><p>Aiming to evaluate the performance of the epidemic threshold to detect epidemics, we studied the sensitivity (Se), specificity (Sp), the positive predictive value (PPV) and the negative predictive value (NPV). The sensitivity consists of the model's ability to categorize epidemic weeks while the specificity is the model's ability to categorize non‐epidemic weeks. According to the cross‐validation analysis described by Vega et al,<xref rid=\"irv12748-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> sensitivity is computed by dividing the number of epidemic weeks above the pre‐epidemic threshold and above the post‐epidemic threshold by the number of epidemic weeks. Specificity is the number of non‐epidemic weeks below the pre‐epidemic threshold and below the post‐epidemic threshold divided by the number of non‐epidemic weeks.</p><p>The number of weeks of the epidemic above the threshold, divided by the number of weeks above the threshold is the positive predictive value, expressing the proportion of epidemic weeks correctly classified by the model. On the other hand, the negative predictive value consists of the number of non‐epidemic weeks below the threshold, divided by the number of weeks below the threshold, corresponding to the proportion of non‐epidemic weeks correctly classified by the model.</p><p>To optimize the goodness of the model, we also looked at the optimum slope parameter to find the value that maximizes the sensitivity and specificity. It is an inner parameter ranging from 2% to 4%.<xref rid=\"irv12748-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> Based on our data, it was 2.8% for both models with the inclusion and the exclusion of pandemic year 2009‐2010. The performance of the model was assessed by the Youden index (J = Sp + Se − 1), which reflects the variation of false positives and false negatives.</p></sec></sec><sec sec-type=\"results\" id=\"irv12748-sec-0010\"><label>3</label><title>RESULTS</title><sec id=\"irv12748-sec-0011\"><label>3.1</label><title>Descriptive analysis of the epidemic movement of influenza in Tunisia</title><p>The beginning, the end and the extent of the epidemic seasons differed from one season to another (Table <xref rid=\"irv12748-tbl-0001\" ref-type=\"table\">1</xref>).</p><table-wrap id=\"irv12748-tbl-0001\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 1</label><caption><p>Characteristics of influenza epidemics in Tunis from 2009‐10 to 2017‐18 seasons</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Season</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Epidemic start</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Epidemic end</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Epidemic duration</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Peak week</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Peak<xref ref-type=\"fn\" rid=\"irv12748-note-0002\">\n<sup>a</sup>\n</xref>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Epidemic %<xref ref-type=\"fn\" rid=\"irv12748-note-0003\">\n<sup>b</sup>\n</xref>\n</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2009‐2010</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">48</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">51</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">30.3%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">74.77%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2010‐2011</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">46</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.2%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">76.63%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2011‐2012</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">46</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.3%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">77.52%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2012‐2013</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">49</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">19</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">15.1%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">76.63%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2013‐2014</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">46</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.5%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">76.62%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2014‐2015</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.3%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">76.61%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2015‐2016</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">47</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.2%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">76.52%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2016‐2017</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">44</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">19</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.9%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">76.51%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2017‐2018</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">51</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.64%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">76.62%</td></tr></tbody></table><table-wrap-foot id=\"irv12748-ntgp-0002\"><fn id=\"irv12748-note-0002\"><label><sup>a</sup></label><p>Proportion of ILI among the total number of consultations at ILI sentinel sites.</p></fn><fn id=\"irv12748-note-0003\"><label><sup>b</sup></label><p>Coverage percentage of the epidemic period.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>In most cases, influenza epidemic started around week 46. The first activity of influenza epidemic was observed in 2017‐2018 at week 43 and the latest one in 2012‐2013 at week 49.</p><p>Influenza epidemic during the pandemic year 2009‐2010 started at week 48 and ended at week 6. The duration of epidemic seasons ranged from 11 weeks (2009‐2010 season) to 23 weeks (2015‐2016 season). Epidemic percentage varied between 74.77% and 77.52%, corresponding to a high coverage of the epidemic period.</p><p>Almost all the seasons were one‐wave seasons except 2010‐2011 and 2014‐2015 with more than one wave. Most often, epidemic peaks were observed between week 3 and week 9. However, some seasons peaked earlier (week 51 in 2009‐2010 and 2017‐2018) and other seasons peaked later (week 12 in 2015‐16). The epidemic seasons seem to follow a pattern with a rapid increase at the beginning and a slowly decrease at the end of the season.</p><p>Besides, Figure <xref rid=\"irv12748-fig-0001\" ref-type=\"fig\">1</xref> displays the curves of all the studied seasons based on the accumulated maximum percentage rate method, specifying the pre‐ and post‐epidemic periods as well as the corresponding epidemic periods. ILI consultation rates differed also with the season, with the highest peak registered during 2009‐2010 (30.3%) and the lowest in 2010‐2011 season (9.2%).</p><fig fig-type=\"FIGURE\" xml:lang=\"en\" id=\"irv12748-fig-0001\" orientation=\"portrait\" position=\"float\"><label>FIGURE 1</label><caption><p>Pre‐epidemic, epidemic and post‐epidemic periods of influenza seasons (2009‐10 to 2017‐18)</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"IRV-14-507-g001\"/></fig><p>Except the pandemic season which was considerate of a very high intensity, most of the seasons were described as low. This was useful to characterize the dynamics of influenza over time.</p></sec><sec id=\"irv12748-sec-0012\"><label>3.2</label><title>Epidemic and intensity thresholds</title><p>The different thresholds and intensity levels were determined using two models; one including and the other excluding the 2009‐10 pandemic season.</p><p>When including the pandemic season, the average duration of influenza epidemic during a typical season was 20 weeks (Figures <xref rid=\"irv12748-fig-0002\" ref-type=\"fig\">2A</xref> and <xref rid=\"irv12748-fig-0003\" ref-type=\"fig\">3A</xref>). This optimal duration of 20 weeks covers 76.61% of total sum of proportions.</p><fig fig-type=\"FIGURE\" xml:lang=\"en\" id=\"irv12748-fig-0002\" orientation=\"portrait\" position=\"float\"><label>FIGURE 2</label><caption><p>Epidemic movement including and excluding 2009‐2010 season. (A) Including 2009‐2010 season and (B) Excluding 2009‐2010 season</p></caption><graphic id=\"nlm-graphic-3\" xlink:href=\"IRV-14-507-g002\"/></fig><fig fig-type=\"FIGURE\" xml:lang=\"en\" id=\"irv12748-fig-0003\" orientation=\"portrait\" position=\"float\"><label>FIGURE 3</label><caption><p>Average curve including and excluding 2009‐10 season. (A) Including 2009‐2010 season and (B) Excluding 2009‐2010 season</p></caption><graphic id=\"nlm-graphic-5\" xlink:href=\"IRV-14-507-g003\"/></fig><p>As a result of this analysis, epidemic threshold was 8.99% if we include the pandemic season and post‐epidemic threshold was 8.25%.</p><p>Medium, high and very high intensity thresholds were 10.48%, 17.69%, and 22.3%, respectively (Table <xref rid=\"irv12748-tbl-0002\" ref-type=\"table\">2</xref>). Regarding the pandemic influenza season 2009‐2010, higher thresholds were observed comparing to the other seasons.</p><table-wrap id=\"irv12748-tbl-0002\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 2</label><caption><p>Influenza epidemic thresholds and intensity levels including and excluding the pandemic year</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"> </th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Epidemic threshold</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Medium intensity threshold</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">High intensity threshold</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Very high intensity threshold</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Including 2009‐2010</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">8.99%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.48%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">17.69%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">22.3%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Excluding 2009‐2010</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6.25%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.74%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">12.05%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">13.24%</td></tr></tbody></table><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>When we excluded the pandemic season 2009‐2010, the average curve lasted 20 weeks and it covered 76.62% of the total rates (Figures <xref rid=\"irv12748-fig-0002\" ref-type=\"fig\">2B</xref> and <xref rid=\"irv12748-fig-0003\" ref-type=\"fig\">3B</xref>). The different parameters and indicators of epidemic threshold calculation decreased.</p><p>The levels of intensity, if we exclude 2009‐2010 season, were established as follows: low, if ILI proportion was below 9.74%, medium below 12.05%; high below 13.27%; and very high above this last proportion. These different intensity thresholds increased to 10.48% for the medium intensity level, to 17.69% for the high intensity level, to 22.3% for the very high threshold when including the pandemic season (Table <xref rid=\"irv12748-tbl-0002\" ref-type=\"table\">2</xref>).</p></sec><sec id=\"irv12748-sec-0013\"><label>3.3</label><title>Cross validation of the model</title><p>Table <xref rid=\"irv12748-tbl-0003\" ref-type=\"table\">3</xref> presents the contribution of the different influenza seasons to calculate the epidemic threshold and how each season can affect this calculation. By proceeding to the comparison of the target season's rate during the epidemic and non‐epidemic period to the thresholds computed and excluding the target season, we observed differences in some seasons and others not. For instance, the pandemic season 2009‐2010 affected the estimate of the epidemic threshold. In fact, its exclusion lead to an important decrease of the epidemic threshold to 6.25% as well as different intensity thresholds.</p><table-wrap id=\"irv12748-tbl-0003\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 3</label><caption><p>Contribution and influence of influenza seasons in the estimate of the influenza epidemic threshold: cross‐validation procedure</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Season</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Peak<xref ref-type=\"fn\" rid=\"irv12748-note-0004\">\n<sup>a</sup>\n</xref> (%)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Peak week</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Epidemic threshold</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Medium threshold</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">High threshold</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Very high threshold</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Level</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Description</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2009/2010</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">30.3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">51</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6.25</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.74</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">12.05</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">13.24</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Very high</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2010/2011</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.2</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.14</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.62</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">17.91</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">22.59</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2011/2012</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.16</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">17.84</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">22.65</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2012/2013</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">15.1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.02</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">16.99</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">21.46</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Medium</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2013/2014</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.14</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.55</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">17.91</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">22.63</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2014/2015</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.12</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.42</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">17.86</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">22.65</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2015/2016</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.2</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.13</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.45</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">17.88</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">22.66</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2016/2017</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.13</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.35</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">17.78</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">22.59</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Medium</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2017/2018</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.6</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">51</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9.09</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">17.9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">22.67</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low</td></tr></tbody></table><table-wrap-foot id=\"irv12748-ntgp-0003\"><fn id=\"irv12748-note-0004\"><label><sup>a</sup></label><p>Proportion of ILI among the total number of consultations at ILI sentinel sites.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>This validation also allowed us to characterize the overall intensity of each season. Out of the nine seasons, six were low, two as medium, and one very high intensity (Table <xref rid=\"irv12748-tbl-0003\" ref-type=\"table\">3</xref>).</p><p>The MEM provided a sensitivity of 85% in detecting the epidemic period. This sensitivity during the overall seasons and for each influenza season increased from 39% to 85% if we exclude the pandemic year. However, the specificity was higher with inclusion of 2009‐10 and decreased from 87% to 69%. Simultaneously, there was a slight change in the VPP but the VPN significantly increased when excluding 2009‐10. Other indicators also increased if we did not consider the 2009‐10 season (Table <xref rid=\"irv12748-tbl-0004\" ref-type=\"table\">4</xref>).</p><table-wrap id=\"irv12748-tbl-0004\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 4</label><caption><p>Goodness of the model</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"> </th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Sensitivity</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Specificity</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Positive predictive value</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Negative predictive value</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Youden index</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Including 2009‐2010</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">39%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">87%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">77%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">52%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">23%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Excluding 2009‐2010</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">85%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">69%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">79%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">76%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">53%</td></tr></tbody></table><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap></sec></sec><sec sec-type=\"discussion\" id=\"irv12748-sec-0014\"><label>4</label><title>DISCUSSION</title><p>For almost two decades, the intensity of influenza activity in Tunisia has been estimated from data of the Sentinel Surveillance System based on a seasonal threshold of 10%, which was set up initially on a combination of criteria. The present study aimed to determine the epidemic threshold of influenza in Tunisia by the moving epidemic method using data of the nine past seasons (from 2009 to 2018).</p><p>This choice was largely motivated by the type of data available by the Tunisian influenza surveillance system. In fact, the basic requirements of the MEM are simple and reliable epidemiological data for a time period between 5 and 10 years, preferably ILI data.<xref rid=\"irv12748-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref>\n</p><p>The MEM is a tool developed to better understand annual influenza epidemics and assess the epidemic status and intensity on a weekly basis.<xref rid=\"irv12748-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref>, <xref rid=\"irv12748-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref> The method was progressively improved and implemented in European documents by the ECDC and WHO.<xref rid=\"irv12748-bib-0015\" ref-type=\"ref\">\n<sup>15</sup>\n</xref> Later on, it became widely used in many countries outside Europe such as USA, Australia, New Zealand, and Canada.<xref rid=\"irv12748-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref>, <xref rid=\"irv12748-bib-0010\" ref-type=\"ref\">\n<sup>10</sup>\n</xref>, <xref rid=\"irv12748-bib-0016\" ref-type=\"ref\">\n<sup>16</sup>\n</xref>, <xref rid=\"irv12748-bib-0017\" ref-type=\"ref\">\n<sup>17</sup>\n</xref> Other countries have opted for the method proposed by WHO and based on the peak mean values of influenza activity.<xref rid=\"irv12748-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref>, <xref rid=\"irv12748-bib-0018\" ref-type=\"ref\">\n<sup>18</sup>\n</xref>, <xref rid=\"irv12748-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref>\n</p><p>The determined epidemic threshold with the exclusion of the pandemic season was 6.25%. It showed a very high sensitivity (85%) and a high specificity (69%). However, when including 2009‐10, the threshold increased to 8.99% with a sensitivity and specificity of 39% and 87%, respectively. The different levels of intensity were also affected with a considerable increase. This is understandable since there was higher ILI rates registered during this year and thus higher pre‐ and post‐epidemic thresholds comparing to the other seasons. This confirms the importance of the pandemic year in the analysis and the need to exclude it when estimating the parameters and indicators of epidemic threshold.<xref rid=\"irv12748-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref>, <xref rid=\"irv12748-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref>\n</p><p>The majority of authors used ILI consultations in primary healthcare settings expressed per 100 000 population <xref rid=\"irv12748-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref>, <xref rid=\"irv12748-bib-0020\" ref-type=\"ref\">\n<sup>20</sup>\n</xref>, <xref rid=\"irv12748-bib-0021\" ref-type=\"ref\">\n<sup>21</sup>\n</xref>, <xref rid=\"irv12748-bib-0022\" ref-type=\"ref\">\n<sup>22</sup>\n</xref> or per 1000,<xref rid=\"irv12748-bib-0016\" ref-type=\"ref\">\n<sup>16</sup>\n</xref> which made the comparison with our results difficult, in addition to other differences in health systems, data collection methods as well as socio‐demographic characteristics, since most studies were from Europe or other developed countries. Studies on establishing influenza thresholds using the MEM in North Africa and the Eastern Mediterranean Region are limited. So far, only one study conducted in Egypt was available but aims and methodology were different.<xref rid=\"irv12748-bib-0023\" ref-type=\"ref\">\n<sup>23</sup>\n</xref> It would therefore be useful to establish one common method for ILI data analysis and interpretation in our region as was done in Europe.<xref rid=\"irv12748-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref>, <xref rid=\"irv12748-bib-0022\" ref-type=\"ref\">\n<sup>22</sup>\n</xref>\n</p><p>Applying the MEM to define the thresholds also allowed us to visualize influenza activity in Tunisia for the past nine seasons. Overall, influenza seasons seem to be mostly one‐wave, homogenous with a seasonal pattern. Epidemics usually start between the 43rd and the 49th week and last from 11 to 23 weeks. The duration of epidemic seasons was comparable to the range reported in other studies (6‐25 weeks)<xref rid=\"irv12748-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref>, <xref rid=\"irv12748-bib-0007\" ref-type=\"ref\">\n<sup>7</sup>\n</xref>, <xref rid=\"irv12748-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref> and more specifically 12‐19 weeks in Europe.<xref rid=\"irv12748-bib-0024\" ref-type=\"ref\">\n<sup>24</sup>\n</xref> Except the pandemic season which was considerate of a very high intensity, most of the seasons were described as low. This was useful to characterize the dynamics of influenza over time. This seasonality and epidemiological patterns are a common thread in neighboring countries and most regions of the Northern Hemisphere sharing the same winter timing.<xref rid=\"irv12748-bib-0024\" ref-type=\"ref\">\n<sup>24</sup>\n</xref>, <xref rid=\"irv12748-bib-0025\" ref-type=\"ref\">\n<sup>25</sup>\n</xref>, <xref rid=\"irv12748-bib-0026\" ref-type=\"ref\">\n<sup>26</sup>\n</xref>\n</p><p>The specificity of the determined epidemic threshold was lower than its sensitivity. Sensitivity is important for detecting epidemics but specificity is crucial to avoid false alerts. In fact, once an epidemic is declared, the media's interest increases and prevention and control measures are implemented, especially vaccination campaigns and antiviral use.<xref rid=\"irv12748-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> That is why it is important to avoid false alerts and to use these attributes wisely trying to find the good balance between specificity and sensitivity.</p><p>Besides, it is important to underline that the specificity of the model is related to the case definition used. The lower is the specificity of the case definition, the lower is the specificity of the model. Although a new case definition was used since 2014, the changes enhanced sensitivity without greatly compromising the specificity.<xref rid=\"irv12748-bib-0027\" ref-type=\"ref\">\n<sup>27</sup>\n</xref> We therefore consider that the specificity of the model did not change over time. The specificity found in our results can be explained by these rather sensitive case definitions, which may increase the identification of other respiratory pathogens especially the respiratory syncytial virus (RSV) and lead to more consultations of acute respiratory infections (ARI) than ILI. Other factors may affect the outpatient rates including public anxiety and excessive awareness and sensitization of physicians in case of false alerts. The continuous training of ILI sites especially on the precise definitions improves the MEM's performance and precision in the epidemic threshold's estimation.</p><p>This low specificity may represent the main limitation of our study. Most authors concluded to models with very high specificity. In Vega's study about establishing thresholds in 19 European countries, the lowest specificity was 89.6% in Kazakhstan with an overall specificity of 95.5%.<xref rid=\"irv12748-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> Other limitations may have resulted of changes in demographics, case reporting and especially ILI case definition, which was modified in 2014, in the second half of the study period.</p><p>The circulation of a new influenza virus, as it was observed during the 2009‐2010 pandemic in many countries, may also generate abnormal epidemiological data and falsely positive results.</p><p>In these situations, additional virological data are necessary to confirm the start of the epidemic period, especially that our results showed a better performance of the MEM model excluding the pandemic season than the one including this season.</p></sec><sec sec-type=\"conclusions\" id=\"irv12748-sec-0015\"><label>5</label><title>CONCLUSION</title><p>In summary, the moving epidemic method is a simple method offering a flexible procedure to calculate epidemic thresholds based on historical epidemiological data. Its strength lies in its ability to also determine different intensity thresholds useful to the weekly monitoring of the season's intensity.</p><p>Our study is the first mathematically based study of seasonal threshold of influenza in Tunisia using historical ILI weekly data. The determined epidemic threshold was 6.25%, differing from the threshold of 10% adopted until now. The high sensitivity and specificity of this threshold in the detection of epidemics make it robust and reliable.</p><p>Indicating the start and assessing the intensity of influenza seasons remain a high priority for Ministries of Health, not only at the national level for timely preventive and control measures but also at the international level by contributing to the pandemic preparedness. The next step is therefore to implement the use of the determined epidemic threshold for public health purposes with monitoring the next seasons.</p></sec></body><back><ref-list content-type=\"cited-references\" id=\"irv12748-bibl-0001\"><title>REFERENCES</title><ref id=\"irv12748-bib-0001\"><label>1</label><mixed-citation publication-type=\"book\" id=\"irv12748-cit-0001\">\n<collab collab-type=\"authors\">WHO</collab>\n. <source xml:lang=\"en\">Influenza (Seasonal) [Internet]</source>. <publisher-name>World Health Organization</publisher-name>; <year>2018</year>\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.who.int/news-room/fact-sheets/detail/influenza-(seasonal)\">http://www.who.int/news‐room/fact‐sheets/detail/influenza‐(seasonal)</ext-link>. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"letter\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"iso-abbrev\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"doi\">10.1111/(ISSN)1750-2659</journal-id><journal-id journal-id-type=\"publisher-id\">IRV</journal-id><journal-title-group><journal-title>Influenza and Other Respiratory Viruses</journal-title></journal-title-group><issn pub-type=\"ppub\">1750-2640</issn><issn pub-type=\"epub\">1750-2659</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32319220</article-id><article-id pub-id-type=\"pmc\">PMC7431643</article-id><article-id pub-id-type=\"doi\">10.1111/irv.12746</article-id><article-id pub-id-type=\"publisher-id\">IRV12746</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Letter to the Editor</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Letters to the Editor</subject></subj-group></article-categories><title-group><article-title>Covid‐19 and Namaste</article-title></title-group><contrib-group><contrib id=\"irv12746-cr-0001\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Kulkarni</surname><given-names>Prashanth</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-2170-2103</contrib-id><xref ref-type=\"aff\" rid=\"irv12746-aff-0001\">\n<sup>1</sup>\n</xref><address><email>docpk77@gmail.com</email></address></contrib><contrib id=\"irv12746-cr-0002\" contrib-type=\"author\"><name><surname>Kodad</surname><given-names>Shruthi</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-8597-1049</contrib-id><xref ref-type=\"aff\" rid=\"irv12746-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12746-cr-0003\" contrib-type=\"author\"><name><surname>Mahadevappa</surname><given-names>Manjappa</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-8710-2397</contrib-id><xref ref-type=\"aff\" rid=\"irv12746-aff-0003\">\n<sup>3</sup>\n</xref></contrib></contrib-group><aff id=\"irv12746-aff-0001\">\n<label><sup>1</sup></label>\n<named-content content-type=\"organisation-division\">Department of Cardiology</named-content>\n<institution>Care Hospitals</institution>\n<city>Hyderabad</city>\n<country country=\"IN\">India</country>\n</aff><aff id=\"irv12746-aff-0002\">\n<label><sup>2</sup></label>\n<named-content content-type=\"organisation-division\">Department of Haematology</named-content>\n<institution>Saskatoon Cancer Centre</institution>\n<city>Saskatoon</city>\n<named-content content-type=\"country-part\">SK</named-content>\n<country country=\"CA\">Canada</country>\n</aff><aff id=\"irv12746-aff-0003\">\n<label><sup>3</sup></label>\n<named-content content-type=\"organisation-division\">Department of Cardiology</named-content>\n<institution>Jagadguru Sri Shivarathreeshwara University</institution>\n<city>Mysuru</city>\n<country country=\"IN\">India</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label><bold>Correspondence</bold><break/>\nKulkarni Prashanth, Department of Cardiology, Care Hospitals, Old Mumbai Highway, Near Cyberabad Commissionerate, Hi‐Tech City, Hyderabad 500032, India.<break/>\nEmail: <email>docpk77@gmail.com</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>21</day><month>4</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>9</month><year>2020</year></pub-date><volume>14</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1111/irv.v14.5</issue-id><fpage>601</fpage><lpage>602</lpage><history><date date-type=\"received\"><day>30</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>02</day><month>4</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 John Wiley & Sons Ltd <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Influenza and Other Respiratory Viruses</italic> Published by John Wiley & Sons Ltd.</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:IRV-14-601.pdf\"/><kwd-group><kwd id=\"irv12746-kwd-0001\">Covid‐19</kwd><kwd id=\"irv12746-kwd-0002\">Namaste</kwd><kwd id=\"irv12746-kwd-0003\">pandemic</kwd></kwd-group><counts><fig-count count=\"1\"/><table-count count=\"0\"/><page-count count=\"2\"/><word-count count=\"454\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>September 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.6 mode:remove_FC converted:18.08.2020</meta-value></custom-meta></custom-meta-group></article-meta><notes><fn-group id=\"irv12746-ntgp-0001\"><fn id=\"irv12746-note-0001\"><p>The peer review history for this article is available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://publons.com/publon/10.1111/irv.12746\">https://publons.com/publon/10.1111/irv.12746</ext-link>\n</p></fn></fn-group></notes></front><body id=\"irv12746-body-0001\"><p>\n<named-content content-type=\"salutation\">To the Editor‐in‐Chief</named-content>\n</p><sec sec-type=\"opening-section\" id=\"irv12746-sec-0001\"><p>The novel coronavirus (SARSCoV‐2) has emerged as a major pandemic stretching the healthcare resources of most countries of the world. In this context, it is imperative that social distancing and good hand hygiene is practised to stem the transmission of this highly contagious virus.</p><p>The WHO’s standard recommendations to prevent infection spread include regular hand washing, covering mouth and nose when coughing and sneezing, thoroughly cooking meat and eggs and to avoid close contact with anyone showing symptoms of this respiratory illness.<xref rid=\"irv12746-bib-0001\" ref-type=\"ref\">\n<sup>1</sup>\n</xref> Also shaking hands or any form of hand‐to‐hand contact should be avoided as cross‐transmission of organisms occurs through contaminated hands.<xref rid=\"irv12746-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref>\n</p><p>In most countries of the World, handshake, fist bump, high five and hugs are some of the different methods of greeting each other, which leads to physical proximity and contact, facilitating rapid propagation of infections such as Covid‐19.</p><p>Alternatively, other non‐physical greeting forms can be explored like Namaste, which is used in Indian subcontinent since hundreds of years to greet people with folded hands, while maintaining a fair distance from each other [Figure <xref rid=\"irv12746-fig-0001\" ref-type=\"fig\">1</xref>]. An individual in addition to saying “Namaste” presses his hands together in front of the chest and respectfully greets the other person. This form of greeting does not involve any physical touch between individuals and gives a sense of parity to all the parties.<xref rid=\"irv12746-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref>\n</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"irv12746-fig-0001\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Namaste</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"IRV-14-601-g001\"/></fig><p>In addition to following general principles of meticulous hand washing, rapid transmission of infections both in hospitals and the community can be overcome by adopting the no‐touch salutation Namaste and other such forms like bowing the head as done in some Asian countries.</p></sec><sec sec-type=\"COI-statement\" id=\"irv12746-sec-0002\"><title>CONFLICT OF INTEREST</title><p>The authors declare no conflict of interest.</p></sec><sec id=\"irv12746-sec-0003\"><title>AUTHORS CONTRIBUTION</title><p>Prashanth Kulkarni: Conceptualization‐Lead, Methodology‐Lead, Resources‐Lead, Software‐Lead, Writing‐original draft‐Lead, Writing‐review & editing‐Equal; Shruthi Kodad: Supervision‐Equal, Validation‐Equal, Visualization‐Equal; Manjappa Mahadevappa: Visualization‐Equal, Writing‐review & editing‐Equal.</p></sec></body><back><ref-list content-type=\"cited-references\" id=\"irv12746-bibl-0001\"><title>REFERENCES</title><ref id=\"irv12746-bib-0001\"><label>1</label><mixed-citation publication-type=\"book\" id=\"irv12746-cit-0001\">\n<collab collab-type=\"authors\">WHO</collab>\n. <source xml:lang=\"en\">WHO Basic Protective Measures Against the New Coronavirus 2019</source>,<publisher-loc>Geneva, Switzerland</publisher-loc>: <collab collab-type=\"authors\">World Health Organization</collab>\n; <year>2019</year>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.who.int/emergencies/diseases/novel-coronavirus-2019/advice-for-public\">https://www.who.int/emergencies/diseases/novel‐coronavirus‐2019/advice‐for‐public</ext-link>. Google Scholar, Accessed March 29, 2020</mixed-citation></ref><ref id=\"irv12746-bib-0002\"><label>2</label><mixed-citation publication-type=\"book\" id=\"irv12746-cit-0002\">\n<collab collab-type=\"authors\">WHO</collab>\n. <source xml:lang=\"en\">WHO Guidelines on Hand Hygiene in Health Care</source>. <publisher-loc>Geneva, Switzerland</publisher-loc>: <publisher-name>World Health Organization</publisher-name>; <year>2009</year>\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.who.int/gpsc/en/\">http://www.who.int/gpsc/en/</ext-link>. Google Scholar Accessed March 29, 2020,</mixed-citation></ref><ref id=\"irv12746-bib-0003\"><label>3</label><mixed-citation publication-type=\"journal\" id=\"irv12746-cit-0003\">\n<string-name>\n<surname>Parmar</surname>\n<given-names>MS</given-names>\n</string-name>. <article-title>Namaste or handshake: time to ponder</article-title>. <source xml:lang=\"en\">BMJ</source>. <year>2009</year>; <volume>339</volume>:<fpage>b2651</fpage>.<pub-id pub-id-type=\"pmid\">19574308</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"iso-abbrev\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"doi\">10.1111/(ISSN)1750-2659</journal-id><journal-id journal-id-type=\"publisher-id\">IRV</journal-id><journal-title-group><journal-title>Influenza and Other Respiratory Viruses</journal-title></journal-title-group><issn pub-type=\"ppub\">1750-2640</issn><issn pub-type=\"epub\">1750-2659</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32579785</article-id><article-id pub-id-type=\"pmc\">PMC7431644</article-id><article-id pub-id-type=\"doi\">10.1111/irv.12755</article-id><article-id pub-id-type=\"publisher-id\">IRV12755</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Original Article</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Original Articles</subject></subj-group></article-categories><title-group><article-title>Clinical phase II and III studies of an AS03‐adjuvanted H5N1 influenza vaccine produced in an EB66<sup>®</sup> cell culture platform</article-title><alt-title alt-title-type=\"left-running-head\">ENDO et al.</alt-title></title-group><contrib-group><contrib id=\"irv12755-cr-0001\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Endo</surname><given-names>Masafumi</given-names></name><xref ref-type=\"aff\" rid=\"irv12755-aff-0001\">\n<sup>1</sup>\n</xref><address><email>endou-ma@kmbiologics.com</email></address></contrib><contrib id=\"irv12755-cr-0002\" contrib-type=\"author\"><name><surname>Tanishima</surname><given-names>Mitsuyoshi</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0003-3289-0127</contrib-id><xref ref-type=\"aff\" rid=\"irv12755-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12755-cr-0003\" contrib-type=\"author\"><name><surname>Ibaragi</surname><given-names>Kayo</given-names></name><xref ref-type=\"aff\" rid=\"irv12755-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12755-cr-0004\" contrib-type=\"author\"><name><surname>Hayashida</surname><given-names>Kenshi</given-names></name><xref ref-type=\"aff\" rid=\"irv12755-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12755-cr-0005\" contrib-type=\"author\"><name><surname>Fukuda</surname><given-names>Tadashi</given-names></name><xref ref-type=\"aff\" rid=\"irv12755-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12755-cr-0006\" contrib-type=\"author\"><name><surname>Tanabe</surname><given-names>Tetsuro</given-names></name><xref ref-type=\"aff\" rid=\"irv12755-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12755-cr-0007\" contrib-type=\"author\"><name><surname>Naruse</surname><given-names>Takeshi</given-names></name><xref ref-type=\"aff\" rid=\"irv12755-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12755-cr-0008\" contrib-type=\"author\"><name><surname>Kino</surname><given-names>Yoichiro</given-names></name><xref ref-type=\"aff\" rid=\"irv12755-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12755-cr-0009\" contrib-type=\"author\"><name><surname>Ueda</surname><given-names>Kohji</given-names></name><xref ref-type=\"aff\" rid=\"irv12755-aff-0003\">\n<sup>3</sup>\n</xref></contrib></contrib-group><aff id=\"irv12755-aff-0001\">\n<label><sup>1</sup></label>\n<institution>KM Biologics Co., Ltd. (KM Biologics)</institution>\n<city>Kumamoto</city>\n<country country=\"JP\">Japan</country>\n</aff><aff id=\"irv12755-aff-0002\">\n<label><sup>2</sup></label>\n<institution>Kino Consulting</institution>\n<city>Kumamoto</city>\n<country country=\"JP\">Japan</country>\n</aff><aff id=\"irv12755-aff-0003\">\n<label><sup>3</sup></label>\n<institution>Kyushu University</institution>\n<city>Fukuoka</city>\n<country country=\"JP\">Japan</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label><bold>Correspondence</bold><break/>\nMasafumi Endo, KM Biologics Co., Ltd (KM Biologics), 869‐1298 Kumamoto, Japan.<break/>\nEmail: <email>endou-ma@kmbiologics.com</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>24</day><month>6</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>9</month><year>2020</year></pub-date><volume>14</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1111/irv.v14.5</issue-id><fpage>551</fpage><lpage>563</lpage><history><date date-type=\"received\"><day>09</day><month>11</month><year>2018</year></date><date date-type=\"rev-recd\"><day>09</day><month>11</month><year>2019</year></date><date date-type=\"accepted\"><day>21</day><month>4</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 John Wiley & Sons Ltd <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Influenza and Other Respiratory Viruses</italic> Published by John Wiley & Sons Ltd.</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:IRV-14-551.pdf\"/><abstract id=\"irv12755-abs-0001\"><title>Abstract</title><sec id=\"irv12755-sec-0001\"><title>Background</title><p>We have developed an AS03‐adjuvanted H5N1 influenza vaccine produced in an EB66<sup>®</sup> cell culture platform (KD‐295).</p></sec><sec id=\"section_ggj_dv3_cmb\"><title>Objectives</title><p>In accordance with Japanese guidelines for development of pandemic prototype vaccines, the phase II study was conducted in a double‐blind, randomized, parallel‐group comparison study and the phase III study was conducted in an open‐label, non‐randomized, uncontrolled study.</p></sec><sec id=\"irv12755-sec-0002\"><title>Methods</title><p>Healthy adult volunteers aged 20 ‐ 64 years enrolled in the phase II and III studies (N = 248 and N = 369) received KD‐295 intramuscularly twice with a 21‐day interval. After administration, immune response and adverse events were evaluated. In the phase II study, four different vaccine formulations were compared: MA (3.75 μg hemagglutinin [HA] antigen + AS03 adjuvant system), MB (3.75 μg HA + 1/2AS03), HA (7.5 μg HA + AS03), and HB (7.5 μg HA + 1/2AS03). In the phase III study, the MA formulation was further evaluated.</p></sec><sec id=\"irv12755-sec-0003\"><title>Results</title><p>In the phase II study, all four vaccine formulations were well‐tolerated and no SAE related to vaccination were observed. The MA formulation was slightly more immunogenic and less reactogenic among the vaccine formulations. Therefore, the MA formulation was selected for the phase III study, and it was well‐tolerated and no serious adverse drug reactions were observed. The vaccine fulfilled the three immunogenicity criteria described in the Japanese guidelines.</p></sec><sec id=\"irv12755-sec-0004\"><title>Conclusions</title><p>These data indicate that the MA formulation of KD‐295 was well‐tolerated and highly immunogenic and it can be considered a useful pandemic and pre‐pandemic influenza vaccine.</p></sec></abstract><kwd-group><kwd id=\"irv12755-kwd-0001\">AS03</kwd><kwd id=\"irv12755-kwd-0002\">EB66<sup>®</sup> cells</kwd><kwd id=\"irv12755-kwd-0003\">H5N1 influenza</kwd></kwd-group><counts><fig-count count=\"0\"/><table-count count=\"6\"/><page-count count=\"13\"/><word-count count=\"8377\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>September 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.6 mode:remove_FC converted:18.08.2020</meta-value></custom-meta></custom-meta-group></article-meta><notes><p content-type=\"self-citation\">\n<mixed-citation publication-type=\"journal\" id=\"irv12755-cit-1001\">\n<string-name>\n<surname>Endo</surname>\n<given-names>M</given-names>\n</string-name>, <string-name>\n<surname>Tanishima</surname>\n<given-names>M</given-names>\n</string-name>, <string-name>\n<surname>Ibaragi</surname>\n<given-names>K</given-names>\n</string-name>, et al. <article-title>Clinical phase II and III studies of an AS03‐adjuvanted H5N1 influenza vaccine produced in an EB66<sup>®</sup> cell culture platform</article-title>. <source xml:lang=\"en\">Influenza Other Respi Viruses</source>. <year>2020</year>;<volume>14</volume>:<fpage>551</fpage>–<lpage>563</lpage>. <pub-id pub-id-type=\"doi\">10.1111/irv.12755</pub-id>\n</mixed-citation>\n</p><fn-group id=\"irv12755-ntgp-0001\"><fn id=\"irv12755-note-0001\"><p>The peer review history for this article is available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://publons.com/publon/10.1111/irv.12755\">https://publons.com/publon/10.1111/irv.12755</ext-link>\n</p></fn><fn fn-type=\"funding\" id=\"irv12755-note-0002\"><p>Funding information</p><p>These studies were sponsored by KM Biologics. Funding was also provided by GlaxoSmithKline Biologicals SA (GSK studies identifier: 202072 & 202073). GlaxoSmithKline Biologicals SA was provided the opportunity to review a preliminary version of this manuscript for factual accuracy but the authors are solely responsible for final content and interpretation.</p></fn></fn-group></notes></front><body id=\"irv12755-body-0001\"><sec id=\"irv12755-sec-0005\"><label>1</label><title>INTRODUCTION</title><p>The most recent influenza pandemic of 2009‐2010 remains fresh in our minds. Contrary to global expectations, the causative agent of the pandemic was an H1N1 virus. In the 2009 pandemic, various vaccines were used, including non‐adjuvanted and adjuvanted subvirion and whole virion vaccines.<xref rid=\"irv12755-bib-0001\" ref-type=\"ref\">\n<sup>1</sup>\n</xref> Among adults, the results of the vaccine use confirmed that even non‐adjuvanted vaccines were highly immunogenic. This is because there was cross‐reactivity in T helper epitopes between the H1N1 pandemic 2009 virus and previous seasonal H1N1 viruses.<xref rid=\"irv12755-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref> In Japan, the local vaccine manufacturers produced monovalent non‐adjuvanted split vaccine. At the same time, the Japanese government imported adjuvanted vaccines as a precaution in case of vaccine shortages, but many of these imported vaccines were left unused. However, pandemic threats, such as H5N1, have not disappeared and nobody knows what virus subtype will cause the next pandemic. At this moment, among the viruses with pandemic potential, viruses of avian origin, including the H7N9 subtypes, are a concern because of sporadic human infection.<xref rid=\"irv12755-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref> As same with H5N1 subtypes, and unlike the H1N1 pandemic 2009 virus, immunogenicity of those viruses is very low in humans, which may be related to predicted poor T‐cell immunogenicity.<xref rid=\"irv12755-bib-0004\" ref-type=\"ref\">\n<sup>4</sup>\n</xref>\n</p><p>Another important condition of a pandemic vaccine is timely manufacturing. In the case of the 2009 pandemic, the causative virus was first isolated in April 2009 and a candidate vaccine virus was generated in May. Usually, seasonal influenza vaccines are produced from spring to summer in Japan; therefore, the transition of production from seasonal vaccine to pandemic vaccine was relatively smooth in 2009. If a pandemic occurs in a period outside of seasonal vaccine production in the egg vaccine platform, more time will be needed to start the vaccine manufacturing because of egg supply. Furthermore, because of the damage of chickens by highly pathogenic avian influenza, there is a risk that egg supply will be stopped. To address these issues, we have been developing an AS03‐adjuvanted vaccine using H5N1 influenza virus antigen derived from a duck cell line (EB66<sup>®</sup>). In the previous phase I study, we confirmed that the vaccine was well‐tolerated and elicited a broadly cross‐reactive antibody response.<xref rid=\"irv12755-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref> In this paper, we report further evaluation of AS03‐adjuvanted H5N1 influenza vaccine formulations produced in an EB66<sup>®</sup> cell culture platform, KD‐295, in phase II and III studies to assess its immunogenicity and safety. In addition, phase II study data were registered and released in JapicCTI‐121788, and phase III study data were registered and released in JapicCTI‐121936.</p></sec><sec sec-type=\"materials-and-methods\" id=\"irv12755-sec-0006\"><label>2</label><title>MATERIALS AND METHODS</title><sec id=\"irv12755-sec-0007\"><label>2.1</label><title>Study designs and subjects</title><p>The phase II study was conducted in adults between the ages of 20 and 64 years in a randomized, double‐blinded (all involved were blinded), comparative fashion from 2 April to 6 November 2012 to further assess immunogenicity and safety of the vaccine, and to determine the appropriate dosage to be evaluated in the phase III study. After selection of one formulation, the phase III study was performed from August 23, 2012 to March 10, 2013 in an unblinded, uncontrolled study enrolling adults between the age of 20 and 64 years.</p><p>In both studies, the selection criteria were healthy adults aged 20‐64 who agreed with written informed consent. Exclusion criteria included no history of H5N1 infection or vaccination. These studies were conducted in Tokyo, Osaka, and Kagoshima in Japan.</p><p>Prior to clinical studies, related documents such as the clinical trial protocol and informed consent form were reviewed by the IRB within each hospital. The studies were conducted in accordance with the Helsinki Declaration, GCP, and other relevant regulations. Written informed consent was obtained from participants prior to enrollment.</p><p>In the phase II study, four different vaccine formulations were evaluated: MA (3.75 μg HA + AS03), HA (7.5 μg HA + AS03), MB (3.75 μg HA + 1/2 AS03), and HB (7.5 μg HA + 1/2 AS03).</p><p>Regarding the allocation method to each group, first the sponsor distributed the investigational drugs allocated randomly to the study sites. The principal investigator or subinvestigator entered the information about subjects as of obtaining written informed consent and about the study sites into the Electronic Data Capture system (Medidata RaveTM). The investigational drug allocation system (Medidata BalanceTM) featured in the Electronic Data Capture system allocated the each subject to MA group, MB group, HA group, and HB group on 1:1:1:1 ratio by using a minimization method. Age, gender, stratification, study, and study sites were used as the adjustment factors.</p><p>In the phase III study, the MA formulation (3.75 μg HA + AS03) was evaluated based on the results of the phase I/II study. In both studies, the investigational vaccine KD‐295 was administered intramuscularly at a dose volume of 0.5 mL given twice at an interval of 21 ± 7 days.</p></sec><sec id=\"irv12755-sec-0008\"><label>2.2</label><title>Vaccines</title><p>KD‐295 was composed of separate vials consisting of hemagglutinin (HA) antigen and AS03 adjuvant (squalene, α‐tocopherol, and Tween 80) and mixed in equal amounts at the time of use. The vaccine virus strain used in this study was A/Indonesia/05/2005(H5N1)/PR8‐IBCDC‐RG2 strain belonging to Clade 2.1.3.2. Briefly, HA antigen was prepared as previously described5, by cultivating the vaccine virus in EB66<sup>®</sup> cells, purifying the virus by sucrose density gradient centrifugation, inactivating with β‐propiolactone and ultraviolet light irradiation, and treating the virus particles with a surfactant.</p></sec><sec id=\"irv12755-sec-0009\"><label>2.3</label><title>Immunological evaluation</title><p>The immunogenicity evaluation protocols of the phase II and III studies were the same. Blood samples were taken before the first vaccination (Day 0), 21 days after the first vaccination (Day 21), and 21 days after the 2nd vaccination (Day 42). To evaluate the immune response to the vaccine strain, HI antibody and neutralizing antibody titers were measured. Measurements of HI antibody and neutralizing antibody titers were conducted with reference to a previously described method.<xref rid=\"irv12755-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref> For the HI test, both horse and chicken erythrocytes were used in the phase II study and only horse erythrocytes in the phase III study. For HI antibody, seroconversion rate (SCR), the geometric mean fold rise (GMFR), and the seroprotection rate (SPR) were calculated. SCR was the percentage of the subjects having an HI antibody titer of less than 1:10 prior to vaccination and 1:40 or higher after vaccination, or an HI titer before vaccination ≥1:10 and at least a 4‐fold increase in HI antibody titers after vaccination. SPR was defined as the percentage of participants who achieved HI titers of 1:40 or more. GMFR was defined as the ratio of change in GMT of HI antibody titers after the vaccination vs prior to the vaccination. The 95% confidence intervals on both sides of SCR and SPR were calculated based on the F‐distribution, and confidence interval of GMFR was calculated based on Student's t‐distribution.</p><p>For evaluation of immunogenicity of the vaccine, we followed the immunogenicity criteria of the Japanese guidelines for development of pandemic prototype vaccines (Japanese guidelines), which is identical to the immunogenicity criteria of the CHMP (Committee for Proprietary Medicinal Products) guidelines in place at the time the study was conducted (CPMP/BWP/214/96). In both studies, for the primary endpoint, we confirmed whether HI antibody (horse erythrocytes only) at Day 42 in a full analysis set (FAS) met the criteria of the Japanese guidelines (SCR >40%, GMFR >2.5, and SPR >70%). FAS was defined as a group of subjects with blood collected after at least one vaccination. The secondary endpoint of the both studies was percentage of subjects with a 4‐fold increase in neutralizing antibody titers at Day 42 in FAS. In addition, in the phase II study, we confirmed whether the chicken HI antibody at Day 42 met the criteria of the Japanese guidelines as a secondary endpoint.</p><p>In the phase II study, the target number of subjects was 50 in each group, for a total of 200, whereas the total number of enrolled subjects was 248, and the FAS included 246 subjects (62 for MA, 61 for HA, 63 for MB, 60 for HB). In the phase III study, the target number of subjects was 300, whereas the total number of subjects was 369, and the FAS included 364 subjects for MA. The target number of subjects in both studies was set according to the Japanese guidelines. Analyses were performed using SAS software version 9.2.</p></sec><sec id=\"irv12755-sec-0010\"><label>2.4</label><title>Safety evaluation</title><p>The safety evaluation protocols of the phase II and III studies were the same.</p><p>Using a health diary, solicited adverse events up to 7 days after each vaccination and unsolicited adverse events up to 21 days after each vaccination were recorded by subjects. Serious adverse events (SAE), important adverse events requiring cessation of vaccination (IAE) or adverse events including autoimmune diseases, and other inflammatory or autoimmune pathogenic neurological diseases (pIMDs: potential immune‐mediated diseases) were recorded during the study for another 6 months after the last vaccination (Day 0‐Day 201). Adverse events were graded in three levels; (a) mild (event that is easily tolerable, accompanied by only slight discomfort and does not interrupt daily activities); (b) moderate (event that interrupts daily activities because of discomfort); and (c) severe (event that makes daily activities impossible). Solicited adverse events were divided into two types, local and systemic adverse events. Solicited local adverse events included injection site erythema, swelling, and induration that occurred in the period following administration of the investigational drug until 6 days after administration. All solicited local adverse events were reactions at the injection site and so were categorized as adverse drug reactions. Solicited systemic adverse events included pyrexia, headache, fatigue, arthralgia, myalgia, chills, and hyperhidrosis that occurred in the period following administration of the investigational drug until 6 days after administration. The potential causal relationship between vaccination and other symptoms or events was determined by the investigator, and if a relationship was recognized, adverse events were categorized as adverse drug reactions. Statistical analyses were performed using SAS software version 9.2.</p></sec><sec id=\"irv12755-sec-0011\"><label>2.5</label><title>Study population</title><p>Demographic data in the FAS of the phase II and III studies are shown in Table <xref rid=\"irv12755-tbl-0001\" ref-type=\"table\">1</xref>. The mean ± SD for age in the FAS was 39.1 ± 11.1 years and 37.8 ± 11.2 years overall in the phase II and III studie,s respectively. No population imbalance was observed among the groups.</p><table-wrap id=\"irv12755-tbl-0001\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 1</label><caption><p>Demographic data in the phase II and III studies full analysis set (FAS)</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"bottom\"><tr style=\"border-bottom:solid 1px #000000\"><th rowspan=\"2\" valign=\"bottom\" colspan=\"1\">Item</th><th align=\"left\" rowspan=\"2\" valign=\"bottom\" colspan=\"1\">Statistics</th><th align=\"left\" colspan=\"9\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Phase II</th><th align=\"left\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Phase III</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">MA group</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">HA group</th><th align=\"left\" colspan=\"5\" valign=\"bottom\" rowspan=\"1\">MB group</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">HB group</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Total</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">MA group</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"2\" rowspan=\"1\">Number of analyzed subjects</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">62</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">61</td><td align=\"left\" colspan=\"5\" rowspan=\"1\">63</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">246</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">364</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"5\" colspan=\"1\">Age (year)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39.3</td><td align=\"left\" colspan=\"5\" rowspan=\"1\">38.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">38.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Standard deviation</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10.4</td><td align=\"left\" colspan=\"5\" rowspan=\"1\">10.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Minimum</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"left\" colspan=\"5\" rowspan=\"1\">21</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Median</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">36.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39.0</td><td align=\"left\" colspan=\"5\" rowspan=\"1\">38.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Maximum</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">64</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">63</td><td align=\"left\" colspan=\"5\" rowspan=\"1\">62</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">64</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">64</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">64</td></tr><tr><td align=\"left\" colspan=\"12\" rowspan=\"1\">Number of subjects （Incidence %）</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">41 y and over</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37 (59.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35(57.4)</td><td align=\"left\" colspan=\"3\" rowspan=\"1\">36 (57.1)</td><td align=\"left\" colspan=\"3\" rowspan=\"1\">35 (58.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">143 (58.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">222 (61.0)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Less than 40 y</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25 (40.3)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">26 (42.6)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">27 (42.9)</td><td align=\"left\" colspan=\"3\" rowspan=\"1\">25 (41.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">103 (41.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">142 (39.0)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Sex</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27 (43.5)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">28 (45.9)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">29 (46.0)</td><td align=\"left\" colspan=\"3\" rowspan=\"1\">28 (46.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">112 (45.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">179 (49.2)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Female</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35 (56.5)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">33 (54.1)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">34 (54.0)</td><td align=\"left\" colspan=\"3\" rowspan=\"1\">32 (53.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">134 (54.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">185 (50.8)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Ethnicity</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Japanese</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">62 (100.0)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">61 (100.0)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">63 (100.0)</td><td align=\"left\" colspan=\"3\" rowspan=\"1\">60 (100.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">246 (100.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">364 (100.0)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Other</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">0 (0.0)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">0 (0.0)</td><td align=\"left\" colspan=\"3\" rowspan=\"1\">0 (0.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0(0.0)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Medical history</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">58 (93.5)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">56 (91.8)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">57 (90.5)</td><td align=\"left\" colspan=\"3\" rowspan=\"1\">55 (91.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">226 (91.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">342 (94.0)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (6.5)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">5 (8.2)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">6 (9.5)</td><td align=\"left\" colspan=\"3\" rowspan=\"1\">5 (8.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20 (8.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22(6.0)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Underlying disease</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">48 (77.4)</td><td align=\"left\" colspan=\"3\" rowspan=\"1\">50 (82.0)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">54 (85.7)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">52 (86.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">204(82.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">285 (78.3)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14 (22.6)</td><td align=\"left\" colspan=\"3\" rowspan=\"1\">11 (18.0)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">9 (14.3)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">8 (13.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">42 (17.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">79 (21.7)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Allergy</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">56 (90.3)</td><td align=\"left\" colspan=\"3\" rowspan=\"1\">51 (83.6)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">57 (90.5)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">54 (90.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">218 (88.6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">289(79.4)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6 (9.7)</td><td align=\"left\" colspan=\"3\" rowspan=\"1\">10 (16.4)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">6 (9.5)</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">6 (10.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">28 (11.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">75 (20.6)</td></tr></tbody></table><table-wrap-foot id=\"irv12755-ntgp-0002\"><title>Note</title><fn id=\"irv12755-note-0003\"><p>MA group; 3.75 μg HA + AS03. HA group; 7.5 μg HA + AS03. MB group; 3.75 μg HA + 1/2 AS03. HB group; 7.5 μg HA + 1/2 AS03.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap></sec></sec><sec sec-type=\"results\" id=\"irv12755-sec-0012\"><label>3</label><title>RESULTS</title><sec id=\"irv12755-sec-0013\"><label>3.1</label><title>Immunogenicity</title><p>HI antibody against the vaccine strain was measured using horse and chicken erythrocytes in the phase II study. In the measurement using horse erythrocytes, the HI antibody response to the vaccine strain after the second vaccination (Day 42) in the FAS fulfilled all three criteria of immunogenicity described in the guidelines in all groups. For measurement using chicken erythrocytes, GMFR fulfilled the guideline criteria in all groups; however, SCR in the MB group and SPR in all groups did not fulfill the criteria. Since horse erythrocytes had higher sensitivity at HI antibody measurement, in the phase III study, only the horse erythrocyte assay was used. Results of the HI test for both studies are shown in Table <xref rid=\"irv12755-tbl-0002\" ref-type=\"table\">2</xref>. Although there was no large difference in the immunogenicity between the different vaccine formulations in the phase II study, the point estimate for GMFR was higher in the groups given the vaccine with the standard amount of AS03 (HA, MA) vs a half dose (MB, HB). The MA formulation was evaluated in the phase III study considering the safety data described later. In the phase III study, the MA formulation was highly immunogenic and it fulfilled all three criteria described in the guidelines.</p><table-wrap id=\"irv12755-tbl-0002\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 2</label><caption><p>Conformance of the parameters of the HI antibody (horse RBCs and chicken RBCs) response to the second vaccination (Day 42) with the three immunogenicity criteria of the guideline</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"bottom\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"2\" valign=\"bottom\" colspan=\"1\">Point estimate (95%CI)</th><th align=\"left\" colspan=\"8\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Phase II</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Phase III</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" colspan=\"2\" valign=\"bottom\" rowspan=\"1\">MA group</th><th align=\"left\" colspan=\"2\" valign=\"bottom\" rowspan=\"1\">HA group</th><th align=\"left\" colspan=\"2\" valign=\"bottom\" rowspan=\"1\">MB group</th><th align=\"left\" colspan=\"2\" valign=\"bottom\" rowspan=\"1\">HB group</th><th align=\"left\" colspan=\"2\" valign=\"bottom\" rowspan=\"1\">MA group</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Number of subjects</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">60</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">59</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">61</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">60</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">364</td></tr><tr><td align=\"left\" colspan=\"11\" rowspan=\"1\">Horse red blood cells</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"2\" colspan=\"1\">\n<p>Seroconversion rate</p>\n<p>>40%</p>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<p>100.0%</p>\n<p>(94.0%‐100.0%)</p>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<p>100.0%</p>\n<p>(93.9%‐100.0%)</p>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>100.0%</p>\n<p>(94.1%‐100.0%)</p>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<p>98.3%</p>\n<p>(91.1%‐100.0%)</p>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<p>100.0%</p>\n<p>(99.0‐100.0)</p>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<p>28.56</p>\n<p>(24.69‐33.04)</p>\n</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"2\" colspan=\"1\">\n<p>Geometric mean fold rise</p>\n<p>>2.5</p>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<p>33.90</p>\n<p>(28.82‐39.88)</p>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<p>40.48</p>\n<p>(34.39‐47.64)</p>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<p>30.55</p>\n<p>(25.44‐36.70)</p>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<p>43.73</p>\n<p>(41.15‐46.47)</p>\n</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">(24.69‐33.04) <p>100.0%</p>\n<p>(94.1%‐100.0%)</p>\n</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">\n<p>Seroprotection rate</p>\n<p>>70%</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>100.0%</p>\n<p>(94.0%‐100.0%)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>100.0%</p>\n<p>(93.9%‐100.0%)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>98.3%</p>\n<p>(91.1%‐100.0%)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>100.0%</p>\n<p>(99.0‐100.0)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">GMT</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>169.5</p>\n<p>(144.1‐199.4)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>202.4</p>\n<p>(171.9‐238.2)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>142.8</p>\n<p>(123.5‐165.2)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>152.8</p>\n<p>(127.2‐183.5)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>220.3</p>\n<p>(207.3‐234.1)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" colspan=\"11\" rowspan=\"1\">Chicken red blood cells</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">\n<p>Seroconversion rate</p>\n<p>>40%</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>55.0%</p>\n<p>(41.6%‐67.9%)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>64.4%</p>\n<p>(50.9%‐76.4%)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>39.3%</p>\n<p>(27.1%‐52.7%)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>56.7%</p>\n<p>(43.2%‐69.4%)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">\n<p>Geometric mean fold rise</p>\n<p>>2.5</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>6.20</p>\n<p>(4.84‐7.96)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>7.37</p>\n<p>(5.80‐9.36)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>4.38</p>\n<p>(3.54‐5.42)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>6.20</p>\n<p>(4.93‐7.81)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref ref-type=\"fn\" rid=\"irv12755-note-0005\">*</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" rowspan=\"2\" colspan=\"1\"/></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">\n<p>Seroprotection rate</p>\n<p>>70%</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>55.0%</p>\n<p>(41.6%‐67.9%)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>64.4%</p>\n<p>(50.9%‐76.4%)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>39.3%</p>\n<p>(27.1%‐52.7%)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>56.7%</p>\n<p>(43.2%‐69.4%)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">GMT</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>31.0</p>\n<p>(24.2‐39.8)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>37.3</p>\n<p>(29.4‐47.2)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>21.9</p>\n<p>(17.7‐27.1)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>31.0</p>\n<p>(24.6‐39.1)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot id=\"irv12755-ntgp-0003\"><title>Note</title><fn id=\"irv12755-note-0004\"><p>Strain measured: A/Indonesia/05/2005(H5N1). Vaccine strain: A/Indonesia/05/2005(H5N1). Subjects analyzed: a group of subjects who was collected blood after the second vaccination. Confidence interval (seroconversion rate, seroprotection rate):lower limit and upper limit of the exact 95% two‐sided confidence interval based on F‐distribution. Confidence interval (rate of change in GMT): lower limit and upper limit of the 95% two‐sided confidence interval based on Student's t‐distribution. MA group; 3.75 μg HA + AS03. HA group; 7.5 μg HA + AS03. MB group; 3.75μg HA+1/2 AS03. HB group; 7.5μg HA+1/2 AS03.</p></fn><fn id=\"irv12755-note-0005\"><label>*</label><p>Fulfilled the immunogenicity criteria of the guideline.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>GMT of neutralizing antibody and percentage of subjects with a 4‐fold increase in neutralizing antibody titers against vaccine strains after the 1st and 2nd vaccination in FAS are shown in Table <xref rid=\"irv12755-tbl-0003\" ref-type=\"table\">3</xref>. Both in the phase II and III studies, after the 1st dose, the seroconversion rates were 20‐30% and rose to nearly 100% after the second vaccination (Day 42). At the same time, GMT increased markedly in all groups after the second vaccination (Day 42).</p><table-wrap id=\"irv12755-tbl-0003\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 3</label><caption><p>Changes in geometric mean titer of neutralizing antibody to the vaccine strain</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"bottom\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"3\" valign=\"bottom\" colspan=\"1\">Timing</th><th align=\"left\" colspan=\"8\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Phase II</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Phase III</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">MA group</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">HA group</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">MB group</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">HB group</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">MA group</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">\n<p>GMT</p>\n<p>(Confidence interval)</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">\n<p>Rates of antibody rise of 4‐fold or higher (%)</p>\n<p>[Confidence interval (%)]</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">\n<p>GMT</p>\n<p>(Confidence interval)</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">\n<p>Rates of antibody rise of 4‐fold or higher (%)</p>\n<p>[Confidence interval (%)]</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">\n<p>GMT</p>\n<p>(Confidence interval)</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">\n<p>Rates of antibody rise of 4‐fold or higher (%)</p>\n<p>[Confidence interval (%)]</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">\n<p>GMT</p>\n<p>(Confidence interval)</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">\n<p>Rates of antibody rise of 4‐fold or higher (%)</p>\n<p>[Confidence interval (%)]</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">\n<p>GMT</p>\n<p>(Confidence interval)</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">\n<p>Rates of antibody rise of 4‐fold or higher (%)</p>\n<p>[Confidence interval (%)]</p>\n</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Number of analyzed subjects</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">62</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">61</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">63</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">60</td><td align=\"left\" colspan=\"2\" rowspan=\"1\">364</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">\n<p>Before vaccination</p>\n<p>(Day 0)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>5.1</p>\n<p>(4.9‐5.2)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">－</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>5.1</p>\n<p>(4.9‐5.2)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">－</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">－</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">－</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">－</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">\n<p>After the 1<sup>st </sup>vaccination</p>\n<p>(Day 21)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>9.8</p>\n<p>(8.0‐12.0)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>27.4</p>\n<p>(16.9‐40.2)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>13.1</p>\n<p>(10.7‐16.2)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>39.3</p>\n<p>(27.1‐52.7)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>8.7</p>(7.3‐10.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>20.6</p>\n<p>(11.5‐32.7)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>9.5</p>\n<p>(7.8‐11.7)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>21.7</p>\n<p>(12.1‐34.2)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>10.2</p>\n<p>(9.5‐11.1)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>27.5</p>\n<p>(22.9‐32.4)</p>\n</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">\n<p>After the 2<sup>nd </sup>vaccination</p>\n<p>(Day 42)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>234.3</p>\n<p>(182.5‐300.7)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>100.0</p>\n<p>(94.0‐100.0)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>265.2</p>\n<p>(205.0‐343.0)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>98.3</p>\n<p>(90.9‐100.0)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>171.3</p>\n<p>(130.9‐224.1)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>100.0</p>\n<p>(94.1‐100.0)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>211.1</p>\n<p>(162.1‐274.9)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>98.3</p>\n<p>(91.1‐100.0)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>301.1</p>\n<p>(275.4‐329.2)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>99.7</p>\n<p>(98.5‐100.0)</p>\n</td></tr></tbody></table><table-wrap-foot id=\"irv12755-ntgp-0004\"><fn id=\"irv12755-note-0006\"><p>Strain measured: A/Indonesia/05/2005(H5N1). Vaccine strain: A/Indonesia/05/2005(H5N1).Subjects analyzed: FAS.GMT: geometric mean antibody titer. Confidence interval: lower limit and upper limit of the 95% two‐sided confidence interval based on Student's t‐distribution. MA group; 3.75 μg HA + AS03. HA group; 7.5 μg HA + AS03. MB group; 3.75 μg HA + 1/2 AS03. HB group; 7.5 μg HA + 1/2 AS03.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap></sec><sec id=\"irv12755-sec-0014\"><label>3.2</label><title>Safety</title><p>Adverse events and adverse drug reactions occurring during the study period (Day 0‐Day 201) are summarized in Table <xref rid=\"irv12755-tbl-0004\" ref-type=\"table\">4</xref>. Two SAEs (thyroid cancer and acute abdomen) and one pIMD (pasuda disease) occurred in the phase 3 trial, but in all cases a causal relationship with vaccination was denied. Through both studies, no serious adverse drug reactions (death, SAE, IAE, and pIMD related to vaccination) were reported. Among unsolicited adverse drug reactions, injection‐site pruritus showed the highest incidence in both studies. The majority of cases of injection site pruritus were grade 1. grade 3 unsolicited adverse drug reactions occurred in one case (dehydration) in the phase II study the MA group and in two cases (positional vertigo and malaise) in the phase III study. The incidence rates were low.</p><table-wrap id=\"irv12755-tbl-0004\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 4</label><caption><p>Safety summary of the phase II and III studies</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"bottom\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"4\" valign=\"bottom\" colspan=\"1\">Classification of adverse events</th><th align=\"left\" rowspan=\"4\" valign=\"bottom\" colspan=\"1\">Investigation periods</th><th align=\"left\" colspan=\"12\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Phase II</th><th align=\"left\" colspan=\"3\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Phase III</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" colspan=\"3\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">MA group</th><th align=\"left\" colspan=\"3\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">HA group</th><th align=\"left\" colspan=\"3\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">MB group</th><th align=\"left\" colspan=\"3\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">HB group</th><th align=\"left\" colspan=\"3\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">MA group</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" colspan=\"3\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Number of subjects with events</th><th align=\"left\" colspan=\"3\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Number of subjects with events</th><th align=\"left\" colspan=\"3\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Number of subjects with events</th><th align=\"left\" colspan=\"3\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Number of subjects with events</th><th align=\"left\" colspan=\"3\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Number of subjects with events</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\"/><th align=\"left\" colspan=\"2\" valign=\"bottom\" rowspan=\"1\">\n<p>Incidence (%)</p>\n<p>(95%CI)</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\"/><th align=\"left\" colspan=\"2\" valign=\"bottom\" rowspan=\"1\">\n<p>Incidence (%)</p>\n<p>(95%CI)</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\"/><th align=\"left\" colspan=\"2\" valign=\"bottom\" rowspan=\"1\">\n<p>Incidence (%)</p>\n<p>(95%CI)</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\"/><th align=\"left\" colspan=\"2\" valign=\"bottom\" rowspan=\"1\">\n<p>Incidence (%)</p>\n<p>(95%CI)</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\"/><th align=\"left\" colspan=\"2\" valign=\"bottom\" rowspan=\"1\">\n<p>Incidence (%)</p>\n<p>(95%CI)</p>\n</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Number of analyzed subjects</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td colspan=\"3\" rowspan=\"1\">62</td><td colspan=\"3\" rowspan=\"1\">62</td><td colspan=\"3\" rowspan=\"1\"> 63 </td><td colspan=\"3\" rowspan=\"1\"> 61 </td><td colspan=\"3\" rowspan=\"1\"> 369 </td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Adverse events</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Death</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 201</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Serious adverse events</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 201</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(0.1‐1.9)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Significant adverse events</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 201</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Potential immune‐mediated diseases</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 201</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(0.0‐1.5)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Solicited local adverse events</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 6, Day 21‐Day 27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">54</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">87.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(76.1‐94.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">52</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">83.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(72.3‐92.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">52</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">82.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(70.9‐90.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">48</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">78.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(66.3‐88.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">330</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">89.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(85.8‐92.4)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Solicited systemic adverse events</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 6, Day 21‐Day 27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">69.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(56.3‐80.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">46</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">74.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(61.5‐84.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">71.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(58.7‐82.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">57.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(44.1‐70.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">247</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">66.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(61.9‐71.7)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Unsolicited adverse events</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 42</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(23.7‐48.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(20.9‐45.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(27.6‐52.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">36.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(24.2‐49.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">124</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(28.8‐38.7)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 43‐Day 201</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(0.0‐1.5)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 201</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(23.7‐48.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(20.9‐45.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(27.6‐52.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">36.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(24.2‐49.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">125</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(29.1‐39.0)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Adverse drug reactions</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Death</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 201</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Serious adverse events</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 201</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Significant adverse events</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 201</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Potential immune‐mediated diseases</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 201</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Solicited local adverse events</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 6, Day 21‐Day 27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">54</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">87.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(76.1‐94.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">52</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">83.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(72.3‐92.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">52</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">82.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(70.9‐90.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">48</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">78.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(66.3‐88.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">330</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">89.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(85.8‐92.4)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Solicited systemic adverse events</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 6, Day 21‐Day 27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">41</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">66.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(53.0‐77.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">44</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">71.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(58.1‐81.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">71.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(58.7‐82.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">55.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(42.4‐68.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">245</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">66.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(61.3‐71.2)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Unsolicited adverse events</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 42</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(18.2‐41.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(15.5‐38.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(14.0‐36.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(11.9‐33.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">99</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(22.4‐31.7)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 43‐Day 201</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Day 0‐Day 201</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(18.2‐41.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(15.5‐38.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(14.0‐36.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(11.9‐33.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">99</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(22.4‐31.7)</td></tr></tbody></table><table-wrap-foot id=\"irv12755-ntgp-0005\"><title>Note</title><fn id=\"irv12755-note-0007\"><p>Sbjects analyzed: safety analysis set. Study period: Day 0 ‐Day 201. Confidence intervalexact 95% two‐sided confidence interval based on F‐distribution. MA group; 3.75 μg HA + AS03. HA group; 7.5 μg HA + AS03. MB group; 3.75 μg HA + 1/2 AS03. HB group; 7.5 μg HA + 1/2 AS03.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>Table <xref rid=\"irv12755-tbl-0005\" ref-type=\"table\">5</xref> and Table <xref rid=\"irv12755-tbl-0006\" ref-type=\"table\">6</xref> show the incidence rates of solicited local adverse events, solicited systemic adverse events, and solicited systemic adverse drug reactions occurring during the investigation periods (Day 0‐Day 6 and Day 21‐Day 27). In the phase II study, the rate of injection site pain, 70%‐90%, was the highest among the solicited local adverse events in all groups. The incidence rates of other solicited local adverse events were between 10% and 30%. The incidence rates of injection site pain and other solicited local adverse events in the phase III study were similar to those in the phase II study. In addition, the incidence rate of grade 3‐solicited adverse events was low.</p><table-wrap id=\"irv12755-tbl-0005\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 5</label><caption><p>Incidence of solicited local adverse events (Day 0‐Day 6 and Day 21‐Day 27)</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"4\" valign=\"top\" colspan=\"1\"/><th align=\"left\" colspan=\"5\" style=\"border-bottom:solid 1px #000000\" valign=\"top\" rowspan=\"1\">No. of subjects with the event</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" colspan=\"5\" style=\"border-bottom:solid 1px #000000\" valign=\"top\" rowspan=\"1\">Incidence(%)(Confidenceinterval)</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" colspan=\"4\" style=\"border-bottom:solid 1px #000000\" valign=\"top\" rowspan=\"1\">Phase II</th><th align=\"left\" style=\"border-bottom:solid 1px #000000\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Phase III</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MA group</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">HA group</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MB group</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">HB group</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MA group</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Number of analyzed subjects</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">62</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">62</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">63</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">61</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">369</td></tr><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">Solicited local adverse events</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">54</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">52</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">52</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">48</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">330</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">87.1% (76.1‐94.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">83.9% (72.3‐92.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">82.5% (70.9‐90.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">78.7% (66.3‐88.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">89.4% (85.8‐92.4)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Pain</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">53</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">52</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">49</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">320</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">85.5% (74.2‐93.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">83.9% (72.3‐92.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">77.8% (65.5‐87.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">73.8% (60.9‐84.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">86.7% (82.8‐90.0)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Erythema</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">19</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">126</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">29.0% (18.2‐41.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">30.6% (19.6‐43.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15.9% (7.9‐27.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">19.7% (10.6‐31.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34.1% (29.3‐39.2)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6% (0.0‐8.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6% (0.0‐8.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.2% (0.9‐4.2)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Swelling</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">106</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">27.4% (16.9‐40.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">24.2% (14.2‐36.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.7% (5.6‐23.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16.4% (8.2‐28.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">28.7% (24.2‐33.6)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6% (0.0‐8.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.1% (0.3‐2.8)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Induration</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">82</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">27.4% (16.9‐40.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22.6% (12.9‐35.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15.9% (7.9‐27.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.0%（9.4‐30.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22.2% (18.1‐26.8)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6% (0.0‐8.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td></tr></tbody></table><table-wrap-foot id=\"irv12755-ntgp-0006\"><title>Note</title><fn id=\"irv12755-note-0008\"><p>Analysis set: Safety analysis set. Period of investigation: Day 0‐Day 6 and Day 21 ‐Day 27.Item: Adverse event. MedDRA/J (Ver15.1). MA group; 3.75 μg HA + AS03. HA group; 7.5 μg HA + AS03. MB group; 3.75 μg HA + 1/2 AS03. HB group; 7.5 μg HA + 1/2 AS03.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><table-wrap id=\"irv12755-tbl-0006\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 6</label><caption><p>Incidence of solicited systemic adverse events and solicited systemic adverse drug reactions (Day 0‐Day 6 and Day 21‐Day 27)</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"bottom\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"4\" valign=\"bottom\" colspan=\"1\">PT</th><th align=\"left\" colspan=\"5\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">No. of subjects with the event</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" colspan=\"5\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Incidence(%)(Confidenceinterval)</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" colspan=\"4\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\">Phase II</th><th align=\"left\" style=\"border-bottom:solid 1px #000000\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Phase III</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">MA group</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">HA group</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">MB group</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">HB group</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">MA group</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Number of analyzed subjects</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">62</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">62</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">63</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">61</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">369</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">46</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">248</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Solicited systemic adverse events</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">69.4% (56.3‐80.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">74.2% (61.5‐84.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">71.4% (58.7‐82.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">57.4% (44.1‐70.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">67.2% (62.2‐72.0)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Pyrexia</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">86</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.9% (5.7‐23.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27.4% (16.9‐40.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.3% (1.8‐15.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.9% (1.0‐13.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23.3% (19.1‐28.0)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.5% (1.8‐15.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6% (0.0‐8.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6% (0.6‐3.5)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Headache</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">132</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.9% (22.3‐47.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">41.9% (29.5‐55.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">41.3% (29.0‐54.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32.8% (21.3‐46.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.8% (30.9‐40.9)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6% (0.0‐8.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3% (0.0‐1.5)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Fatigue</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">157</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">58.1% (44.8‐70.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">58.1% (44.8‐70.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">42.9% (30.5‐ 56.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">44.3% (31.5‐57.6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">42.5% (37.4‐47.8)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6% (0.0‐8.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Arthralgia</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">97</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">29.0% (18.2‐41.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27.4% (16.9‐40.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17.5% (9.1‐29.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13.1% (5.8‐24.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26.3% (21.9‐31.1)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3% (0.0‐1.5)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Myalgia</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">124</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.1% (25.2‐50.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.1% (25.2‐50.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.3% (22.0‐46.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34.4% (22.7‐47.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.6% (28.8‐38.7)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Chills</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">93</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">21.0% (11.7‐33.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27.4% (16.9‐40.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.1% (4.6‐21.6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.2% (2.7‐18.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25.2% (20.9‐30.0)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6% (0.0‐8.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.3% (0.0‐1.5)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Hyperhidrosis</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">44</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.5% (1.8‐15.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.3% (4.7‐21.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20.6% (11.5‐32.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.2% (2.7‐18.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.9% (8.8‐15.7)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td></tr><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">Solicited systemic adverse drug reactions</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">41</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">44</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">245</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">66.1% (53.0‐77.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">71.0% (58.1‐81.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">71.4% (58.7‐82.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">55.7% (42.4‐68.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">66.4% (61.3‐71.2)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Pyrexia</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">85</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.9% (5.7‐23.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27.4% (16.9‐40.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.3% (1.8‐15.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.9% (1.0‐13.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23.0% (18.8‐27.7)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.5% (1.8‐15.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6% (0.0‐8.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6% (0.6‐3.5)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Headache</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">131</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">32.3% (20.9‐45.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">40.3% (28.1‐53.6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39.7% (27.6‐52.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32.8% (21.3‐46.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.5% (30.6‐40.6)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6% (0.0‐8.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3% (0.0‐1.5)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Fatigue</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">156</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">58.1% (44.8‐70.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">54.8% (41.7‐67.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">42.9% (30.5‐ 56.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">41.0% (28.6‐54.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">42.3% (37.2‐47.5)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6% (0.0‐8.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Arthralgia</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">96</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">29.0% (18.2‐41.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27.4% (16.9‐40.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17.5% (9.1‐29.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.5% (4.7‐22.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26.0% (21.6‐30.8)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3% (0.0‐1.5)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Myalgia</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">122</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.5% (23.7‐48.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.1% (25.2‐50.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.3% (22.0‐46.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32.8% (21.3‐46.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.1% (28.3‐38.1)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Chills</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">93</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">19.4% (10.4‐31.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27.4% (16.9‐40.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.1% (4.6‐21.6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.2% (2.7‐18.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25.2% (20.9‐30.0)</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6% (0.0‐8.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3% (0.0‐1.5)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" colspan=\"6\" rowspan=\"1\">Hyperhidrosis</td></tr><tr><td align=\"left\" style=\"padding-left:15%\" rowspan=\"2\" colspan=\"1\">Total</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">44</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.5% (1.8‐15.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.3% (4.7‐21.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20.6% (11.5‐32.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.2% (2.7‐18.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.9% (8.8‐15.7)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"2\" colspan=\"1\">Grade 3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td><td rowspan=\"1\" colspan=\"1\">0%</td></tr></tbody></table><table-wrap-foot id=\"irv12755-ntgp-0007\"><title>Note</title><fn id=\"irv12755-note-0009\"><p>Analysis set: Safety analysis set. Period of investigation: Days 0‐6 and Days 21‐27.Item: Adverse event, Adverse drug reaction. MedDRA/J (Ver15.1)</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>Of the solicited systemic adverse events and solicited systemic adverse drug reactions, that with the highest incidence was fatigue, which was expressed in 40%‐60% of patients in each group. The next highest were headache and myalgia (30%‐40%). In addition, the incidence rate of grade 3‐solicited systemic adverse events and solicited systemic adverse drug reactions was low.</p></sec></sec><sec sec-type=\"discussion\" id=\"irv12755-sec-0015\"><label>4</label><title>DISCUSSION</title><p>The phase II and phase III clinical studies revealed that MA formulation of KD‐295 (3.75 μg HA + AS03) was well‐tolerated and highly immunogenic and that no serious adverse drug reactions were observed. Therefore, the MA formulation can be considered as a useful pandemic and pre‐pandemic influenza vaccine.</p><p>Although most human cases of avian influenza to date have been associated with direct contact with infected birds, as causative agents for the next pandemic, viruses including H5, H7, and H9 subtypes are still of concern as viruses with pandemic potential.<xref rid=\"irv12755-bib-0007\" ref-type=\"ref\">\n<sup>7</sup>\n</xref> Among them, the H5 subtype was the first target for vaccine development, and since then many types of vaccine have been developed. The first developed H5N1 vaccine was a non‐adjuvanted split vaccine, and it was reported that two doses of 90 μg HA of the vaccine‐induced neutralization antibody titers reaching 1:40 or greater in 54 percent of study subjects.<xref rid=\"irv12755-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> Non‐adjuvanted and adjuvanted whole virion vaccines were then developed, and their immunogenicity in humans with 7.5‐15 μg HA antigen dose was much better than that of the split vaccine.<xref rid=\"irv12755-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref> We also conducted a clinical study with an egg‐derived, alum‐adjuvanted whole virion H5N1 vaccine.<xref rid=\"irv12755-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref> However, although the vaccine was immunogenic, it could not meet one of the three criteria of CHMP guidelines. Therefore, we decided to develop a more immunogenic vaccine with a platform other than chicken eggs to have flexibility in the vaccine production.</p><p>As a result of immunological evaluation in the phase II study, all vaccine groups fulfilled the three required criteria described in the Japanese guidelines based on the HI antibody titers measured using horse erythrocytes after administration of two doses of the vaccine.</p><p>When vaccine strain derived from avian influenza virus such as H5N1 is used as an antigen, HI antibody titers' measurement using horse erythrocytes has more sensitive in detection of the antigen than using chicken erythrocytes.<xref rid=\"irv12755-bib-0010\" ref-type=\"ref\">\n<sup>10</sup>\n</xref> This is why HI antibody titers measured using not chicken, but horse erythrocytes were used for primary evaluation.</p><p>Although not statistically significant, GMFR was higher in the groups given the vaccine with standard AS03 (HA, MA) dose. Although all the vaccine formulations were well‐tolerated, the MA formulations were less incidence of solicited systemic adverse events (pyrexia, headache, and chills) than the HA formulations. Therefore, the MA formulation was selected to be evaluated in the phase III study.</p><p>In the phase III study, the MA formulation containing a standard dose of AS03 and 3.75 μg HA antigen was further confirmed to be highly immunogenic and fulfilled the all criteria of the Japanese guidelines. The results of clinical studies, including those of the present study, confirm that the AS03 adjuvant is potent and the antigen dose could be reduced to 3.75 μg HA. Regarding an H5N1 vaccine with antigen derived from EB66<sup>®</sup> cells and formulated with AS03, Schuind et al<xref rid=\"irv12755-bib-0011\" ref-type=\"ref\">\n<sup>11</sup>\n</xref> reported similar results to ours in their clinical phase I study. Chada et al<xref rid=\"irv12755-bib-0012\" ref-type=\"ref\">\n<sup>12</sup>\n</xref> conducted a meta‐analysis and concluded that vaccines with emulsion‐type adjuvants could induce broad cross‐clade antibodies and are suitable for stockpiling. Feldstein et al<xref rid=\"irv12755-bib-0013\" ref-type=\"ref\">\n<sup>13</sup>\n</xref> compared human immunogenity data of several H5N1 vaccines and concluded that adjuvanted H5N1 vaccines induced high theoretical efficacy and that AS03‐adjuvanted vaccine was more immunogenic than MF59‐adjuvanted vaccine. It has also been confirmed that the AS03 could increase immunogenicity of H7N1, H7N9, and H9N2 antigens.<xref rid=\"irv12755-bib-0014\" ref-type=\"ref\">\n<sup>14</sup>\n</xref>, <xref rid=\"irv12755-bib-0015\" ref-type=\"ref\">\n<sup>15</sup>\n</xref>, <xref rid=\"irv12755-bib-0016\" ref-type=\"ref\">\n<sup>16</sup>\n</xref>, <xref rid=\"irv12755-bib-0017\" ref-type=\"ref\">\n<sup>17</sup>\n</xref> Based on these data, this study has limitations because it does not directly compare KD‐295 with other vaccines, but KD‐295 appears to be more effective than other licensed H5N1 vaccines.</p><p>Marichal et al proposed that alum‐adjuvant induces neutrophil migration and cell death, and subsequently DNA released from the host cell activates innate immunity as DAMPs.<xref rid=\"irv12755-bib-0018\" ref-type=\"ref\">\n<sup>18</sup>\n</xref> AS03‐adjuvant reportedly activates not only innate immunity, but also adaptive immunity comprehensively, induces production of various cytokines, and contributes to enhancing the antigen‐specific antibody production of B cells.<xref rid=\"irv12755-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref>, <xref rid=\"irv12755-bib-0020\" ref-type=\"ref\">\n<sup>20</sup>\n</xref> It also reported that α‐tocopherol plays an important role in these immune responses. In fact, omission of α‐tocopherol from AS03 modified the innate immune response and lead to lower antibody responses.<xref rid=\"irv12755-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref> Therefore, at this time, vaccines with emulsion‐type adjuvant, including AS03 with α‐tocopherol, would be the promising choice for both pre‐pandemic and pandemic avian influenza vaccines.</p><p>When compared with the safety profile of our alum‐adjuvanted H5N1 whole virion vaccine,<xref rid=\"irv12755-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref> the greatest difference between the two vaccines is local (injection site) pain. With the alum‐adjuvanted vaccine, 10%‐45% of participants reported local pain after administration of the vaccine, whereas 70%‐80% of subjects reported it with the AS03‐adjuvanted vaccine. Local pain is a common adverse drug reaction with emulsion‐type adjuvanted of vaccines.<xref rid=\"irv12755-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref> However, it is self‐limiting and leads to no further complication. The incidence rates of other local and systemic adverse events observed in the current studies were generally higher than those of alum‐adjuvanted vaccines; however, most events were grades 1 and 2, and the highest rate of grade 3 was pyrexia yet at only 1.6% which led to no further complication. In general, as was the case in the phase I study, the vaccine was well‐tolerated in both the phase II and III studies.</p><p>Finally, for the possible next pandemic, global cooperation will be essential for an effective response. For this purpose, the WHO established the Pandemic Influenza Preparedness (PIP) framework for the sharing of influenza viruses and access to vaccines and other benefits. The WHO PIP documents<xref rid=\"irv12755-bib-0021\" ref-type=\"ref\">\n<sup>21</sup>\n</xref> state that member states should urge vaccine manufacturers to set aside a portion of each production cycle of pandemic influenza vaccine for use by developing countries. Therefore, in the event of a pandemic, several kinds of vaccines will be distributed at the same time in those countries. As described previously, immunogenicity of the licensed pandemic vaccines is varied, and vaccines with the emulsion‐type adjuvants are the most immunogenic. Leroux‐Roels et al<xref rid=\"irv12755-bib-0022\" ref-type=\"ref\">\n<sup>22</sup>\n</xref> reported that priming with AS03‐adjuvanted H5N1 influenza vaccine improves the immune response of a heterologous booster vaccination. They also reported that priming with non‐adjuvanted vaccine appears to inhibit the response to subsequent vaccination; therefore, KD‐295 also should be evaluated in combination of several vaccines with different immunogenicity. In conclusion, although it remains necessary to evaluate in pediatric and elderly populations, the MA (3.75 μg HA + AS03) formulation was well‐tolerated and highly immunogenic. KD‐295 can be considered as a useful pandemic and pre‐pandemic influenza vaccine. In addition, KD‐295 is currently approved in Japan.</p></sec><sec sec-type=\"COI-statement\" id=\"irv12755-sec-0017\"><title>CONFLICT OF INTEREST</title><p>Kohji Ueda received a fee from KAKETSUKEN, currently KM Biologics Co., Ltd., for the implementation of this study. Yoichiro Kino, which belongs to Kino Consulting, was an employee of KAKETSUKEN at the time of the study period. The other authors have no conflicts of interest of declare.</p></sec></body><back><ack id=\"irv12755-sec-0016\"><title>ACKNOWLEDGEMENT</title><p>We thank Dr Setsuo Hasegawa, Dr Hirotaka nagashima, Dr Kazuhiro Takenaka, Dr Hiroyuki Fukase, Dr Osamu Matsuoka, Dr Hirotaka Yasuba and Dr Hirofumi Tahara for his great contributions to the clinical trials described in this paper and Valneva providing EB66 cell line, and the GSK vaccines development staff for their great support of this work.</p></ack><ref-list content-type=\"cited-references\" id=\"irv12755-bibl-0001\"><title>REFERENCES</title><ref id=\"irv12755-bib-0001\"><label>1</label><mixed-citation publication-type=\"journal\" id=\"irv12755-cit-0001\">\n<string-name>\n<surname>Lansbury</surname>\n<given-names>LE</given-names>\n</string-name>, <string-name>\n<surname>Smith</surname>\n<given-names>S</given-names>\n</string-name>, <string-name>\n<surname>Beyer</surname>\n<given-names>W</given-names>\n</string-name>, et al. 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Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32410384</article-id><article-id pub-id-type=\"pmc\">PMC7431645</article-id><article-id pub-id-type=\"doi\">10.1111/irv.12747</article-id><article-id pub-id-type=\"publisher-id\">IRV12747</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Original Article</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Original Articles</subject></subj-group></article-categories><title-group><article-title>Evaluation of a mobile health approach to improve the Early Warning System of influenza surveillance in Cameroon</article-title><alt-title alt-title-type=\"left-running-head\">MONAMELE et al.</alt-title></title-group><contrib-group><contrib id=\"irv12747-cr-0001\" contrib-type=\"author\"><name><surname>Monamele</surname><given-names>Chavely Gwladys</given-names></name><xref ref-type=\"aff\" rid=\"irv12747-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12747-cr-0002\" contrib-type=\"author\"><name><surname>Messanga Essengue</surname><given-names>Loique Landry</given-names></name><xref ref-type=\"aff\" rid=\"irv12747-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12747-cr-0003\" contrib-type=\"author\"><name><surname>Ripa Njankouo</surname><given-names>Mohamadou</given-names></name><xref ref-type=\"aff\" rid=\"irv12747-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12747-cr-0004\" contrib-type=\"author\"><name><surname>Munshili Njifon</surname><given-names>Hermann Landry</given-names></name><xref ref-type=\"aff\" rid=\"irv12747-aff-0003\">\n<sup>3</sup>\n</xref></contrib><contrib id=\"irv12747-cr-0005\" contrib-type=\"author\"><name><surname>Tchatchueng</surname><given-names>Jules</given-names></name><xref ref-type=\"aff\" rid=\"irv12747-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12747-cr-0006\" contrib-type=\"author\"><name><surname>Tejiokem</surname><given-names>Mathurin Cyrille</given-names></name><xref ref-type=\"aff\" rid=\"irv12747-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12747-cr-0007\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Njouom</surname><given-names>Richard</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0003-3112-6370</contrib-id><xref ref-type=\"aff\" rid=\"irv12747-aff-0001\">\n<sup>1</sup>\n</xref><address><email>njouom@pasteur-yaounde.org</email></address></contrib></contrib-group><aff id=\"irv12747-aff-0001\">\n<label><sup>1</sup></label>\n<named-content content-type=\"organisation-division\">Laboratory of Virology</named-content>\n<institution>Centre Pasteur of Cameroon</institution>\n<city>Yaoundé</city>\n<country country=\"CM\">Cameroon</country>\n</aff><aff id=\"irv12747-aff-0002\">\n<label><sup>2</sup></label>\n<named-content content-type=\"organisation-division\">Laboratory of Epidemiology</named-content>\n<institution>Centre Pasteur of Cameroon</institution>\n<city>Yaoundé</city>\n<country country=\"CM\">Cameroon</country>\n</aff><aff id=\"irv12747-aff-0003\">\n<label><sup>3</sup></label>\n<institution>Centre Pasteur of Cameroon</institution>\n<institution>Annex of Garoua</institution>\n<city>Garoua</city>\n<country country=\"CM\">Cameroon</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label><bold>Correspondence</bold><break/>\nRichard Njouom, Centre Pasteur of Cameroon, PO Box 1274, Yaounde, Cameroon.<break/>\nEmail: <email>njouom@pasteur-yaounde.org</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>14</day><month>5</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>9</month><year>2020</year></pub-date><volume>14</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1111/irv.v14.5</issue-id><fpage>491</fpage><lpage>498</lpage><history><date date-type=\"received\"><day>17</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>12</day><month>4</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 John Wiley & Sons Ltd <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Influenza and Other Respiratory Viruses</italic> Published by John Wiley & Sons Ltd.</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:IRV-14-491.pdf\"/><abstract id=\"irv12747-abs-0001\"><title>Abstract</title><sec id=\"irv12747-sec-0001\"><title>Background</title><p>Rapid reporting of surveillance data is essential to better inform national prevention and control strategies.</p></sec><sec id=\"irv12747-sec-0002\"><title>Objectives</title><p>We compare the newly implemented smartphone‐based system to the former paper‐based and short message service (SMS) for collecting influenza epidemiological data in Cameroon.</p></sec><sec id=\"irv12747-sec-0003\"><title>Methods</title><p>Of the 13 sites which collect data from persons with influenza‐like illness (ILI), six sites send data through the EWS, while seven sites make use of the paper‐based system and SMS. We used four criteria for the comparison of the data collection tools: completeness, timeliness, conformity and cost.</p></sec><sec id=\"irv12747-sec-0004\"><title>Results</title><p>Regarding the different collection tools, data sent by the EWS were significantly more complete (97.6% vs 81.6% vs 44.8%), prompt (74.4% vs n/a vs 60.7%) and of better quality (93.7% vs 76.1% vs 84.0%) than data sent by the paper‐based system and SMS, respectively. The average cost of sending a datum by a sentinel site per week was higher for the forms (5.0 USD) than for the EWS (0.9 USD) and SMS (0.1 USD). The number of outpatient visits and subsequently all surveillance data decreased across the years 2017‐2019 together with the influenza positivity rate from 30.7% to 28.3%. Contrarily, the proportion of influenza‐associated ILI to outpatient load was highest in the year 2019 (0.37 per 100 persons vs 0.28 and 0.26 in the other 2 years).</p></sec><sec id=\"irv12747-sec-0005\"><title>Conclusion</title><p>All sentinel sites and even other disease surveillance systems are expected to use this tool in the near term future due to its satisfactory performance and cost.</p></sec></abstract><kwd-group><kwd id=\"irv12747-kwd-0001\">Cameroon</kwd><kwd id=\"irv12747-kwd-0002\">data collection</kwd><kwd id=\"irv12747-kwd-0003\">Early Warning System</kwd><kwd id=\"irv12747-kwd-0004\">influenza</kwd><kwd id=\"irv12747-kwd-0005\">paper‐based system</kwd><kwd id=\"irv12747-kwd-0006\">short message service</kwd></kwd-group><funding-group><award-group id=\"funding-0001\"><funding-source>WHO PIP Implementation project in Cameroon</funding-source></award-group><award-group id=\"funding-0002\"><funding-source><institution-wrap><institution>U.S. Department of Health and Human Services </institution><institution-id institution-id-type=\"open-funder-registry\">10.13039/100000016</institution-id></institution-wrap></funding-source><award-id>6 DESP060001‐01‐01</award-id></award-group></funding-group><counts><fig-count count=\"2\"/><table-count count=\"2\"/><page-count count=\"8\"/><word-count count=\"4897\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>September 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.6 mode:remove_FC converted:18.08.2020</meta-value></custom-meta></custom-meta-group></article-meta><notes><p content-type=\"self-citation\">\n<mixed-citation publication-type=\"journal\" id=\"irv12747-cit-1001\">\n<string-name>\n<surname>Monamele</surname>\n<given-names>CG</given-names>\n</string-name>, <string-name>\n<surname>Messanga Essengue</surname>\n<given-names>LL</given-names>\n</string-name>, <string-name>\n<surname>Ripa Njankouo</surname>\n<given-names>M</given-names>\n</string-name>, et al. <article-title>Evaluation of a mobile health approach to improve the Early Warning System of influenza surveillance in Cameroon</article-title>. <source xml:lang=\"en\">Influenza Other Respi Viruses</source>. <year>2020</year>;<volume>14</volume>:<fpage>491</fpage>–<lpage>498</lpage>. <pub-id pub-id-type=\"doi\">10.1111/irv.12747</pub-id>\n</mixed-citation>\n</p></notes></front><body id=\"irv12747-body-0001\"><sec id=\"irv12747-sec-0006\"><label>1</label><title>INTRODUCTION</title><p>In recent years, influenza surveillance that was essentially virological expanded to include more epidemiological information to complement the virological data collected by the Global Influenza Surveillance and Response System (GISRS).<xref rid=\"irv12747-bib-0001\" ref-type=\"ref\">\n<sup>1</sup>\n</xref> The 2009 influenza pandemics highlighted the need for rapid reporting of cases to assess the severity of the disease, define risk factors for severe outcome and to better inform national prevention and control strategies. This has urged many countries to establish surveillance systems for the early detection of public health emergencies and detection of potential pandemic influenza strains.<xref rid=\"irv12747-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref>\n</p><p>Reporting of surveillance data has mostly made use of paper‐based systems, mobile phone–based systems and Web‐based systems. Among these, mobile and Internet technologies have been successfully used for EWS in several countries and settings.<xref rid=\"irv12747-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref>, <xref rid=\"irv12747-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref>, <xref rid=\"irv12747-bib-0004\" ref-type=\"ref\">\n<sup>4</sup>\n</xref>, <xref rid=\"irv12747-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref> In Cameroon, there has been progress in the collection tools for influenza epidemiological data from forms to SMS (short message service) to smartphone using the Internet in order to improve on the timeliness of data collected. The implementation of the EWS, a Web‐based system that makes use of smartphones, within the influenza surveillance in 2017 started with a few sentinel sites in Cameroon for more real‐time analyses of data collected and in the preparedness of a future pandemic event.</p><p>We evaluate here the performance of the EWS as compared to prior tools for collecting influenza epidemiological data and estimate the annual proportion of influenza‐associated illness among total outpatient visits in Cameroon.</p></sec><sec sec-type=\"methods\" id=\"irv12747-sec-0007\"><label>2</label><title>METHODS</title><sec id=\"irv12747-sec-0008\"><label>2.1</label><title>Description of the influenza surveillance system</title><p>For more than a decade, the Centre Pasteur of Cameroon has been designated the National Influenza Centre of Cameroon by the Ministry of Health and by the World Health Organization. In 2019, the influenza surveillance system comprised 16 sites distributed in 7 of the 10 administrative regions of the country. Among these, 13 sites collect data from outpatients, while 3 sites collect data from hospitalized patients with a severe acute respiratory infection (SARI). This surveillance system generates two main types of data: epidemiological data from sentinel sites and virological data from laboratory analysis of samples collected. Epidemiological data are collected weekly from sentinel sites and comprise information on the number of consultations, number of febrile illness, number of acute respiratory infections (ARI), number of influenza‐like illness (ILI) and number of samples collected. Meanwhile, virological data obtained mostly comprise the influenza status of each individual sample collected. Nasopharyngeal and/or oropharyngeal swabs collected from the sites are analysed for the presence of influenza virus using the gold standard assay, rRT‐PCR, as previously described.<xref rid=\"irv12747-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref>\n</p></sec><sec id=\"irv12747-sec-0009\"><label>2.2</label><title>Evolution in the tools for collecting epidemiological data</title><p>Tools for the collection of epidemiological data from sentinel sites have gradually evolved over the years from forms (paper‐based system) to SMS to the smartphones (EWS). Initially, all epidemiological data were sent through the paper‐based system together with the respiratory samples. However, some major issues encountered with this system were the lack of complete data and timeliness. In September 2012, weekly reporting by SMS started at the sentinel sites in addition to the paper‐based system. Data sent by SMS comprised reduced information as compared to the forms, with two parameters reported by age groups, that is number of consultations and number of ILI. This reduced reporting via SMS was implemented to enable timely reporting of the minimum essential data in the WHO FluID platform (<ext-link ext-link-type=\"uri\" xlink:href=\"https://extranet.who.int/fluid/Login.aspx?ReturnUrl=%252ffluid%252f\">https://extranet.who.int/fluid/Login.aspx?ReturnUrl=%2ffluid%2f</ext-link>) since sentinel sites located in distant regions had difficulties sending the forms on time. Data sent by SMS could be received by one of the two telephone devices located at the NIC. Once the form or SMS data are received, they are entered manually in an Excel database.</p><p>Recently, reporting via the EWS with smartphones was initiated in a few sentinel sites in order to improve still on the timeliness of data received. The EWS makes use of <italic>Event Capture</italic>, an Android application which enables to capture and submit events (<ext-link ext-link-type=\"uri\" xlink:href=\"https://play.google.com/store/apps/details?id=org.hisp.dhis.android.eventcapture%26hl=en\">https://play.google.com/store/apps/details?id=org.hisp.dhis.android.eventcapture&hl=en</ext-link>). This system first started in January 2017 with sites located in the same town as the NIC (Yaounde) for a better coordination of this novel tool, and then was extended to sites located in the Northern region of Cameroon (Garoua) in August 2018. The EWS started with weekly reporting, but changed during the second phase of implementation to daily reporting for a better preparedness to a future pandemic event or in case of any unusual rise in influenza activity. Daily data sent through the EWS are aggregated into weekly data and extracted automatically in the server at the NIC.</p><p>Of the 13 sites which collect data from persons with ILI, 6 sites send data through the EWS, while the remaining 7 sites make use of forms and SMS. Of the 6 sites supposed to send data through the EWS, one had not sent any data and was discarded in the analysis.</p></sec><sec id=\"irv12747-sec-0010\"><label>2.3</label><title>Method of comparison of collection tools</title><p>We used four criteria for the comparison of the epidemiological data collection tools: completeness, timeliness, conformity and cost. Proportions of each criterion were compared among all three tools. Completeness refers to data of the 52 epidemiological weeks that was successfully sent. For the EWS, completeness also involved sending all five or six daily data corresponding to the working days of the week.<disp-formula id=\"irv12747-disp-0001\"><mml:math id=\"nlm-math-1\"><mml:mrow><mml:mtext>Completeness</mml:mtext><mml:mspace width=\"0.277778em\"/><mml:mfenced close=\")\" open=\"(\"><mml:mo>%</mml:mo></mml:mfenced><mml:mo>=</mml:mo><mml:mtext>Number of data received</mml:mtext><mml:mo stretchy=\"false\">/</mml:mo><mml:mtext>Number of data expected</mml:mtext><mml:mo>×</mml:mo><mml:mn>100</mml:mn></mml:mrow></mml:math></disp-formula>\n</p><p>Timeliness refers to data that were sent timely, that is within three days following the end of the reporting period.<disp-formula id=\"irv12747-disp-0002\"><mml:math id=\"nlm-math-2\"><mml:mrow><mml:mtext>Timeliness</mml:mtext><mml:mspace width=\"0.277778em\"/><mml:mfenced close=\")\" open=\"(\"><mml:mo>%</mml:mo></mml:mfenced><mml:mo>=</mml:mo><mml:mtext>Number of data received on time</mml:mtext><mml:mo stretchy=\"false\">/</mml:mo><mml:mtext>Number of data received</mml:mtext><mml:mo>×</mml:mo><mml:mn>100</mml:mn></mml:mrow></mml:math></disp-formula>\n</p><p>Conformity refers to data that had no errors. We considered here as errors data with totals of each parameter wrongly calculated, incoherence of data (number of ILI > number of ARI OR number of febrile illness > number of consultations), errors in selecting the epidemiological week and presence of missing values in data sent.<disp-formula id=\"irv12747-disp-0003\"><mml:math id=\"nlm-math-3\"><mml:mrow><mml:mtext>Conformity</mml:mtext><mml:mspace width=\"0.277778em\"/><mml:mfenced close=\")\" open=\"(\"><mml:mo>%</mml:mo></mml:mfenced><mml:mo>=</mml:mo><mml:mtext>Number of quality data received</mml:mtext><mml:mo stretchy=\"false\">/</mml:mo><mml:mtext>Number of data received</mml:mtext><mml:mo>×</mml:mo><mml:mn>100</mml:mn></mml:mrow></mml:math></disp-formula>\n</p><p>Cost corresponds to the average cost in USD of sending one datum by a sentinel site per week. The cost of sending one datum through the EWS comprised the weekly cost of Internet provision necessary to send the data. The cost of sending one datum through the SMS comprised the cost of the SMS in accordance with the network provisioner. The cost of sending data through the paper‐based system comprised the transport cost for sending the notification forms alongside the collected samples. We exclusively use 2019 data for comparisons among the different tools to ease analyses and to minimize bias.</p></sec><sec id=\"irv12747-sec-0011\"><label>2.4</label><title>Statistical analysis</title><p>Comparison of proportions of the different collection tools was performed using the chi‐square test in IBM SPSS statistical software version 22.0 and considering the proportions obtained with the EWS as reference values. Meanwhile, the Student <italic>t</italic> test was used to compare means. The annual proportional contribution of influenza‐associated ILI to outpatient load (<italic>P</italic>) was calculated using the method described in WHO's Manual for Estimating Disease Burden Associated with Influenza.<xref rid=\"irv12747-bib-0007\" ref-type=\"ref\">\n<sup>7</sup>\n</xref> The burden of influenza‐associated ILI to annual outpatient load was calculated by estimating the proportion of the total number of influenza‐associated ILI visits among all outpatient visits. For more adequate analyses, virological data were considered for the sites that had consistently collected at least 75% of complete epidemiological data during the years 2017‐2019.<disp-formula id=\"irv12747-disp-0004\"><mml:math id=\"nlm-math-4\"><mml:mrow><mml:mi>P</mml:mi><mml:mspace width=\"0.277778em\"/><mml:mfenced close=\")\" open=\"(\"><mml:mo>%</mml:mo></mml:mfenced><mml:mo>=</mml:mo><mml:mrow><mml:mtext>Number of influenza</mml:mtext><mml:mspace width=\"0.333333em\"/><mml:mo>-</mml:mo><mml:mspace width=\"0.333333em\"/><mml:mtext>associated ILI visits</mml:mtext></mml:mrow><mml:mo stretchy=\"false\">/</mml:mo><mml:mtext>Total number of outpatient visits at the sentinel site</mml:mtext><mml:mo>×</mml:mo><mml:mn>100</mml:mn></mml:mrow></mml:math></disp-formula>\n</p></sec></sec><sec sec-type=\"results\" id=\"irv12747-sec-0012\"><label>3</label><title>RESULTS</title><sec id=\"irv12747-sec-0013\"><label>3.1</label><title>Description of epidemiological data collected from 2017‐2019</title><p>The number of outpatient visits and subsequently all surveillance data decreased across the years 2017‐2019. The proportion of febrile illness, ARI and ILI with respect to the number of consultations was highest in the 1‐4 years age group in all 3 years, whereas the lowest proportions of the three epidemiological data were observed in the ≥ 50 years age group (Table <xref rid=\"irv12747-tbl-0001\" ref-type=\"table\">1</xref>).</p><table-wrap id=\"irv12747-tbl-0001\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 1</label><caption><p>Epidemiological data collected with respect to virological data</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Age group</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Consultation<sup>a</sup>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Febrile illness</p>\n<p>N (%)</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>ARI</p>\n<p>N (%)</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>ILI<sup>b</sup>\n</p>\n<p>N (%)</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">No. tested</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Influenza positive</p>\n<p>N (%)<sup>c</sup>\n</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Inf.‐associated ILI cases<sup>d</sup>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Inf.‐associated ILI to outpatient load per 100 persons (%)<sup>e</sup>\n</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"9\" rowspan=\"1\">2017</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\"><1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">18 196</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4917 (27.0)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">836 (4.6)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">303 (1.7)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">286</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">47 (16.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.27</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">1‐4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">20 715</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6117 (29.5)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1006 (4.9)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">547 (2.6)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">518</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">185 (35.7)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">195</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.94</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">5‐14</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">17 246</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4918 (28.5)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">435 (2.5)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">164 (1.0)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">155</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">67 (43.2)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">71</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.41</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">15‐49</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">65 908</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6984 (10.6)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">702 (1.1)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">249 (0.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">189</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">52 (27.5)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">69</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.10</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">≥50</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">24 824</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1793 (7.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">258 (1.0)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">81 (0.3)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">62</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">14 (22.6)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.07</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Unknown</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">86</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">33 (38.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">/</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">/</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">146 889</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">24 729 (16.8)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3237 (2.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1344 (0.9)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1296</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">398 (30.7)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">413</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.28</td></tr><tr><td align=\"left\" colspan=\"9\" rowspan=\"1\">2018</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\"><1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">18 046</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4636 (25.7)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">924 (5.1)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">260 (1.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">200</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">50 (25.0)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">65</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.36</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">1‐4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">18 542</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5953 (32.1)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1173 (6.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">491 (2.6)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">354</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">109 (30.8)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">151</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.82</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">5‐14</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">16 079</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5073 (31.6)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">520 (3.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">177 (1.1)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">138</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">45 (32.6)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">58</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.36</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">15‐49</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">66 579</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">8251 (12.4)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">967 (1.5)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">229 (0.3)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">162</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">57 (35.2)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">81</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.12</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">≥50</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">20 952</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2072 (9.9)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">311 (1.5)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">77 (0.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">48</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">15 (31.3)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">24</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.11</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Unknown</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (7.7)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">/</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">/</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">140 198</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">25 985 (18.5)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3895 (2.8)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1234 (0.9)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">928</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">278 (30.0)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">370</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.26</td></tr><tr><td align=\"left\" colspan=\"9\" rowspan=\"1\">2019</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\"><1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">12 452</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3206 (25.7)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">771 (6.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">298 (2.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">158</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">32 (20.3)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.48</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">1‐4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">12 433</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4122 (33.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">862 (6.9)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">421 (3.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">240</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">73 (30.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">128</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.03</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">5‐14</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10 423</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3004 (28.8)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">488 (4.7)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">235 (2.3)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">102</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">42 (41.2)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">97</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.93</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">15‐49</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">45 307</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6024 (13.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">806 (1.8)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">231 (0.5)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">105</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">33 (31.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">73</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.16</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">≥50</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">14 227</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1476 (10.4)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">198 (1.4)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">54 (0.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">9 (24.3)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.09</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Unknown</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">140</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">32 (22.9)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">/</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">/</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">94 842</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">17 832 (18.8)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3125 (3.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1239 (1.3)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">782</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">221 (28.3)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">351</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.37</td></tr></tbody></table><table-wrap-foot id=\"irv12747-ntgp-0001\"><title>Note</title><fn id=\"irv12747-note-0001\"><p>d = (b) × (c); e = (d)/(a) × 100.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>Influenza positivity rate decreased across the years from 30.7% to 28.3% with predominant age group varying from one year to another. Contrarily, the proportion of influenza‐associated ILI to outpatient load was highest in the year 2019 (0.37 per 100 persons vs 0.28 and 0.26 in the other 2 years). In all 3 years, the proportion of influenza‐associated ILI to outpatient load was highest in the 1‐4 years age group and lowest in the ≥ 50 years age group.</p></sec><sec id=\"irv12747-sec-0014\"><label>3.2</label><title>Trends in epidemiological and virological surveillance data</title><p>Figure <xref rid=\"irv12747-fig-0001\" ref-type=\"fig\">1</xref> shows the epidemiological trends and weekly distribution of influenza virus during the years 2017‐2019. Globally, we noted some visual correlation between influenza positivity rate and the epidemiological data collected. In 2017, influenza positivity rate correlated with number of ARI and ILI. Meanwhile, in 2018‐2019, influenza positivity rate correlated with numbers of ARI and ILI between week 37 and week 52. Moreover, periods with consistently high ILI levels (>20) were associated with increased influenza activity. Higher influenza activity was observed at the end of the year between week 37 and week 52. Meanwhile, a small peak in influenza activity between week 11 and week 21 did not correlate with any epidemiological data.</p><fig fig-type=\"FIGURE\" xml:lang=\"en\" id=\"irv12747-fig-0001\" orientation=\"portrait\" position=\"float\"><label>FIGURE 1</label><caption><p>Epidemiological trends and weekly distribution of influenza virus</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"IRV-14-491-g001\"/></fig></sec><sec id=\"irv12747-sec-0015\"><label>3.3</label><title>Comparison of the tools for collecting surveillance data</title><p>Concerning the tools used in collecting epidemiological data; of the 364 data that were expected to be sent by forms, 81.6% were eventually sent to the NIC, 76.1% of which were conform. Meanwhile, data sent by SMS were 44.8% complete, 60.7% prompt and 84.0% conform. Data sent via the EWS on the other hand was complete at 97.6%, with a timeliness of 74.4% and conformity of 89.5%. Completeness of daily data collected via the EWS was moderate at 77.3% (Table <xref rid=\"irv12747-tbl-0002\" ref-type=\"table\">2</xref>). Regarding the reasons for non‐conformity of data reported; errors in the forms and SMS were mostly due to calculation of the totals of each parameter (32.9% vs 71.4%), incoherence of data (64.5% vs 10.7%) and errors in selecting the epidemiological week (2.6% vs 17.9%). Non‐conformity observed with the EWS was essentially due to missing values in data sent. The average cost of sending a datum by a sentinel site per week was higher for the forms (5.0 USD) than for the EWS (0.9 USD) and SMS (0.1 USD).</p><table-wrap id=\"irv12747-tbl-0002\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 2</label><caption><p>Comparison of completeness, timeliness and conformity of collection tools</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"2\" valign=\"top\" colspan=\"1\"/><th align=\"left\" rowspan=\"2\" valign=\"top\" colspan=\"1\">\n<p>EWS (Ref)</p>\n<p>N = 260</p>\n</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"top\" rowspan=\"1\">\n<p>Forms</p>\n<p>N = 364</p>\n</th><th align=\"left\" colspan=\"2\" style=\"border-bottom:solid 1px #000000\" valign=\"top\" rowspan=\"1\">\n<p>SMS</p>\n<p>N = 364</p>\n</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">N (%)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>‐value</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">N (%)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>‐value</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Completeness (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">254 (97.6)/201 (77.3)<xref ref-type=\"fn\" rid=\"irv12747-note-0003\">\n<sup>a</sup>\n</xref>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">297 (81.6)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"><.001</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">163 (44.8)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"><.001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Timeliness (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">192 (74.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">n/a</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">99 (60.7)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Conformity</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">238 (93.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">226 (76.1)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"><.001</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">137 (84.0)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.025</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Average cost/week (USD)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"><.001</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"><.001</td></tr></tbody></table><table-wrap-foot id=\"irv12747-ntgp-0002\"><title>Note</title><fn id=\"irv12747-note-0002\"><p>N = expected data. n/a: not applicable; <italic>P</italic>‐values are related to comparison of proportions or average with respect to the EWS considered here as reference.</p></fn><fn id=\"irv12747-note-0003\"><label><sup>a</sup></label><p>Completeness related to sending the 5 or 6 daily data via the EWS.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>Regarding the performance of the collection tools by sentinel sites, the majority of data sent by the EWS were ≥94% complete (weekly data), 70%‐94% had complete daily data, 70%‐90% were sent on time, and 86%‐100% were of good quality. Data sent by forms were 85%‐100% complete for the majority; meanwhile, two sites (BASB and EBHR) had 27% and 65% complete data. Conformity of data was 71%‐96% for five of the seven sites; meanwhile, DOAG and BASB had 34% and 64% conformity, respectively. Data sent by SMS were 35%‐77% complete for most sites; meanwhile, one site (DOAG) had 13% complete data. Timeliness of data sent by SMS was 64%‐86% for five sites with one site having 6% timeliness (BASB). SMS conformity on the other hand was 86%‐93% for most sites and 62% for BASB. One of the sentinel sites sent no SMS data. Figure <xref rid=\"irv12747-fig-0002\" ref-type=\"fig\">2</xref> shows the performance of each sentinel site based on the three data collection tools.</p><fig fig-type=\"FIGURE\" xml:lang=\"en\" id=\"irv12747-fig-0002\" orientation=\"portrait\" position=\"float\"><label>FIGURE 2</label><caption><p>Sentinel site performance based on the different data collection tools. Sentinel sites are denoted by four‐letter codes; YAAF = CMS Ambassade de France (Yaounde); YAET = CSI d'Etoudi (Yaounde); GAFO = Hôpital de Foulbere (Garoua); GARO = CSI de Roumde Adjia (Garoua); GAHR = Garoua Regional Hospital (Garoua); BJSE = CSI de Bandjoun (Bandjoun); FOKU = CSI de Kueka (Foumban); DOAG = Hôpital Albert le Grand (Douala); DOCL = Hôpital Catholique de Log Pom (Douala); BUMM = Mount Mary Hospital (Buea); BASB = Polyclinic St Blaise (Bamenda); EBHR = Ebolowa Regional Hospital</p></caption><graphic id=\"nlm-graphic-3\" xlink:href=\"IRV-14-491-g002\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"irv12747-sec-0016\"><label>4</label><title>DISCUSSION</title><p>This study aimed to compare the performance of the EWS to the paper‐based system and to the SMS in reporting influenza epidemiological data with respect to four selected criteria. Results showed that the EWS had significantly better performance in sending complete, prompt and conform data at a low cost.</p><p>Regarding completeness of data, SMS had the lowest proportion of complete data. This can be attributed in part to the disruption of the mobile network for over 3 months in the main telephone device through which the SMS should be sent. Also, some focal points raised the work overload as a reason for not sending SMS data and preferred making snapshots of the epidemiological forms which they consider easier to send via mobile applications (WhatsApp). Another reason for the low proportion of complete data received is the security issue faced by two regions in which the sentinel sites are located. Focal points in these regions (BASB and BUMM) reported facing difficulties conducting their daily activity including the surveillance activity. Meanwhile, not all forms containing epidemiological data were sent to the NIC. The main reason for this is the small number of persons involved in the influenza surveillance activity at sentinel sites and the high workload. As reported by the influenza surveillance team in Zambia, having a dedicated surveillance staff may increase enrolment rates. However, hiring new staff would decrease the sustainability of the surveillance system.<xref rid=\"irv12747-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref>\n</p><p>Routinely, timeliness of the paper‐based system in Cameroon is generally low and is not evaluated due to the fact that sentinel sites located in further regions do not send data when there are no samples accompanying it. Meanwhile, timeliness of SMS data was lower than the EWS although both had moderately good values. The workload has been reported by the focal points as the main reason for not sending timely data. Timeliness of the SMS was lower than reported by other influenza surveillance systems in Africa<xref rid=\"irv12747-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref>, <xref rid=\"irv12747-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref> but higher than that observed in 2014‐2015 with the IDSR in Madagascar.<xref rid=\"irv12747-bib-0010\" ref-type=\"ref\">\n<sup>10</sup>\n</xref>\n</p><p>Regarding data quality, there were fewer errors in data sent through the EWS than data sent by forms or SMS. This is not surprising since the most commonly noted sources of error with the forms and SMS were corrected during the implementation and programming of the EWS. However, some data presented with missing information in the EWS, and this was corrected automatically in the system once the error was identified. A similar study in Kenya reported as well less errors in smartphones compared to the paper‐based questionnaire.<xref rid=\"irv12747-bib-0011\" ref-type=\"ref\">\n<sup>11</sup>\n</xref> Generating automated weekly bulletins for reporting performance, trends and summary of data collected by each site could help identify erroneous data rapidly, improve on site performance and help in driving public health actions as noted by other EWS.<xref rid=\"irv12747-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref>\n</p><p>The average cost of sending a datum by a sentinel site per week was lower for the SMS (0.1 USD) than for the forms (5 USD) and EWS (0.9 USD). However, SMS data still need to be entered manually in the database and this could be a potential source of error. The cost of sending data by the paper‐based system was high because the forms are generally sent together with the samples. Meanwhile, the annual average cost for sending data through the EWS did not take into consideration the cost of setting up the electronic data collection system which is greater due to the high cost of electronic equipment and operating software. However, once these initial expenses have been handled, the EWS remains more cost‐effective than using the paper‐based system and SMS especially considering the possibility of analysing the data on real time. Similar findings were reported in Kenya where the EWS was found to be more cost‐effective than the paper‐based system.<xref rid=\"irv12747-bib-0011\" ref-type=\"ref\">\n<sup>11</sup>\n</xref>\n</p><p>The estimated incidence of influenza‐associated ILI outpatient visits in 2019 (0.37) was lower than that observed in Senegal within the cumulative period of 2013‐2015 (0.9/100 population), in the USA (8.7/1000 population) and in Thailand (14.2/1000 population).<xref rid=\"irv12747-bib-0004\" ref-type=\"ref\">\n<sup>4</sup>\n</xref>, <xref rid=\"irv12747-bib-0012\" ref-type=\"ref\">\n<sup>12</sup>\n</xref>, <xref rid=\"irv12747-bib-0013\" ref-type=\"ref\">\n<sup>13</sup>\n</xref> Our results might underestimate the burden of influenza‐associated ILI in Cameroon since the majority of patients with ILI do not refer to any health facility for treatment. Also, a hospital admission survey is essential in order to have more accurate burden of disease estimate using the catchment population.<xref rid=\"irv12747-bib-0007\" ref-type=\"ref\">\n<sup>7</sup>\n</xref> Nevertheless, the 1‐4 and 5‐14 years age groups had higher proportions of influenza‐associated ILI outpatient visits (1.03 and 0.93) confirming that there are risk groups on which targeted prevention strategies should be addressed. A previous study from Cameroon has indeed confirmed higher transmission rates of influenza virus in this age groups probably due to high contact rates in schools.<xref rid=\"irv12747-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref> There was one peak of influenza activity in 2019 between week 39 and week 52 and this was slightly correlated with ILI levels. This could be used in setting up the alert thresholds in the EWS. This result corroborates with previous findings which showed that the major period for influenza activity in Cameroon is between the months of September to December.<xref rid=\"irv12747-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref> Although ILI and ILI% are better indicators for use in EWS, as they are easily generated, these indicators may results in bias since illnesses other than influenza may present with ILI.<xref rid=\"irv12747-bib-0014\" ref-type=\"ref\">\n<sup>14</sup>\n</xref>, <xref rid=\"irv12747-bib-0015\" ref-type=\"ref\">\n<sup>15</sup>\n</xref>\n</p></sec><sec sec-type=\"conclusions\" id=\"irv12747-sec-0017\"><label>5</label><title>CONCLUSION</title><p>At the end of this study, which aimed to evaluate the performance of the EWS in collecting epidemiological data as compared to the paper‐based system and the SMS, we found that the EWS had significantly satisfactory performance based on the four selected criteria for evaluation. Also, after implementation, considering the low cost of approximately 0.9 USD for sending one complete surveillance data per site, this tool could be proposed for national surveillance systems. All sentinel sites and even other disease surveillance systems are expected to use this tool in the near term future due to its satisfactory performance and cost. The next step in the EWS is to integrate alert threshold for influenza virus circulation in Cameroon based on previous surveillance data.</p></sec><sec sec-type=\"COI-statement\" id=\"irv12747-sec-0019\"><title>CONFLICT OF INTEREST</title><p>The authors declare that they have no competing interests.</p></sec><sec id=\"irv12747-sec-0020\"><title>AUTHOR CONTRIBUTIONS</title><p>\n<bold>Chavely Gwladys Monamele: </bold>Formal analysis (lead); methodology (lead); writing‐original draft (lead); writing‐review & editing (equal). <bold>Loique Landry Messanga Essengue: </bold>Data curation (equal); formal analysis (supporting); software (equal); writing‐review & editing (equal). <bold>Mohamadou Ripa Njankouo: </bold>Investigation (equal); writing‐review & editing (equal). <bold>Hermann Landry Munshili Njifon: </bold>Investigation (equal); writing‐review & editing (equal); <bold>Jules Tchatchueng: </bold>Data curation (equal); software (equal); writing‐review & editing (equal); <bold>Mathurin Cyrille Tejiokem: </bold>Conceptualization (supporting); methodology (supporting); supervision (equal); writing‐review & editing (equal). <bold>Richard Njouom: </bold>Conceptualization (lead); funding acquisition (lead); methodology (supporting); supervision (equal); validation (lead); writing‐review & editing (equal).</p></sec></body><back><ack id=\"irv12747-sec-0018\"><title>ACKNOWLEDGEMENTS</title><p>We are grateful to all the focal points of the influenza surveillance system in Cameroon for their involvement in data collection. This work received a grant from the US Department of Health and Human Services, DHHS (Grant Number 6 DESP060001‐01‐01), and from the WHO PIP Implementation project in Cameroon.</p></ack><ref-list content-type=\"cited-references\" id=\"irv12747-bibl-0001\"><title>REFERENCES</title><ref id=\"irv12747-bib-0001\"><label>1</label><mixed-citation publication-type=\"book\" id=\"irv12747-cit-0001\">\n<collab collab-type=\"authors\">WHO</collab>\n. <source xml:lang=\"en\">Protocol for National Influenza Sentinel Surveillance</source>; <year>2015</year>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.afro.who.int/sites/default/files/2017-06/97892%252090232889.pdf\">https://www.afro.who.int/sites/default/files/2017‐06/97892%2090232889.pdf</ext-link>. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"iso-abbrev\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"doi\">10.1111/(ISSN)1750-2659</journal-id><journal-id journal-id-type=\"publisher-id\">IRV</journal-id><journal-title-group><journal-title>Influenza and Other Respiratory Viruses</journal-title></journal-title-group><issn pub-type=\"ppub\">1750-2640</issn><issn pub-type=\"epub\">1750-2659</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32445270</article-id><article-id pub-id-type=\"pmc\">PMC7431647</article-id><article-id pub-id-type=\"doi\">10.1111/irv.12757</article-id><article-id pub-id-type=\"publisher-id\">IRV12757</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Original Article</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Original Articles</subject></subj-group></article-categories><title-group><article-title>Epidemiological trends in notified influenza cases in Australia’s Northern Territory, 2007‐2016</article-title><alt-title alt-title-type=\"left-running-head\">WEINMAN et al.</alt-title></title-group><contrib-group><contrib id=\"irv12757-cr-0001\" contrib-type=\"author\"><name><surname>Weinman</surname><given-names>Aaron L.</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-5177-9614</contrib-id><xref ref-type=\"aff\" rid=\"irv12757-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12757-cr-0002\" contrib-type=\"author\"><name><surname>Sullivan</surname><given-names>Sheena G.</given-names></name><xref ref-type=\"aff\" rid=\"irv12757-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12757-cr-0003\" contrib-type=\"author\"><name><surname>Vijaykrishna</surname><given-names>Dhanasekaran</given-names></name><xref ref-type=\"aff\" rid=\"irv12757-aff-0003\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12757-aff-0004\">\n<sup>4</sup>\n</xref></contrib><contrib id=\"irv12757-cr-0004\" contrib-type=\"author\"><name><surname>Markey</surname><given-names>Peter</given-names></name><xref ref-type=\"aff\" rid=\"irv12757-aff-0005\">\n<sup>5</sup>\n</xref></contrib><contrib id=\"irv12757-cr-0005\" contrib-type=\"author\"><name><surname>Levy</surname><given-names>Avram</given-names></name><xref ref-type=\"aff\" rid=\"irv12757-aff-0006\">\n<sup>6</sup>\n</xref></contrib><contrib id=\"irv12757-cr-0006\" contrib-type=\"author\"><name><surname>Miller</surname><given-names>Adrian</given-names></name><xref ref-type=\"aff\" rid=\"irv12757-aff-0007\">\n<sup>7</sup>\n</xref></contrib><contrib id=\"irv12757-cr-0007\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Tong</surname><given-names>Steven Y. C.</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-1368-8356</contrib-id><xref ref-type=\"aff\" rid=\"irv12757-aff-0008\">\n<sup>8</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12757-aff-0009\">\n<sup>9</sup>\n</xref><address><email>steven.tong@mh.org.au</email></address></contrib></contrib-group><aff id=\"irv12757-aff-0001\">\n<label><sup>1</sup></label>\n<named-content content-type=\"organisation-division\">Doherty Department</named-content>\n<named-content content-type=\"organisation-division\">Peter Doherty Institute for Infection and Immunity</named-content>\n<institution>University of Melbourne</institution>\n<city>Melbourne</city>\n<named-content content-type=\"country-part\">Victoria</named-content>\n<country country=\"AU\">Australia</country>\n</aff><aff id=\"irv12757-aff-0002\">\n<label><sup>2</sup></label>\n<named-content content-type=\"organisation-division\">Doherty Department</named-content>\n<named-content content-type=\"organisation-division\">WHO Collaborating Centre for Reference and Research on Influenza</named-content>\n<named-content content-type=\"organisation-division\">Royal Melbourne Hospital</named-content>\n<named-content content-type=\"organisation-division\">Peter Doherty Institute for Infection and Immunity</named-content>\n<institution>University of Melbourne</institution>\n<city>Melbourne</city>\n<named-content content-type=\"country-part\">Victoria</named-content>\n<country country=\"AU\">Australia</country>\n</aff><aff id=\"irv12757-aff-0003\">\n<label><sup>3</sup></label>\n<named-content content-type=\"organisation-division\">Department of Microbiology</named-content>\n<named-content content-type=\"organisation-division\">Biomedicine Discovery Institute</named-content>\n<institution>Monash University</institution>\n<city>Clayton</city>\n<named-content content-type=\"country-part\">Victoria</named-content>\n<country country=\"AU\">Australia</country>\n</aff><aff id=\"irv12757-aff-0004\">\n<label><sup>4</sup></label>\n<named-content content-type=\"organisation-division\">WHO Collaborating Centre for Reference and Research on Influenza</named-content>\n<named-content content-type=\"organisation-division\">Royal Melbourne Hospital</named-content>\n<institution>Peter Doherty Institute for Infection and Immunity</institution>\n<city>Melbourne</city>\n<named-content content-type=\"country-part\">Victoria</named-content>\n<country country=\"AU\">Australia</country>\n</aff><aff id=\"irv12757-aff-0005\">\n<label><sup>5</sup></label>\n<institution>Northern Territory Centre for Disease Control</institution>\n<city>Casuarina</city>\n<named-content content-type=\"country-part\">Northern Territory</named-content>\n<country country=\"AU\">Australia</country>\n</aff><aff id=\"irv12757-aff-0006\">\n<label><sup>6</sup></label>\n<institution>PathWest Laboratory Medicine</institution>\n<city>Nedlands</city>\n<named-content content-type=\"country-part\">Western Australia</named-content>\n<country country=\"AU\">Australia</country>\n</aff><aff id=\"irv12757-aff-0007\">\n<label><sup>7</sup></label>\n<named-content content-type=\"organisation-division\">Centre for Indigenous Health and Equity Research</named-content>\n<institution>CQUniversity</institution>\n<city>Townsville</city>\n<named-content content-type=\"country-part\">Queensland</named-content>\n<country country=\"AU\">Australia</country>\n</aff><aff id=\"irv12757-aff-0008\">\n<label><sup>8</sup></label>\n<named-content content-type=\"organisation-division\">Doherty Department</named-content>\n<named-content content-type=\"organisation-division\">Victorian Infectious Diseases Service</named-content>\n<named-content content-type=\"organisation-division\">The Royal Melbourne Hospital</named-content>\n<named-content content-type=\"organisation-division\">Peter Doherty Institute for Infection and Immunity</named-content>\n<institution>University of Melbourne</institution>\n<city>Melbourne</city>\n<named-content content-type=\"country-part\">Victoria</named-content>\n<country country=\"AU\">Australia</country>\n</aff><aff id=\"irv12757-aff-0009\">\n<label><sup>9</sup></label>\n<institution>Menzies School of Health Research</institution>\n<city>Darwin</city>\n<named-content content-type=\"country-part\">Northern Territory</named-content>\n<country country=\"AU\">Australia</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label>\nCorrespondence<break/>\nSteven Y. C. TongVictorian Infectious Diseases Service, Royal Melbourne Hospital, and Doherty Department, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Victoria, 3000, Australia.<break/>\nEmail: <email>steven.tong@mh.org.au</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>23</day><month>5</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>9</month><year>2020</year></pub-date><volume>14</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1111/irv.v14.5</issue-id><fpage>541</fpage><lpage>550</lpage><history><date date-type=\"received\"><day>15</day><month>2</month><year>2020</year></date><date date-type=\"rev-recd\"><day>21</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>23</day><month>4</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 John Wiley & Sons Ltd <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Influenza and Other Respiratory Viruses</italic> Published by John Wiley & Sons Ltd.</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:IRV-14-541.pdf\"/><abstract id=\"irv12757-abs-0001\"><title>Abstract</title><sec id=\"irv12757-sec-0100\"><title>Background</title><p>The Northern Territory (NT) of Australia has a mix of climates, sparsely distributed population and a large proportion of the populace are Indigenous Australians, and influenza is known to have a disproportionate impact upon this group. Understanding the epidemiology of influenza in this region would inform public health strategies.</p></sec><sec id=\"irv12757-sec-0001\"><title>Objectives</title><p>To assess if there are consistent patterns in characteristics of influenza outbreaks in the NT.</p></sec><sec id=\"irv12757-sec-0002\"><title>Methods</title><p>Laboratory confirmed influenza cases in the NT are notified to the NT Centre for Disease Control. We conducted analyses on notified cases from 2007‐2016 to determine incidence rates (by age group, Indigenous status and area), seasonality of cases and spatial distribution of influenza types. Notified cases were linked to laboratory datasets to update information on influenza type or subtype</p></sec><sec id=\"irv12757-sec-0003\"><title>Results</title><p>The disparity in Indigenous and non‐Indigenous notification rates varied by age group, with rate ratios for Indigenous versus non‐Indigenous ranging from 1.58 (95% CI:1.39, 1.80) for ages 15‐24 to 5.56 (95% CI: 4.71, 6.57) for ages 55‐64. The disparity between Indigenous and non‐Indigenous notification rates appeared higher in the Central Australia region. Indigenous versus non‐Indigenous hospitalisation and mortality rate ratios were 6.51 (95% CI: 5.91, 7.18) and 5.46 (95% CI: 2.40, 12.71) respectively. Inter‐seasonal peaks during February and March occurred in 2011, 2013 and 2014, and were due to influenza activity in the tropical north of the NT.</p></sec><sec id=\"irv12757-sec-0004\"><title>Conclusions</title><p>Our results highlight the importance of influenza vaccination across all age groups for Indigenous Australians. An early vaccination campaign targeted against outbreaks in February‐March would be best focused on the tropical north.</p></sec></abstract><kwd-group><kwd id=\"irv12757-kwd-0001\">epidemics</kwd><kwd id=\"irv12757-kwd-0002\">epidemiology</kwd><kwd id=\"irv12757-kwd-0003\">influenza</kwd><kwd id=\"irv12757-kwd-0004\">Northern Territory</kwd></kwd-group><counts><fig-count count=\"5\"/><table-count count=\"2\"/><page-count count=\"10\"/><word-count count=\"5943\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>September 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.6 mode:remove_FC converted:18.08.2020</meta-value></custom-meta></custom-meta-group></article-meta><notes><p content-type=\"self-citation\">\n<mixed-citation publication-type=\"journal\" id=\"irv12757-cit-1001\">\n<string-name>\n<surname>Weinman</surname>\n<given-names>AL</given-names>\n</string-name>, <string-name>\n<surname>Sullivan</surname>\n<given-names>SG</given-names>\n</string-name>, <string-name>\n<surname>Vijaykrishna</surname>\n<given-names>D</given-names>\n</string-name>, et al. <article-title>Epidemiological trends in notified influenza cases in Australia’s Northern Territory, 2007‐2016</article-title>. <source xml:lang=\"en\">Influenza Other Respi Viruses</source>. <year>2020</year>;<volume>14</volume>:<fpage>541</fpage>–<lpage>550</lpage>. <pub-id pub-id-type=\"doi\">10.1111/irv.12757</pub-id>\n</mixed-citation>\n</p><fn-group id=\"irv12757-ntgp-0001\"><fn id=\"irv12757-note-0001\"><p>The peer review history for this article is available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://publons.com/publon/10.1111/IRV.12757\">https://publons.com/publon/10.1111/IRV.12757</ext-link>.</p></fn></fn-group></notes></front><body id=\"irv12757-body-0001\"><sec id=\"irv12757-sec-0005\"><label>1</label><title>INTRODUCTION</title><p>Seasonal epidemics of the influenza virus represent a continual global challenge. These epidemics usually occur in the winter months of temperate regions, but in tropical regions, influenza is reported to circulate widely outside this season.<xref rid=\"irv12757-bib-0001\" ref-type=\"ref\">\n<sup>1</sup>\n</xref> Particular groups in the population, such as the elderly and pregnant women, are known to be at higher risk of severe outcomes from influenza infection.<xref rid=\"irv12757-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref> The virus has also caused the breakout of pandemics, the most recent being the 2009 H1N1 pandemic.<xref rid=\"irv12757-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref>\n</p><p>Australia's Northern Territory has several features which are likely to make the epidemiology of influenza in this area unique. First, climatic conditions are variable across the Northern Territory. The northern section of the Northern Territory (known as the “Top End”) has a tropical climate, whereas the southern half of the Northern Territory (known as the “Central Australia” region) has a desert climate. The potential for circulation of the virus outside the typical influenza season in this tropical region could impact upon the optimal time for vaccination in this area.</p><p>Second, 30% of the Northern Territory’s population are Indigenous Australians, making the proportion of Indigenous Australians living in the Northern Territory over 5 times higher than that of any other jurisdiction in Australia.<xref rid=\"irv12757-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref> Influenza has had a particularly severe impact on Indigenous Australians. For example, serosurveys conducted to determine rates of A(H1N1)pdm09 in the Northern Territory estimated that 22.9% of the Indigenous population in the region experienced A(H1N1)pdm09 infection in 2009, compared to only 12.4% of the non‐Indigenous population.<xref rid=\"irv12757-bib-0004\" ref-type=\"ref\">\n<sup>4</sup>\n</xref> Furthermore, hospital admissions for A(H1N1)pdm09 in the Top End region were 12 times higher for Indigenous compared to non‐Indigenous Australians.<xref rid=\"irv12757-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref>\n</p><p>Given the increased rates of influenza infection and severe outcomes from infection amongst the Indigenous population, beginning in 1999, the Australian Government funded influenza vaccination for Indigenous Australians aged 50 and older and those 15 and older that had a comorbid medical condition.<xref rid=\"irv12757-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref> Funded vaccination was expanded in 2010 to include all Indigenous Australians aged 15 and older, and expanded again in 2015 to include Indigenous Australians aged 6 months to under 5 years.<xref rid=\"irv12757-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref> Due to the disproportionate impact influenza has had upon Indigenous Australians and the large Indigenous population in the region, it is important that public health measures to counter the spread of influenza in the Northern Territory be optimised to mitigate the impact of future outbreaks. Previous surveillance of influenza activity in the Northern Territory has highlighted increased rates of influenza cases and severe outcomes of infection in the Indigenous population.<xref rid=\"irv12757-bib-0007\" ref-type=\"ref\">\n<sup>7</sup>\n</xref> However, the full impact of influenza, through pandemic and non‐pandemic periods, in the Indigenous and non‐Indigenous population has not been quantified. There also has not been a long‐term post‐pandemic comparison of the areas in which the disparity in rates of influenza is greatest. There have been reports of outbreaks of influenza activity in the Top End region of the Northern Territory outside the typical influenza season,<xref rid=\"irv12757-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> but the regions of the Northern Territory these outbreaks affect have not been assessed in all years. Year‐by‐year analyses of the divisions of the Northern Territory in which influenza rates are highest are also lacking, as are comparisons of the distribution of influenza cases between the major towns and remote areas. A better understanding of the epidemiology of influenza in this region could lead to the development of more optimally targeted vaccination campaigns. In this study, laboratory‐confirmed influenza notifications were assessed to understand whether there are consistent patterns in the timing, geographic characteristics and groups affected by influenza in the Northern Territory.</p></sec><sec id=\"irv12757-sec-0006\"><label>2</label><title>MATERIALS AND METHODS</title><sec id=\"irv12757-sec-0007\"><label>2.1</label><title>Data source</title><p>In the Northern Territory, laboratory‐confirmed influenza cases are notified to the Northern Territory Centre for Disease Control. The Centre for Disease Control collects meta‐data for each case including details about the patient’s age, sex, Indigenous status, residential location, influenza type, diagnosis date, hospitalisation status, dates of hospital stays (for hospitalised cases) and mortality. We analysed notifications from 2007 to 2016.</p><p>Following laboratory confirmation of influenza infection by pathology providers, further analysis of samples is carried out by PathWest Laboratory Medicine, Perth, or the WHO Collaborating Centre for Reference and Research on Influenza, Melbourne. Results of typing and subtyping performed by these groups on samples corresponding to notified cases were accessed.</p><p>Population denominator data were obtained from the Australian Bureau of Statistics.</p></sec><sec id=\"irv12757-sec-0008\"><label>2.2</label><title>Data cleaning and analysis</title><p>Duplicate notifications were deleted. Notifications for patients who resided outside the Northern Territory were excluded from the calculations of rates but included in analyses that used raw count data. Hospital admissions that occurred >14 days after or more than 3 days before influenza diagnosis were considered not to be influenza associated. Notifications missing hospitalisation dates were excluded from analysis of hospitalisations.</p><p>The location of each patient was classified into 2016 Statistical Area 2 (SA2) level groupings, as defined by the Australian Bureau of Statistics,<xref rid=\"irv12757-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref> based on the 2016 Australian Statistical Geography Standard Coding Indexes<xref rid=\"irv12757-bib-0010\" ref-type=\"ref\">\n<sup>10</sup>\n</xref> where possible. Locations of notified cases not found on the coding index were assigned manually using interactive maps supplied by the Australian Bureau of Statistics<xref rid=\"irv12757-bib-0011\" ref-type=\"ref\">\n<sup>11</sup>\n</xref> and the Northern Territory Places Names Register.<xref rid=\"irv12757-bib-0012\" ref-type=\"ref\">\n<sup>12</sup>\n</xref> SA2 areas that were recognised to form the suburban areas of the major cities of Darwin and Alice Springs were combined to form the Darwin and Alice Springs regions in analyses.</p><p>The notification data set was linked to corresponding patient samples stored at PathWest Laboratory Medicine, Perth, or the WHO Collaborating Centre for Reference and Research on Influenza, Melbourne. Notification data were linked to samples by matching sample date and at least one of the fields of patient name and date of birth. Information present on the databases of these laboratories was used to update information on influenza type or subtype in the notifications data set. For analyses of spatial distribution and seasonality, cases of co‐infections with type A and B were counted as separate notifications for each type.</p><p>Case notification rates in each age group in the Northern Territory by Indigenous status and age group were calculated. Rates of hospitalisation and death from influenza infection were calculated and standardised by age group to the 2007 population of the Northern Territory. Plots of numbers of cases by month and proportion of each year’s cases by month and region were constructed. Case notification rates per 100 000 population and proportion of influenza A and B cases in each region were mapped. To examine which areas consistently showed higher notification rates, each area’s incidence rate was ranked, and the median rank for each area over the 10‐year period was calculated. Maps showing the ratio of the Indigenous case notification rate to the non‐Indigenous case notification rate were constructed by pooling remote SA2 areas into broad areas due to low case numbers. Only areas that contained at least 5 notifications in the broad area for a given year and a notification for an Indigenous and non‐Indigenous population member were analysed. Statistical analyses were performed using Stata version 15.1 (StataCorp).</p></sec><sec id=\"irv12757-sec-0009\"><label>2.3</label><title>Ethics approval</title><p>Ethical approval for the study was obtained from the Human Research Ethics Committee of the Northern Territory Department of Health and Menzies School of Health Research (reference number: 2017‐2894), Central Australia Human Research Ethics Committee (reference number: CA‐18‐3031), the West Australian Aboriginal Health Ethics Committee (reference number: 851) and Melbourne Health Human Research Ethics Committee (reference number: 2018.119). A waiver of consent was approved by the reviewing committees. This study was conducted in accordance with the Australian Code for the Responsible Conduct of Research.<xref rid=\"irv12757-bib-0013\" ref-type=\"ref\">\n<sup>13</sup>\n</xref>\n</p></sec></sec><sec sec-type=\"results\" id=\"irv12757-sec-0010\"><label>3</label><title>RESULTS</title><sec id=\"irv12757-sec-0011\"><label>3.1</label><title>Notified cases and notification rates</title><p>Between 2007 and 2016, there were 6891 notified cases of influenza in the Northern Territory. The annual population during this period averaged 233 162. Influenza A made up of 5650 (82.0%) and influenza B 1222 (17.7%) of the cases. The remainder were co‐infections (9 cases) or untyped (10 cases). The subtype or lineage of 3756 (54.5%) of cases was known. Notification rates after 2009 were consistently higher than notification rates prior to 2009 (Table <xref rid=\"irv12757-sup-0001\" ref-type=\"supplementary-material\">S1</xref>).</p><p>In most years and age groups, Indigenous Australians had higher notification rates than non‐Indigenous Australians (Figure <xref rid=\"irv12757-fig-0001\" ref-type=\"fig\">1</xref>). The greatest disparity between Indigenous and non‐Indigenous notification rates throughout the study period was for older adults, although children aged between 0 and 4 were also disproportionately affected (Table <xref rid=\"irv12757-tbl-0001\" ref-type=\"table\">1</xref>).</p><fig fig-type=\"FIGURE\" xml:lang=\"en\" id=\"irv12757-fig-0001\" orientation=\"portrait\" position=\"float\"><label>FIGURE 1</label><caption><p>Rates of laboratory‐confirmed influenza cases per 100 000 population members in the Northern Territory from 2007 to 2016 by Indigenous status. Note case rates for 2009 presented with different scales on vertical axis</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"IRV-14-541-g001\"/></fig><table-wrap id=\"irv12757-tbl-0001\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 1</label><caption><p>Ratios (95% CI) of Indigenous influenza case notification rate to non‐Indigenous influenza case notification rate in the Northern Territory from 2007 to 2016 by age group. Categories marked ND indicate that there were no notifications for Indigenous or non‐Indigenous Australians that year</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Age group</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2007</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2008</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2009</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2010</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2011</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2012</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2013</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2014</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2015</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2016</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2007‐2016</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0‐4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.34 (2.85, 18.88)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">16.84 (6.10, 46.49)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5.56 (3.90, 7.92)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6.11 (3.26, 11.47)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">12.63 (6.76, 23.60)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.73 (2.29, 6.08)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.39 (2.06, 5.59)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.86 (2.59, 5.73)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.38 (1.59, 3.55)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.23 (0.74, 2.07)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4.30 (3.69, 5.00)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">5‐14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.55 (0.47, 5.08)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.22 (0.87, 5.64)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4.49 (3.49, 5.77)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.34 (1.53, 3.58)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.97 (2.40, 6.56)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.36 (0.83, 2.24)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.95 (1.13, 3.38)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.60 (2.28, 5.66)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.75 (0.53,1.06)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.77 (0.50, 1.19)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.25 (1.97, 2.56)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">15‐24</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.59 (0.26, 1.34)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.97 (1.61, 2.42)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.80 (1.16, 2.79)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.04 (1.87, 4.94)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.49 (0.86, 2.56)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.85 (1.57, 5.17)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.17 (1.43, 3.28)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.98 (0.62, 1.55)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.68 (0.43, 1.07)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.58 (1.39, 1.80)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">25‐34</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.20 (0.51, 2.81)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.54 (0.16, 1.88)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.95 (2.35, 3.71)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.20 (0.73, 1.98)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4.71 (3.01, 7.37)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.94 (0.53, 1.65)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.44 (1.40, 4.26)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.12 (2.14, 4.56)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.47 (0.94, 2.30)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.91 (0.60, 1.39)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.07 (1.81, 2.36)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">35‐44</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.04 (0.44, 2.45)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.17 (0.45, 3.00)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6.04 (4.67, 7.83)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.93 (2.32, 6.65)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">8.24 (5.08, 13.38)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.57 (0.91, 2.71)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4.58 (2.78, 7.54)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.29 (2.27, 4.76)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.23 (1.38, 3.60)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.00 (1.27, 3.17)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.71 (3.24, 4.26)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">45‐54</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.46 (0.46, 4.58)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.01 (0.81, 4.97)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7.42 (5.64, 9.75)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4.48 (2.67, 7.50)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">10.20 (6.25, 16.64)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.68 (2.07, 6.56)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7.90 (5.06, 12.35)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5.62 (3.75, 8.41)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.50 (1.60, 3.91)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.13 (1.42, 3.19)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4.97 (4.32, 5.71)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">55‐64</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.90 (1.24, 12.30)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6.35 (2.13, 18.90)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">8.88 (6.05, 13.04)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5.02 (2.48, 10.15)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">13.46 (6.91, 26.21)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.12 (1.51, 6.42)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5.28 (3.23, 8.63)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4.89 (3.36, 7.11)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.89 (2.35, 6.46)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.55 (2.29, 5.49)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5.56 (4.71, 6.57)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">65+</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.29 (0.42, 12.49)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5.95 (3.44, 10.28)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.66 (1.27, 10.54)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">14.41 (6.75, 30.74)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.00 (1.62, 5.57)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">9.12 (4.37, 19.03)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4.06 (2.56, 6.44)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.82 (1.62, 4.90)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.74 (1.05, 2.88)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.79 (3.10, 4.62)</td></tr></tbody></table><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap></sec><sec id=\"irv12757-sec-0012\"><label>3.2</label><title>Notification rates by area</title><p>Maps of notification rates by SA2 groupings demonstrated considerable variation by area and year (Figure <xref rid=\"irv12757-fig-0002\" ref-type=\"fig\">2</xref>). Tanami and the Tiwi Islands consistently ranked as having the highest rates. Notification rates in the 5 major towns of the Northern Territory (Darwin, Alice Springs, Katherine, Tennant Creek and Nhulunbuy) were broadly similar to notification rates in the surrounding remote areas.</p><fig fig-type=\"FIGURE\" xml:lang=\"en\" id=\"irv12757-fig-0002\" orientation=\"portrait\" position=\"float\"><label>FIGURE 2</label><caption><p>Influenza notification rates per 100 000 population in SA2 divisions of the Northern Territory, 2007‐2016. SA2 areas that were recognised to form the suburban areas of Darwin and Alice Springs were combined to form the Darwin and Alice Springs regions. The areas of Darwin, Alice Springs, Katherine, Nhulunbuy and Tennant Creek represent the major town areas in the Northern Territory. The map of 2007 was drawn larger to allow for labels to be resolved</p></caption><graphic id=\"nlm-graphic-3\" xlink:href=\"IRV-14-541-g002\"/></fig><p>The majority of geographical regions had higher influenza notification rates in the Indigenous population (Figure <xref rid=\"irv12757-fig-0003\" ref-type=\"fig\">3</xref>, Table <xref rid=\"irv12757-tbl-0002\" ref-type=\"table\">2</xref>). Based on raw rate ratios alone, the Central Australia region had the greatest disparity in influenza case notification rate between the Indigenous and non‐Indigenous populations. The wide confidence intervals present are likely due to small case numbers.</p><fig fig-type=\"FIGURE\" xml:lang=\"en\" id=\"irv12757-fig-0003\" orientation=\"portrait\" position=\"float\"><label>FIGURE 3</label><caption><p>Maps of the ratio of the rate of influenza notifications in the Indigenous population to the rate of influenza notifications in the non‐Indigenous population. Areas with < 5 total notifications or no notifications in either the Indigenous or non‐Indigenous population were considered to have no valid data for this analysis. The map of 2009 was drawn larger to allow for labels to be resolved</p></caption><graphic id=\"nlm-graphic-5\" xlink:href=\"IRV-14-541-g003\"/></fig><table-wrap id=\"irv12757-tbl-0002\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 2</label><caption><p>Ratios (95% CI) of Indigenous influenza case notification rate to non‐Indigenous influenza case notification rate in the Northern Territory from 2009 to 2016 by area. Categories marked ND indicate that there were no notifications for Indigenous or non‐Indigenous Australians that year or <5 total notifications in the area</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Area</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2009</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2010</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2011</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2012</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2013</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2014</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2015</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2016</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Darwin</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.03 (2.54, 3.62)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.04 (2.22, 4.16)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.22 (3.48, 7.84)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.26 (1.50, 3.40)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.54 (2.46, 5.08)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.72 (1.25, 2.38)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.14 (0.82, 1.59)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.04 (0.77, 1.41)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Alice Springs</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">8.56 (6.38, 11.48)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.59 (3.07, 6.87)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.76 (1.86, 4.08)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.87 (2.22, 6.74)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.28 (2.42, 4.46)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.27 (1.47, 3.52)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.91 (1.90, 4.47)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Tennant Creek</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">12.8 (3.08, 53.22)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.37 (0.07, 2.00)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.51 (1.24,72.69)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.39 (0.98, 19.61)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.93 (0.33, 26.18)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.36 (0.55, 3.32)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.95 (0.16, 5.68)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Nhulunbuy</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.68 (1.11, 6.47)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.81 (4.10, 14.89)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.59 (3.99, 18.52)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.60 (0.08, 4.43)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.26 (1.33, 8.01)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.45 (0.48, 4.42)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.45 (0.30, 7.00)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Katherine</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.96 (2.77, 5.65)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.77 (0.28, 2.12)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.72 (2.93, 7.62)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.91 (0.48, 1.73)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.32 (1.26, 4.24)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.92 (0.95, 3.89)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.80 (0.47, 1.39)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.11 (2.25, 7.48)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Central Australia</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">9.44 (5.04, 17.71)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20.52 (2.85, 147.52)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.18 (1.50, 25.45)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">9.73 (1.34, 70.95)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">9.29 (2.96, 29.18)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.16 (0.77, 6.04)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.26 (0.85, 45.99)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Tiwi‐West Arnhem</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.52 (1.62, 3.92)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.42 (1.34, 4.35)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.15 (2.07, 12.79)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.13 (0.53, 2.43)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.27 (1.57, 6.82)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.05 (1.68, 5.55)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.11 (1.34, 7.24)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.27 (0.69, 2.35)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">East Arnhem</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.26 (0.67, 2.37)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.37 (1.61, 11.88)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10.45 (1.45, 75.46)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.90 (0.20, 4.11)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.27 (0.91, 5.64)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.85 (0.67, 5.08)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.74 (0.86, 8.72)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.89 (0.27, 2.97)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Lower Top End</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4.11 (2.18, 7.76)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5.68 (0.77, 41.88)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.80 (0.42, 7.77)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.61 (0.62, 34.13)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ND</td></tr></tbody></table><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap></sec><sec id=\"irv12757-sec-0013\"><label>3.3</label><title>Rates of severe outcomes in Indigenous and non‐Indigenous Australians</title><p>Hospitalisations were 6.51 times higher (95% CI: 5.91, 7.18) amongst Indigenous Australians (with the Indigenous and non‐Indigenous rates being 237.01 and 36.40 per 100 000, respectively). Mortality was 5.46 times higher (95% CI: 2.40, 12.71) than that for non‐Indigenous Australians (with the Indigenous and non‐Indigenous rates being 3.14 and 0.57 per 100 000, respectively).</p></sec><sec id=\"irv12757-sec-0014\"><label>3.4</label><title>Variation in influenza seasonality</title><p>For most seasons, a typical Southern Hemisphere influenza epidemic curve is seen with peaks in August‐September (Figure <xref rid=\"irv12757-fig-0004\" ref-type=\"fig\">4A</xref>), with influenza A predominating in most time periods. However, in 2011, 2013 and 2014, an additional peak was seen during February‐March. In seasons with multiple peaks, most of the earlier cases occurred in the Top End (Figure <xref rid=\"irv12757-fig-0004\" ref-type=\"fig\">4B</xref>).</p><fig fig-type=\"FIGURE\" xml:lang=\"en\" id=\"irv12757-fig-0004\" orientation=\"portrait\" position=\"float\"><label>FIGURE 4</label><caption><p>A, Laboratory‐confirmed influenza cases in the Northern Territory by month. B, Histograms of percentage of laboratory‐confirmed influenza cases seen each month in the Top End and Central Australia regions of the Northern Territory for years in which a bimodal peak in the influenza season is seen. The total number of laboratory‐confirmed influenza cases for each of these years was 2011‐Top End 447, Central Australia: 189; 2013‐Top End 407, Central Australia: 94; 2014‐Top End 543, Central Australia: 318</p></caption><graphic id=\"nlm-graphic-7\" xlink:href=\"IRV-14-541-g004\"/></fig></sec><sec id=\"irv12757-sec-0015\"><label>3.5</label><title>Spatial distribution of influenza A and B</title><p>The proportion of type A and type B cases in each SA2 area by year is shown in Figure <xref rid=\"irv12757-fig-0005\" ref-type=\"fig\">5</xref>. While influenza A was broadly predominant in some years (2007, 2009, 2011), for most years there was geographical heterogeneity in the predominance of influenza A and B in different regions (eg 2012 and 2015). The predominant types in the town areas are broadly reflective of the predominant type in the surrounding remote area.</p><fig fig-type=\"FIGURE\" xml:lang=\"en\" id=\"irv12757-fig-0005\" orientation=\"portrait\" position=\"float\"><label>FIGURE 5</label><caption><p>Maps of the proportion of laboratory‐confirmed influenza type A and B cases in each SA2 area of the Northern Territory from 2007 to 2016. SA2 areas that were recognised to form the suburban areas of Darwin and Alice Springs were combined to form the Darwin and Alice Springs regions. The areas of Darwin, Alice Springs, Katherine, Nhulunbuy and Tennant Creek represent the major town areas in the Northern Territory</p></caption><graphic id=\"nlm-graphic-9\" xlink:href=\"IRV-14-541-g005\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"irv12757-sec-0016\"><label>4</label><title>DISCUSSION</title><p>Our analysis of notifications of laboratory‐confirmed influenza cases has revealed consistently higher rates of influenza cases in the Indigenous population of the Northern Territory. Influenza had a particularly disproportionate impact upon older Indigenous adults and Indigenous children aged 0‐4. We have also demonstrated that influenza outbreaks in the February‐March period in the Northern Territory are occurring in the tropical Top End.</p><p>The increased influenza rates within the Indigenous population seen in this study during both pandemic and non‐pandemic periods (Figures <xref rid=\"irv12757-fig-0001\" ref-type=\"fig\">1</xref> and <xref rid=\"irv12757-fig-0003\" ref-type=\"fig\">3</xref>) could be associated with increased crowding within Indigenous households, which is more common in remote areas. A study which modelled an influenza‐like illness outbreak and incorporated data on household structures found that the illness would impact 90% of the population of a remote Indigenous community, 75% of an urban Indigenous community and 25% of a non‐Indigenous urban population.<xref rid=\"irv12757-bib-0014\" ref-type=\"ref\">\n<sup>14</sup>\n</xref>\n</p><p>The higher rates of severe outcomes from infection are consistent with previous data that Indigenous populations are disproportionately affected by influenza. A study examining severe influenza A(H1N1)pdm09 cases in the Indigenous and non‐Indigenous populations of countries across the Americas and the Pacific found that rates of hospitalisations from A(H1N1)pdm09 were 3.0‐ to 7.7‐fold higher for the Indigenous population compared to the corresponding non‐Indigenous population.<xref rid=\"irv12757-bib-0015\" ref-type=\"ref\">\n<sup>15</sup>\n</xref> Furthermore, this study revealed that mortality rates from A(H1N1)pdm09 were between 3.4‐ and 5.3‐fold higher in the Indigenous population again compared to the corresponding non‐Indigenous population.<xref rid=\"irv12757-bib-0015\" ref-type=\"ref\">\n<sup>15</sup>\n</xref> Seasonal influenza too has been reported to have had a disproportionate impact upon Indigenous Australians at a national level, with studies reporting that Indigenous hospitalisation rates from influenza were 2.3‐3.9 times higher than rates for non‐Indigenous Australians.<xref rid=\"irv12757-bib-0016\" ref-type=\"ref\">\n<sup>16</sup>\n</xref>, <xref rid=\"irv12757-bib-0017\" ref-type=\"ref\">\n<sup>17</sup>\n</xref>, <xref rid=\"irv12757-bib-0018\" ref-type=\"ref\">\n<sup>18</sup>\n</xref>\n</p><p>Genetic factors may contribute to the higher rates of severe outcomes from influenza infection in the Indigenous population.<xref rid=\"irv12757-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref> Analysis of HLA types found in Aboriginal Australians found that alleles of the A*24 type, which are thought to be less efficient at presenting conserved regions of the virus, were found at a higher frequency in Aboriginal Australians.<xref rid=\"irv12757-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref> The A*24 allele was also detected at higher frequency in Native American populations, indicating this genetic susceptibility to severe influenza outcomes is not merely restricted to Indigenous Australian populations.<xref rid=\"irv12757-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref> HLA types efficient at presenting conserved regions of the H7N9 subtype were found at lower frequencies in Aboriginal Australians compared to Caucasians, making this group more vulnerable to a subtype already known to be highly pathogenic.<xref rid=\"irv12757-bib-0020\" ref-type=\"ref\">\n<sup>20</sup>\n</xref>\n</p><p>Comorbid medical conditions are reported to be more prominent in the Indigenous population than the non‐Indigenous population in this region,<xref rid=\"irv12757-bib-0021\" ref-type=\"ref\">\n<sup>21</sup>\n</xref> and this too may contribute to the higher rates of severe outcomes from influenza infection. For example, it was reported that 40% of Indigenous adults in the Northern Territory had chronic kidney disease compared to only 8.7% of non‐Indigenous Australians.<xref rid=\"irv12757-bib-0021\" ref-type=\"ref\">\n<sup>21</sup>\n</xref> In 2015, the prevalence of rheumatic heart disease was reported to be 37 times higher in Indigenous Australians than non‐Indigenous Australians in the Northern Territory.<xref rid=\"irv12757-bib-0021\" ref-type=\"ref\">\n<sup>21</sup>\n</xref>\n</p><p>The data provide some guidance as to priorities for vaccination strategies. Given the consistently higher rates of notifications seen in the Indigenous population, it is important that good vaccination coverage is maintained in the Indigenous population. The extremely large difference in notification rates seen in the 0‐4 age bracket and older adults highlights the importance of maintaining high immunisation rates amongst this group. Since the change in government policy in 2015 to cover the cost of vaccination in Indigenous Australians aged 6 months to under 5 years, vaccine uptake for Indigenous Australians in the Northern Territory in this age group has increased, from <10% in 2007‐2014, to over 50% in 2015 and 2016.<xref rid=\"irv12757-bib-0022\" ref-type=\"ref\">\n<sup>22</sup>\n</xref> Although the notification rates in the 0‐4 age bracket remained higher for Indigenous Australians in 2015 and 2016, the fold difference in case rates was comparatively lower than in other years, with the fold difference in case rates in 2016 being the lowest ever seen for this age group (Table <xref rid=\"irv12757-tbl-0001\" ref-type=\"table\">1</xref>). This could be an early indication that the change in immunisation policy has helped reduce the disparity in influenza rates. Nevertheless, it is too soon to draw conclusions as to the effectiveness of expanding subsidised vaccination to Indigenous Australians in the 0‐4 age group based on the duration of data presented here; continued monitoring is needed. The higher notification rates seen in the Tanami region and the Tiwi Islands (Figure <xref rid=\"irv12757-fig-0002\" ref-type=\"fig\">2</xref>) also emphasise that maintaining high vaccination coverage in these areas is important.</p><p>The peak in influenza activity outside the typical influenza season seen in the northern Top End of the Northern Territory bears similarity to tropical countries such as Columbia, Ecuador, Costa Rica and Thailand which show 2 peaks in influenza activity.<xref rid=\"irv12757-bib-0023\" ref-type=\"ref\">\n<sup>23</sup>\n</xref>\n</p><p>Influenza activity during the February‐March period presents challenges to effective vaccination. In the Northern Territory, influenza vaccinations are available to the public from March‐April onwards. There is growing evidence to suggest a decline in the effectiveness of the influenza vaccine over time but the rate at which the effectiveness of the vaccine declines is unclear.<xref rid=\"irv12757-bib-0024\" ref-type=\"ref\">\n<sup>24</sup>\n</xref> Nevertheless, a meta‐analysis of 14 studies revealed that significant decreases in the effectiveness of the vaccine against H3N2 and type B viruses were seen 91‐180 days after vaccination.<xref rid=\"irv12757-bib-0025\" ref-type=\"ref\">\n<sup>25</sup>\n</xref> This suggests that during outbreaks in the February‐March period, recipients of the influenza vaccine from the previous season are likely to have reduced levels of protection than during the typical influenza season. It is also possible that the strains that circulate during the February‐March period may differ from the strains that circulated during the previous epidemic peak.</p><p>The possibility of re‐vaccinating individuals against potential outbreaks in February‐March could be considered. A randomised controlled trial in Singapore found that semi‐annual vaccination led to a significant increase in the proportion of participants with antibody titres ≥1:40 for H1N1 virus only.<xref rid=\"irv12757-bib-0026\" ref-type=\"ref\">\n<sup>26</sup>\n</xref> Despite there not being an increase seen in the proportion of participants with antibody titres ≥1:40 for the H3N2 or B viruses, there may still be some clinical benefits for biannual vaccination.<xref rid=\"irv12757-bib-0026\" ref-type=\"ref\">\n<sup>26</sup>\n</xref> A lower proportion of participants that received biannual vaccination reported influenza‐like illnesses, although it was noted that the sample used in this study was too small to detect whether there was a reduction in laboratory‐confirmed influenza.<xref rid=\"irv12757-bib-0026\" ref-type=\"ref\">\n<sup>26</sup>\n</xref>\n</p><p>Given that influenza outbreaks in the February‐March period are occurring in the Top End, a vaccination campaign against these outbreaks would be best focused on this region. However, it appears difficult to judge in which years an outbreak in February‐March would occur. It would be useful if forecasting models were developed to allow for the detection of interseasonal outbreaks, thus allowing for better informed decisions to be made on whether to revaccinate individuals in the Top End against influenza later in the year. However, recent attempts at forecasting influenza outbreaks in tropical areas have had limited success.<xref rid=\"irv12757-bib-0027\" ref-type=\"ref\">\n<sup>27</sup>\n</xref> Attempts at modelling an influenza outbreak in a tropical region suggested accurate forecasts can be developed 3 weeks in advance,<xref rid=\"irv12757-bib-0028\" ref-type=\"ref\">\n<sup>28</sup>\n</xref> but this clearly not enough time to promote and conduct a vaccination campaign in this region. Whether the predominant circulating subtype or lineage during the February‐March is the same as that during the August‐September peak also needs to be investigated.</p><p>There are some limitations associated with this data set. First, the data set is restricted to patients who present to medical care and receive a laboratory test for influenza. Since not all those infected with influenza will seek medical care and few are swabbed for influenza, the true influenza burden is underestimated. Furthermore, comparison of rates between the different regions is only valid if patients in the different regions are equally as likely to present to medical care and receive laboratory tests when presenting with influenza‐like illnesses. Laboratory testing practices have changed over the period of the study, with consistently higher rates of notifications in post‐pandemic years (Table <xref rid=\"irv12757-sup-0001\" ref-type=\"supplementary-material\">S1</xref>). Finally, the resident location supplied with the notification is that of the patient’s home location, not where the patient presented to medical care. Thus, it is possible that a patient was infectious while present in an area different to that listed with the notification, although analysing location data by SA2 area does decrease the likelihood of misclassifying a patient’s location. It should also be noted that when examining notification rates by area, the population of many remote areas is small, and in areas where the population is small, a few cases can lead to more dramatic fluctuations in the rate than in areas where the population is large.</p><p>By analysing notifications of influenza infection in the Northern Territory, we have highlighted the unequal burden of influenza on the Indigenous population and thus the importance of maintaining good vaccination coverage, especially for those in the areas of Tanami and the Tiwi Islands. Furthermore, by demonstrating that influenza outbreaks in the Northern Territory during the February‐March period are occurring in the tropical Top End, we have established which area of the Northern Territory to target vaccination campaigns towards in order to prevent outbreaks during this period.</p></sec><sec sec-type=\"COI-statement\" id=\"irv12757-sec-0018\"><title>CONFLICT OF INTEREST</title><p>Dr. Tong reports grants from National Health and Medical Research Council, during the conduct of the study.</p></sec><sec id=\"irv12757-sec-0019\"><title>AUTHOR CONTRIBUTIONS</title><p>\n<bold>Aaron Lawson Weinman:</bold> Formal analysis (lead); Investigation (lead); Project administration (lead); Visualization (lead); Writing‐original draft (lead); Writing‐review & editing (supporting). <bold>Sheena Sullivan:</bold> Formal analysis (equal); Investigation (equal); Supervision (equal); Visualization (supporting); Writing‐review & editing (equal). <bold>Dhanasekaran Vijaykrishna:</bold> Supervision (equal); Writing‐review & editing (equal). <bold>Peter Markey:</bold> Data curation (lead); Resources (lead). <bold>Avram Levy:</bold> Resources (supporting); Writing‐review & editing (supporting). <bold>Adrian Miller:</bold> Supervision (supporting). <bold>Steven Tong:</bold> Conceptualization (lead); Formal analysis (equal); Funding acquisition (lead); Investigation (equal); Project administration (supporting); Supervision (lead); Visualization (supporting); Writing‐review & editing (lead).</p></sec><sec sec-type=\"supplementary-material\"><title>Supporting information</title><supplementary-material content-type=\"local-data\" id=\"irv12757-sup-0001\"><caption><p>Table S1</p></caption><media xlink:href=\"IRV-14-541-s001.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"irv12757-sec-0017\"><title>ACKNOWLEDGEMENTS</title><p>We would like to thank staff at the WHO Collaborating Centre for Reference and Research on Influenza for their work in linking the notification data to the Centre's database. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"iso-abbrev\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"doi\">10.1111/(ISSN)1750-2659</journal-id><journal-id journal-id-type=\"publisher-id\">IRV</journal-id><journal-title-group><journal-title>Influenza and Other Respiratory Viruses</journal-title></journal-title-group><issn pub-type=\"ppub\">1750-2640</issn><issn pub-type=\"epub\">1750-2659</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32449300</article-id><article-id pub-id-type=\"pmc\">PMC7431648</article-id><article-id pub-id-type=\"doi\">10.1111/irv.12754</article-id><article-id pub-id-type=\"publisher-id\">IRV12754</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Original Article</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Original Articles</subject></subj-group></article-categories><title-group><article-title>Severity and mortality of respiratory syncytial virus vs influenza A infection in hospitalized adults in China</article-title><alt-title alt-title-type=\"left-running-head\">ZHANG et al.</alt-title></title-group><contrib-group><contrib id=\"irv12754-cr-0001\" contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Yulin</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-3231-953X</contrib-id><xref ref-type=\"aff\" rid=\"irv12754-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12754-cr-0002\" contrib-type=\"author\"><name><surname>Wang</surname><given-names>Yeming</given-names></name><xref ref-type=\"aff\" rid=\"irv12754-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12754-cr-0003\" contrib-type=\"author\"><name><surname>Zhao</surname><given-names>Jiankang</given-names></name><xref ref-type=\"aff\" rid=\"irv12754-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12754-cr-0004\" contrib-type=\"author\"><name><surname>Xiong</surname><given-names>Zhujia</given-names></name><xref ref-type=\"aff\" rid=\"irv12754-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12754-cr-0005\" contrib-type=\"author\"><name><surname>Fan</surname><given-names>Yanyan</given-names></name><xref ref-type=\"aff\" rid=\"irv12754-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12754-cr-0006\" contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Wang</given-names></name><xref ref-type=\"aff\" rid=\"irv12754-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12754-cr-0007\" contrib-type=\"author\"><name><surname>Zou</surname><given-names>Xiaohui</given-names></name><xref ref-type=\"aff\" rid=\"irv12754-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12754-cr-0008\" contrib-type=\"author\"><name><surname>Wang</surname><given-names>Chunlei</given-names></name><xref ref-type=\"aff\" rid=\"irv12754-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12754-cr-0009\" contrib-type=\"author\"><name><surname>Han</surname><given-names>Jiajing</given-names></name><xref ref-type=\"aff\" rid=\"irv12754-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12754-cr-0010\" contrib-type=\"author\"><name><surname>Li</surname><given-names>Binbin</given-names></name><xref ref-type=\"aff\" rid=\"irv12754-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12754-cr-0011\" contrib-type=\"author\"><name><surname>Lu</surname><given-names>Binghuai</given-names></name></contrib><contrib id=\"irv12754-cr-0012\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Cao</surname><given-names>Bin</given-names></name><xref ref-type=\"aff\" rid=\"irv12754-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12754-aff-0002\">\n<sup>2</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12754-aff-0003\">\n<sup>3</sup>\n</xref><address><email>caobin_ben@163.com</email></address></contrib><contrib id=\"irv12754-cr-0013\" contrib-type=\"author\"><collab collab-type=\"authors\">CAP-China Network</collab></contrib></contrib-group><aff id=\"irv12754-aff-0001\">\n<label><sup>1</sup></label>\n<named-content content-type=\"organisation-division\">Department of Pulmonary and Critical Care Medicine</named-content>\n<named-content content-type=\"organisation-division\">Laboratory of Clinical Microbiology and Infectious Diseases</named-content>\n<named-content content-type=\"organisation-division\">Center for Respiratory Diseases</named-content>\n<named-content content-type=\"organisation-division\">National Clinical Research Center of Respiratory Diseases</named-content>\n<institution>China‐Japan Friendship Hospital</institution>\n<city>Beijing</city>\n<country country=\"CN\">China</country>\n</aff><aff id=\"irv12754-aff-0002\">\n<label><sup>2</sup></label>\n<named-content content-type=\"organisation-division\">Clinical Center for Pulmonary Infections</named-content>\n<institution>Capital Medical University</institution>\n<city>Beijing</city>\n<country country=\"CN\">China</country>\n</aff><aff id=\"irv12754-aff-0003\">\n<label><sup>3</sup></label>\n<institution>Tsinghua University‐Peking University Joint Center for Life Sciences</institution>\n<city>Beijing</city>\n<country country=\"CN\">China</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label><bold>Correspondence</bold><break/>\nBin Cao, Department of Pulmonary and Critical Care Medicine, Center for Respiratory Diseases, National Clinical Research Center of Respiratory Diseases, China‐Japan Friendship Hospital, Beijing, China.<break/>\nEmail: <email>caobin_ben@163.com</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>25</day><month>5</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>9</month><year>2020</year></pub-date><volume>14</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1111/irv.v14.5</issue-id><fpage>483</fpage><lpage>490</lpage><history><date date-type=\"received\"><day>11</day><month>10</month><year>2019</year></date><date date-type=\"rev-recd\"><day>19</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>21</day><month>4</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 John Wiley & Sons Ltd <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Influenza and Other Respiratory Viruses</italic> Published by John Wiley & Sons Ltd.</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:IRV-14-483.pdf\"/><abstract id=\"irv12754-abs-0001\"><title>Abstract</title><sec id=\"irv12754-sec-0001\"><title>Background </title><p>Respiratory syncytial virus (RSV) is an important cause of medically attended acute respiratory illnesses in older adults but awareness of the relevance of RSV in older people remains lower than that of influenza, which exhibits similar clinical characteristics to those of RSV.</p></sec><sec id=\"irv12754-sec-0022\"><title>Objectives</title><p>This study was performed to assess the clinical significance of RSV in respiratory samples from hospitalized adults.</p></sec><sec id=\"irv12754-sec-0002\"><title>Methods</title><p>Characteristics and outcomes in adults (≥18 years) hospitalized for RSV infection (n = 51) were compared with a cohort hospitalized for influenza A infection (n = 279) in a single‐center retrospective cohort study in Beijing, China.</p></sec><sec id=\"irv12754-sec-0003\"><title>Results</title><p>Respiratory syncytial virus patients were slightly older, with no significant differences in underlying chronic conditions. Lower respiratory tract infection and cardiovascular complications were more frequent (<italic>P</italic> < .05) in RSV patients. Rates of mortality in the RSV cohorts were significantly higher within 30 days (13.7% vs 5.0%, <italic>P</italic> = .019) and 60 days (17.6% vs 7.5%, <italic>P</italic> = .021). Bacterial co‐infection in respiratory samples was associated with reduced survival among RSV patients (log rank, <italic>P</italic> = .013).</p></sec><sec id=\"irv12754-sec-0004\"><title>Conclusions</title><p>Respiratory syncytial virus is a common cause of serious illness among hospitalized adults in China with greater mortality than influenza A. Increased awareness and the availability of antiviral agents might increase the scope for successful management.</p></sec></abstract><kwd-group><kwd id=\"irv12754-kwd-0001\">bacterial co‐infection</kwd><kwd id=\"irv12754-kwd-0002\">cardiovascular complications</kwd><kwd id=\"irv12754-kwd-0003\">in‐hospital mortality</kwd><kwd id=\"irv12754-kwd-0004\">viral infection</kwd></kwd-group><funding-group><award-group id=\"funding-0001\"><funding-source>Intramural Research Program of China‐Japan Friendship Hospital</funding-source><award-id>2018‐2‐QN‐22 </award-id></award-group><award-group id=\"funding-0002\"><funding-source>National Science Fund for Distinguished Young Scholars</funding-source><award-id> 81425001/H0104</award-id></award-group><award-group id=\"funding-0003\"><funding-source>Innovation Fund for Medical Sciences</funding-source><award-id>CIFMS 2018‐I2M‐1‐003</award-id></award-group></funding-group><counts><fig-count count=\"2\"/><table-count count=\"4\"/><page-count count=\"8\"/><word-count count=\"5462\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>September 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.6 mode:remove_FC converted:18.08.2020</meta-value></custom-meta></custom-meta-group></article-meta><notes><p content-type=\"self-citation\">\n<mixed-citation publication-type=\"journal\" id=\"irv12754-cit-1001\">\n<string-name>\n<surname>Zhang</surname>\n<given-names>Y</given-names>\n</string-name>, <string-name>\n<surname>Wang</surname>\n<given-names>Y</given-names>\n</string-name>, <string-name>\n<surname>Zhao</surname>\n<given-names>J</given-names>\n</string-name>, et al; <collab collab-type=\"authors\">CAP‐China network</collab>\n. <article-title>Severity and mortality of respiratory syncytial virus vs influenza A infection in hospitalized adults in China</article-title>. <source xml:lang=\"en\">Influenza Other Respi Viruses</source>. <year>2020</year>;<volume>14</volume>:<fpage>483</fpage>–<lpage>490</lpage>. <pub-id pub-id-type=\"doi\">10.1111/irv.12754</pub-id>\n</mixed-citation>\n</p><fn-group id=\"irv12754-ntgp-0001\"><fn id=\"irv12754-note-0001\"><p>The peer review history for this article is available at https://publons.com/publon/10.1111/IRV.12754.</p></fn><fn fn-type=\"funding\" id=\"irv12754-note-0002\"><p>\n<bold>Funding information</bold>\n</p><p>This work was supported by the National Science Fund for Distinguished Young Scholars [grant number 81425001/H0104 to Dr Bin Cao], CAMS Innovation Fund for Medical Sciences (CIFMS 2018‐I2M‐1‐003 to Dr Bin Cao), and the Intramural Research Program of China‐Japan Friendship Hospital [grant number 2018‐2‐QN‐22 to Yulin Zhang].</p></fn></fn-group></notes></front><body id=\"irv12754-body-0001\"><sec id=\"irv12754-sec-0005\"><label>1</label><title>INTRODUCTION</title><p>Respiratory syncytial virus (RSV) used to be known primarily as a respiratory pathogen of young children and many laudable projects such as the World Health Organization RSV surveillance platform focus on pregnant women and young children.<xref rid=\"irv12754-bib-0001\" ref-type=\"ref\">\n<sup>1</sup>\n</xref> However, in recent decades, awareness has grown of the importance of RSV infection to the health of older adults. In the United States, RSV infections occur at an annual rate of up to 10% in older adults, a rate which can exceed that observed for influenza in this population group.<xref rid=\"irv12754-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref> In the older adult population, RSV infection can have serious consequences: RSV is responsible for around 12% of all medically attended acute respiratory illnesses in older adults<xref rid=\"irv12754-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref> and the incidence of RSV‐associated hospitalization increases with age.<xref rid=\"irv12754-bib-0004\" ref-type=\"ref\">\n<sup>4</sup>\n</xref> Notably, whereas earlier data used a cutoff point of ≥65 years to define “older adult,”<xref rid=\"irv12754-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref> a recent study suggested that increased risk of severe RSV disease may commence at early as at 50 years of age.<xref rid=\"irv12754-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref> Among older adults hospitalized with RSV, a mortality rate of 6%‐8% has been reported.<xref rid=\"irv12754-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref> RSV infection was shown to lead to severe lower respiratory complications and even respiratory failure in elderly in Hong Kong, with a mortality rate up to 11.9% within 60 days.<xref rid=\"irv12754-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref> It is likely that even these alarming numbers represent an underestimation of the burden of RSV infection in older adults.<xref rid=\"irv12754-bib-0007\" ref-type=\"ref\">\n<sup>7</sup>\n</xref>\n</p><p>Despite these numbers, awareness of the relevance of RSV in older people remains lower than that of influenza, which exhibits similar clinical characteristics to those of RSV and which has been recognized for generations as cause of severe morbidity and mortality in older adults.<xref rid=\"irv12754-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> The need for awareness and distinction between the two diseases is illustrated by the fact that some 200 000 hospitalizations annually are associated with RSV infection compared with 300 000 hospitalizations secondary to influenza in the same population.<xref rid=\"irv12754-bib-0004\" ref-type=\"ref\">\n<sup>4</sup>\n</xref>, <xref rid=\"irv12754-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref>\n</p><p>Low awareness is also reflected in a dearth of international data. Most of the available studies were performed in the United States. Particularly for China, with the world's largest population and an increasing proportion of elderly individuals, more data are urgently needed on the prevalence, clinical manifestations, complications, and outcomes of severe RSV infections in hospitalized adults.<xref rid=\"irv12754-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref>, <xref rid=\"irv12754-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref>, <xref rid=\"irv12754-bib-0010\" ref-type=\"ref\">\n<sup>10</sup>\n</xref> The recent progress on antiviral treatments for RSV<xref rid=\"irv12754-bib-0011\" ref-type=\"ref\">\n<sup>11</sup>\n</xref>, <xref rid=\"irv12754-bib-0012\" ref-type=\"ref\">\n<sup>12</sup>\n</xref> has given such data unprecedented relevance to clinicians.</p><p>We performed a retrospective single‐center study of a large cohort of adults hospitalized with laboratory‐confirmed RSV infections in Beijing, China, between January 2017 and June 2018. Data were gathered on characteristics, complications, and outcomes and used to compare with patients admitted for influenza A virus infection between August 2017 and June 2018.</p></sec><sec sec-type=\"methods\" id=\"irv12754-sec-0006\"><label>2</label><title>METHODS</title><sec id=\"irv12754-sec-0007\"><label>2.1</label><title>Study population</title><p>This retrospective cohort study analyzed patients aged ≥18 years admitted to the China‐Japan Friendship Hospital, Beijing with laboratory‐confirmed RSV and FA infection in 2017‐2018. A total of 51 RSV‐infected patients between January 2017 and June 2018 were enrolled in this study. All influenza A patients admitted to the center between August 2017 and June 2018 were used as the comparator group, excluding the patients who had a mixed infection of influenza A virus and RSV. The study was approved by the China‐Japan Friendship Hospital Medical Ethical Committee.</p></sec><sec id=\"irv12754-sec-0008\"><label>2.2</label><title>Clinical data collection and definitions</title><p>Electronic and written medical records were reviewed for all subjects. Data collected included demographic details, comorbid illnesses, presenting symptoms and signs, antiviral and antibiotic use, corticosteroid treatments received (intravenous or oral steroids), intensive care unit (ICU) admission, hospital length of stay, occurrence of complications, requirement for ventilatory support, exacerbation of chronic conditions, and all‐cause death within 30 days and 60 days. Medical complications associated with RSV infection were defined as a new or exacerbated medical condition as confirmed by laboratory and radiographic studies. Lower respiratory complications were defined as radiologically confirmed pneumonia or exacerbation of asthma/bronchitis/chronic obstructive pulmonary disease (COPD). Cardiovascular complications were defined as the occurrence or exacerbation of cardiac symptoms (coronary syndrome, arrhythmia, myocarditis, and decompensated heart failure) and/or acute cerebrovascular events.<xref rid=\"irv12754-bib-0013\" ref-type=\"ref\">\n<sup>13</sup>\n</xref>, <xref rid=\"irv12754-bib-0014\" ref-type=\"ref\">\n<sup>14</sup>\n</xref> Bacterial superinfection is defined as the isolation of one or more bacterial pathogen from nasopharyngeal swabs, sputum, bronchoalveolar lavage fluid and/or blood and/or urine samples.</p></sec><sec id=\"irv12754-sec-0009\"><label>2.3</label><title>Virus identification</title><p>Respiratory syncytial virus and influenza A virus infection were confirmed by analysis of nasopharyngeal swabs, sputum, bronchoalveolar lavage fluid and/or blood and/or urine samples using RSV Nucleic Acid Detection Kit (Liferiver) and Influenza A Virus Nucleic Acid Detection kit (Liferiver), respectively.<xref rid=\"irv12754-bib-0015\" ref-type=\"ref\">\n<sup>15</sup>\n</xref>\n</p></sec><sec id=\"irv12754-sec-0010\"><label>2.4</label><title>Statistical analysis</title><p>Categorical variables are presented as frequencies and percentages. Continuous variables are described as mean, standard deviation, and range. Comparisons of proportions were performed with chi‐square and Fisher's exact tests; continuous variables were compared using Student's <italic>t</italic> test. All probabilities were 2‐tailed, with statistical significance defined as <italic>P</italic> ≤ .05. Binary logistic regression was performed to estimate the odds ratio (OR) and 95% confidence interval (CI) for clinical hospitalization outcomes in RSV‐infected patients compared with influenza A virus‐infected cohorts. Survival curves were generated using the Kaplan‐Meier method and compared using the log‐rank test. All analyses were performed using PASW Statistics software, version 18.0.</p></sec></sec><sec sec-type=\"results\" id=\"irv12754-sec-0011\"><label>3</label><title>RESULTS</title><sec id=\"irv12754-sec-0012\"><label>3.1</label><title>Study population</title><p>Demographic characteristics and comorbidities prior to admission of all hospitalized patients are presented in Table <xref rid=\"irv12754-tbl-0001\" ref-type=\"table\">1</xref>. The proportions of women and patient smoking status were similar in the two cohorts. The median ages of RSV and influenza A virus‐infected patients were 64.1 years (SD 15.6, range 21.0‐85.0) and 60.2 years (SD 16.3, range 19.0‐94.0), respectively (<italic>P</italic> > .05, chi‐square test). The proportion of subjects aged >60 years was significantly greater in the RSV cohort than in the influenza A virus cohort: 66.7% vs 51.3% <italic>P</italic> = .042, (chi‐square test; Figure <xref rid=\"irv12754-fig-0001\" ref-type=\"fig\">1</xref>). There were no significant differences between the groups in rates of comorbid conditions at admission although cardiac disease, respiratory disease, and cerebrovascular disease were more common in RSV‐infected populations than those in influenza A‐infected cohorts (Table <xref rid=\"irv12754-tbl-0001\" ref-type=\"table\">1</xref>).</p><table-wrap id=\"irv12754-tbl-0001\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 1</label><caption><p>Baseline characteristics of hospitalized adults with RSV and Influenza A infection</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Patient characteristics</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">RSV (N = 51)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Influenza A (N = 279)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic>P‐</italic>value<xref ref-type=\"fn\" rid=\"irv12754-note-0004\">\n<sup>a</sup>\n</xref>\n</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age (years) at admission (mean, sd, range)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">64.1, 15.6, 21.0‐85.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">60.2, 16.3, 19.0‐94.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.116</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">18‐30 y (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3 (5.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15 (5.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">31‐40 y (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3 (5.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20 (7.2)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">41‐50 y (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (3.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37 (13.3)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">51‐60 y (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9 (17.6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">64 (22.9)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">>60 y (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34 (66.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">143 (51.3)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Male (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">28 (54.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">146 (52.3)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.735</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">Smoking</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Non‐smoker (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">48 (94.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">230 (82.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Current smoker (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3 (5.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">49 (‐17.6)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.058</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Comorbidities prior to admission</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">47 (92.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">231 (82.8)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.139</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Cardiac disease (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">19 (37.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">68 (24.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.055</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">COPD (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7 (13.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22 (7.9)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.176</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Bronchial asthma (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (3.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (1.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.233</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Hypertension (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20 (39.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">120 (43.0)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.614</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Chronic kidney disease (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (7.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21 (7.5)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Any solid cancer (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5 (9.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">28 (10.0)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.960</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Diabetes (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13 (25.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">83 (29.7)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.538</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Cerebrovascular disease (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14 (27.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32 (11.5)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.002</td></tr></tbody></table><table-wrap-foot id=\"irv12754-ntgp-0002\"><fn id=\"irv12754-note-0003\"><p>Abbreviations: COPD: chronic obstructive pulmonary disease; RSV, respiratory syncytial virus; sd, standard deviation.</p></fn><fn id=\"irv12754-note-0004\"><label><sup>a</sup></label><p>\n<italic>P</italic>‐value from chi‐square test, <italic>t</italic> test, Fisher's exact test, as appropriate.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><fig fig-type=\"FIGURE\" xml:lang=\"en\" id=\"irv12754-fig-0001\" orientation=\"portrait\" position=\"float\"><label>FIGURE 1</label><caption><p>Age distribution of hospitalized adults with RSV and influenza A infection</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"IRV-14-483-g001\"/></fig></sec><sec id=\"irv12754-sec-0013\"><label>3.2</label><title>Clinical presentation and outcomes</title><p>Clinical symptoms and outcomes in the cohorts are presented in Table <xref rid=\"irv12754-tbl-0002\" ref-type=\"table\">2</xref>. Fever, cough, and sputum production were the most frequent presenting signs in both cohorts, but RSV cases were less likely than influenza A cases to report fever (<italic>P</italic> < .001; chi‐square test) and cough (<italic>P</italic> = .026; chi‐square test). The rates of bacterial superinfection in each kind of samples (respiratory samples or blood samples or urine samples) were similar between RSV‐infected patients and influenza A patients (<italic>P</italic> > .05; chi‐square test). The median time from admission to diagnosis of RSV infection was longer than for influenza A virus infections: 4 vs 3 days, <italic>P</italic> = .049 (<italic>t</italic> test).</p><table-wrap id=\"irv12754-tbl-0002\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 2</label><caption><p>In‐hospital characteristics of hospitalized adults with RSV and Influenza A infection</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Patient characteristics</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">RSV (N = 51)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Influenza A (N = 279)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>‐value<xref ref-type=\"fn\" rid=\"irv12754-note-0006\">\n<sup>a</sup>\n</xref>\n</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">Symptoms</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Fever (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32 (62.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">242 (86.7)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"><.001</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Cough (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34 (66.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">225 (80.6)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.026</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Sputum production (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32 (62.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">192 (68.8)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.393</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Hemoptysis (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (3.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15 (5.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.930</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Myalgia (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5 (9.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">53 (19.0)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.113</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Weakness (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8 (15.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">71 (25.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.133</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Days from admission to diagnosis (median, IQR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4, 1‐9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3, 1‐6</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.049</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Any antiviral drug use during hospitalization (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21 (41.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">242 (86.7)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"><.001</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Oseltamivir use (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12 (23.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">242 (86.7)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"><.001</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Ribavirin use (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8 (15.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">\n<italic><</italic>.001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Any antibiotic drug use during hospitalization (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">45 (88.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">235 (84.2)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.463</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>Any intravenous or oral steroid use during</p>\n<p>hospitalization (%)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27 (52.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">107 (38.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.051</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Intravenous steroid use (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21 (41.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">71 (25.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.021</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Oral steroid use (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6 (11.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">36 (12.9)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.823</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">Bacterial superinfection<xref ref-type=\"fn\" rid=\"irv12754-note-0007\">\n<sup>b</sup>\n</xref> (%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Blood samples</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3 (5.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6 (2.2)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.300</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Respiratory samples</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16 (31.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">98 (35.1)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.604</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Urine samples</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (7.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8 (2.9)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.181</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">Complication/outcome</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Lower respiratory tract complications<xref ref-type=\"fn\" rid=\"irv12754-note-0008\">\n<sup>c</sup>\n</xref> (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32 (62.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">126 (45.2)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.021</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Cardiovascular complications<xref ref-type=\"fn\" rid=\"irv12754-note-0009\">\n<sup>d</sup>\n</xref> (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26 (51.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">96 (34.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.024</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Pneumonia (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">30 (58.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">107 (38.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.006</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Need for intensive care (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12 (23.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">70 (25.1)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.813</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Need for invasive mechanical ventilation (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12 (23.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">48 (17.2)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.282</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">In‐hospital mortality (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9 (17.6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21 (7.5)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.021</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">30‐d mortality (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7 (13.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14 (5.0)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.019</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">60‐d mortality (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9 (17.6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21 (7.5)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.021</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Time to death (days) (median, IQR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10, 8.5‐14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11, 8‐18.5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.762</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Duration of hospitalization for survivors (days) (median, IQR)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15, 13‐22</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14, 10‐19</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.148</td></tr></tbody></table><table-wrap-foot id=\"irv12754-ntgp-0003\"><fn id=\"irv12754-note-0005\"><p>Abbreviations: IQR, interquartile range; RSV, respiratory syncytial virus.</p></fn><fn id=\"irv12754-note-0006\"><label><sup>a</sup></label><p>\n<italic>P</italic>‐value from chi‐square test, <italic>t</italic> test, Fisher's exact test, as appropriate.</p></fn><fn id=\"irv12754-note-0007\"><label><sup>b</sup></label><p>Bacterial superinfection is defined as the isolation of one or more bacterial pathogen from respiratory samples (nasopharyngeal swabs, sputum, and bronchoalveolar lavage fluid) and/or blood and/or urine samples.</p></fn><fn id=\"irv12754-note-0008\"><label><sup>c</sup></label><p>Lower respiratory complications included radiologically confirmed pneumonia or exacerbation of asthma/bronchitis/chronic obstructive pulmonary disease [13,14].</p></fn><fn id=\"irv12754-note-0009\"><label><sup>d</sup></label><p>Cardiovascular complications included the occurrence or exacerbation of cardiac symptoms (coronary syndrome, arrhythmia, myocarditis, and decompensated heart failure) and/or acute cerebrovascular events [13,14].</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>During hospitalization lower respiratory tract complications occurred in 62.7% of RSV cases and 45.2% of influenza A cases, respectively (<italic>P</italic> = .021; chi‐square test). Cardiovascular complications during hospitalization were also more frequent in the RSV group than in the cohort with influenza A virus infection: 51.0% vs 34.4%, <italic>P</italic> = .024 (chi‐square test), as was pneumonia: (58.8% vs 38.4%, <italic>P</italic> = .006; chi‐square test).</p><p>Use of antibiotics as well as of intravenous or oral corticosteroids during the hospitalization period was similar in the two cohorts. Among RSV cases, oseltamivir was prescribed significantly less commonly to RSV‐infected than to influenza A‐infected patients (23.5% vs 86.7%, <italic>P</italic> < .001; chi‐square test) but whole ribavirin was prescribed only for RSV infections (15.7% vs 0.0%, <italic>P</italic> < .001; chi‐square test). Use of invasive mechanical ventilation was similar in both cohorts. There were no differences in rates of ICU admission between the cohorts.</p><p>Rates of mortality in the RSV cohorts were significantly greater than that for influenza A‐infected patients within 30 days (13.7% vs 5.0%, <italic>P</italic> = .019; chi‐square test) and 60 days (17.6% vs 7.5%, <italic>P</italic> = .021; chi‐square test) respectively. There were no differences in median time from admission to death between the groups nor in the median duration of hospitalization for survivors. In the binary logistic regression analyses, the odds of hospitalization outcomes (cardiovascular complications, pneumonia, lower respiratory tract complications, the need for invasive mechanical ventilation and 60‐day mortality) in RSV cases were higher than in those hospitalized with influenza A infection, but the 95% CI crossed the boundary for all variables except for cardiovascular complications (Table <xref rid=\"irv12754-tbl-0003\" ref-type=\"table\">3</xref>).</p><table-wrap id=\"irv12754-tbl-0003\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 3</label><caption><p>Binary logistic regression analyses associated with clinical hospitalization outcomes in hospitalized adults with RSV and Influenza A infection</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Hospitalization outcomes<xref ref-type=\"fn\" rid=\"irv12754-note-0011\">\n<sup>a</sup>\n</xref>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">OR</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">95% CI</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Lower respiratory tract complications</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.6‐3.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Pneumonia</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.8‐4.6</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cardiovascular complications</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.2‐6.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Need for invasive mechanical ventilation</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.6‐3.4</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">60‐d mortality</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.6‐4.6</td></tr></tbody></table><table-wrap-foot id=\"irv12754-ntgp-0004\"><fn id=\"irv12754-note-0010\"><p>Abbreviations: CI, confidence interval; OR, odd ratio.</p></fn><fn id=\"irv12754-note-0011\"><label><sup>a</sup></label><p>Hospitalization outcomes included lower respiratory tract complications, pneumonia, cardiovascular complications, the need for invasive mechanical ventilation and 60‐d mortality.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap></sec><sec id=\"irv12754-sec-0014\"><label>3.3</label><title>Analysis of RSV cases with fatal outcomes</title><p>Nine patients with RSV infection died during hospitalization. A comparison with survivors showed no differences in sex, comorbidities, blood biochemical indices, symptoms, and signs; notably though, bacterial superinfection in respiratory samples (nasopharyngeal swabs, sputum, or bronchoalveolar lavage fluid) was more common among non‐survivors than that in survivors (<italic>P</italic> = .021, chi‐square test; Table <xref rid=\"irv12754-tbl-0004\" ref-type=\"table\">4</xref>) and was showed to be related to lower survival (Figure <xref rid=\"irv12754-fig-0002\" ref-type=\"fig\">2</xref>). Mortality within 60 days in patients with bacteria and RSV co‐infection in respiratory samples was up to 37.5%. Injected or oral corticosteroid use was more frequent in deceased than in surviving patients (88.9% vs 45.2%, <italic>P</italic> = .044 (chi‐square test) as was use of invasive mechanical ventilation (66.7% vs 14.3%, <italic>P</italic> = .003; chi‐square test) and ICU admission (66.7% vs 14.3%, <italic>P</italic> = .003; chi‐square test; Table <xref rid=\"irv12754-tbl-0004\" ref-type=\"table\">4</xref>). Survivors tended to have less cardiac disease and lower respiratory tract complications, but these differences did not reach statistical significance.</p><table-wrap id=\"irv12754-tbl-0004\" xml:lang=\"en\" content-type=\"TABLE\" orientation=\"portrait\" position=\"float\"><label>TABLE 4</label><caption><p>Patients characteristics of RSV‐infected adults with and without survivor</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Patient characteristics</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Dead cases (N = 9)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Survivors (N = 42)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>‐value<xref ref-type=\"fn\" rid=\"irv12754-note-0013\">\n<sup>a</sup>\n</xref>\n</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Male (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5 (55.6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23 (54.8)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age (years) (mean, sd, range)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">73.3, 9.3, 54.0‐84.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">62.1, 16.1, 21.0‐85.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.049</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">Smoking</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Current smoker (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3 (7.1)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Current non‐smoker (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9 (100.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39 (92.9)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">Comorbidities prior to admission</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Hypertension (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (44.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16 (38.1)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Diabetes (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (22.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11 (26.2)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Cerebrovascular disease (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (44.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10 (23.8)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.397</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Chronic kidney disease (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (22.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (4.8)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.278</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Any solid cancer (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1 (11.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (9.5)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">COPD (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (22.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5 (11.9)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.778</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Bronchial asthma (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (4.8)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">Complication/outcome</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Cardiac disease<xref ref-type=\"fn\" rid=\"irv12754-note-0014\">\n<sup>b</sup>\n</xref> (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5 (55.6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14 (33.3)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.384</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Pneumonia (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7 (77.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23 (54.8)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.368</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Lower respiratory tract complications<xref ref-type=\"fn\" rid=\"irv12754-note-0015\">\n<sup>c</sup>\n</xref> (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7 (77.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25 (59.5)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.517</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">Symptoms and signs</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Temperature (mean, sd, range)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">38.6, 1.4, 36.4‐40.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.6, 1.2, 36.0‐41.0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.025</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Cough (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7 (77.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27 (64.3)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.697</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Hemoptysis (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (4.8)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Sputum production (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6 (66.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26 (61.9)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Myalgia (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1 (11.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (9.5)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Weakness (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3 (33.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5 (11.9)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.272</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Need for invasive mechanical ventilation (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6 (66.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6 (14.3)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.003</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">Bacterial superinfection<xref ref-type=\"fn\" rid=\"irv12754-note-0016\">\n<sup>d</sup>\n</xref> (%)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Blood samples</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1 (2.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Respiratory samples</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6 (66.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9 (21.4)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.021</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Urine samples</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (22.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (4.8)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.278</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Need for intensive care (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6 (66.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6 (14.3)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.003</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Any antiviral drug use during hospitalization (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (44.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17 (40.5)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Oseltamivir use (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (44.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8 (19.0)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0.231</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Ribavirin use (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8 (19.0)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.322</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Any injection or oral steroid use during hospitalization (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8（88.9）</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">19（45.2）</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.044</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Any antibiotic drug use during hospitalization (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9（100.0）</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">36（85.7）</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.575</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">Blood biochemical indexes</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Serum alanine aminotransferase Concentration, IU/L (mean)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">606.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">49.9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.370</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Bilirubin (mean)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.557</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Serum creatinine</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">203.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">102.5</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.425</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Glucose</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">.933</td></tr></tbody></table><table-wrap-foot id=\"irv12754-ntgp-0005\"><fn id=\"irv12754-note-0012\"><p>Abbreviations: COPD: chronic obstructive pulmonary disease; IQR, interquartile range; RSV, respiratory syncytial virus; sd, standard deviation.</p></fn><fn id=\"irv12754-note-0013\"><label><sup>a</sup></label><p>\n<italic>P</italic>‐value from chi‐square test, <italic>t</italic> test, Fisher's exact test, as appropriate;</p></fn><fn id=\"irv12754-note-0014\"><label><sup>b</sup></label><p>Cardiac disease included the occurrence or exacerbation of cardiac symptoms (coronary syndrome, arrhythmia, myocarditis, and decompensated heart failure) [13,14];</p></fn><fn id=\"irv12754-note-0015\"><label><sup>c</sup></label><p>Lower respiratory complications included radiologically confirmed pneumonia or exacerbation of asthma/bronchitis/chronic obstructive pulmonary disease [13,14];</p></fn><fn id=\"irv12754-note-0016\"><label><sup>d</sup></label><p>Bacterial superinfection is defined as the isolation of one or more bacterial pathogen from respiratory samples (nasopharyngeal swabs, sputum, and bronchoalveolar lavage fluid) and/or blood and/or urine samples.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><fig fig-type=\"FIGURE\" xml:lang=\"en\" id=\"irv12754-fig-0002\" orientation=\"portrait\" position=\"float\"><label>FIGURE 2</label><caption><p>Kaplan‐Meier survival curves for patients with RSV infection with (n = 16) and without (n = 35) bacterial co‐infection in respiratory samples (nasopharyngeal swabs, sputum, or bronchoalveolar lavage fluid), respectively</p></caption><graphic id=\"nlm-graphic-3\" xlink:href=\"IRV-14-483-g002\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"irv12754-sec-0015\"><label>4</label><title>DISCUSSION</title><p>With RSV‐specific antiviral therapy advancing in clinical development, the question of differentiating RSV infection from that of influenza in adult populations will likely become highly relevant to care decisions worldwide.<xref rid=\"irv12754-bib-0012\" ref-type=\"ref\">\n<sup>12</sup>\n</xref>, <xref rid=\"irv12754-bib-0016\" ref-type=\"ref\">\n<sup>16</sup>\n</xref> The current retrospective study is the first, to our knowledge, to describe and compare populations of adults hospitalized with RSV or influenza A virus infection in China. The results show RSV infection to be an important cause of morbidity and mortality in this, the largest population in the world. The comparisons with influenza A are relevant to clinicians faced with adults hospitalized with respiratory tract infection beyond China.</p><p>Some observations such as the greater age, higher rates of complications, and greater mortality of RSV‐infected subjects compared with influenza A virus infection confirm the serious nature of RSV that has been reported from other countries, mostly the United States.<xref rid=\"irv12754-bib-0017\" ref-type=\"ref\">\n<sup>17</sup>\n</xref>, <xref rid=\"irv12754-bib-0018\" ref-type=\"ref\">\n<sup>18</sup>\n</xref>, <xref rid=\"irv12754-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref>, <xref rid=\"irv12754-bib-0020\" ref-type=\"ref\">\n<sup>20</sup>\n</xref>, <xref rid=\"irv12754-bib-0021\" ref-type=\"ref\">\n<sup>21</sup>\n</xref> Additionally, in consistent with the previous studies, higher proportions of patients with cardiac disease, respiratory disease, and cerebrovascular disease were observed in RSV‐infected populations than those in influenza A‐infected cohorts although there is no significant difference.<xref rid=\"irv12754-bib-0020\" ref-type=\"ref\">\n<sup>20</sup>\n</xref>, <xref rid=\"irv12754-bib-0021\" ref-type=\"ref\">\n<sup>21</sup>\n</xref> The higher underlying conditions might be the cause of higher rates of lower respiratory tract infection and cardiovascular complications in these cohorts. Other findings diverge from earlier reports. The RSV‐infected hospitalized adults in the present study were younger than those described in other studies,<xref rid=\"irv12754-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref>, <xref rid=\"irv12754-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref>, <xref rid=\"irv12754-bib-0020\" ref-type=\"ref\">\n<sup>20</sup>\n</xref> but the mortality was higher. These findings may reflect low awareness of the seriousness of RSV infection, as has been observed in other countries.<xref rid=\"irv12754-bib-0022\" ref-type=\"ref\">\n<sup>22</sup>\n</xref>, <xref rid=\"irv12754-bib-0023\" ref-type=\"ref\">\n<sup>23</sup>\n</xref> Similarly, although RSV patients were twice as likely as those with influenza A infection to present with lower respiratory tract and cardiovascular complications, there were no differences in the use of inhaled, oral or intravenous steroids, ICU admission, and invasive mechanical ventilation in the hospital.</p><p>We found a high rate of bacterial co‐infection in respiratory samples among non‐survivors with RSV infection and a likely correlation with mortality. As the data are from a retrospective analysis, a causal connection between bacterial infection and excess mortality cannot be definitively demonstrated; this would need further studies. It is possible that the respiratory tract microbiome influences host responses to RSV, modulating inflammation, and disease severity<xref rid=\"irv12754-bib-0024\" ref-type=\"ref\">\n<sup>24</sup>\n</xref> although the immunopathogenesis of co‐infection remains unknown. Whatever the causal relationship, the findings support the recommendation that RSV‐infected patients with any bacterial infection during hospitalization should be promptly identified and treated.<xref rid=\"irv12754-bib-0025\" ref-type=\"ref\">\n<sup>25</sup>\n</xref>\n</p><p>The differences in presentation at admission between RSV and influenza A infection are of interest. Fever and cough were less common among RSV cases, but rates of lower respiratory tract and cardiovascular complications, especially pneumonia, were greater in the RSV‐infected population. The latter complications may partially explain the higher rates of mortality in this group. It is also possible that lower respiratory tract disease progression is more rapid in RSV infection, although further research into the mechanisms and natural history may be necessary.<xref rid=\"irv12754-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref>, <xref rid=\"irv12754-bib-0026\" ref-type=\"ref\">\n<sup>26</sup>\n</xref>\n</p><p>The seasonal pattern of RSV infection in children in China has recently been shown to be very similar to those in the United States,<xref rid=\"irv12754-bib-0027\" ref-type=\"ref\">\n<sup>27</sup>\n</xref> and it is reasonable to assume that this would also be the case for adult disease. The development of efficacious interventions against RSV should be a high priority as they could reduce mortality and morbidity, and burdens on the healthcare system. Furthermore, if early identification and diagnosis of RSV infection in hospitalized adults with bacterial co‐infection enabled the timely implementation of appropriate therapies to reduce complications, this would reduce mortality, morbidity, and healthcare costs. An economic analysis in the United States estimated the average cost of RSV hospitalizations to be more than twice that of influenza A.<xref rid=\"irv12754-bib-0028\" ref-type=\"ref\">\n<sup>28</sup>\n</xref> No economic data are available for our cohort but a substantial economic burden can be inferred from the demonstrated severity of the RSV‐infected population.</p><p>There are limitations to this study. It was a single‐center analysis with modest sample size, and the features of the setting may not be representative of China as a whole. As a retrospective study, causation, for example, between bacterial co‐infection and mortality cannot be definitively determined. There was no analysis of RSV genotypes, which would be important for future epidemiological studies as well as to possibly assess future anti‐RSV therapies.<xref rid=\"irv12754-bib-0029\" ref-type=\"ref\">\n<sup>29</sup>\n</xref>\n</p><p>In conclusion, RSV infection is a common cause of serious illness among hospitalized Chinese adults, with greater morbidity and mortality than influenza A virus infection. Greater awareness of the serious nature of RSV infection among healthcare professionals would enable adult RSV‐infected patients, particularly those with bacterial infection or prior cardiac and pulmonary disease to be recognized in time and given appropriate treatments on admission.<xref rid=\"irv12754-bib-0030\" ref-type=\"ref\">\n<sup>30</sup>\n</xref> If recent reports of successful antiviral treatment for RSV<xref rid=\"irv12754-bib-0012\" ref-type=\"ref\">\n<sup>12</sup>\n</xref> are confirmed in further clinical trials these needs will take on a heightened relevance.</p></sec><sec sec-type=\"COI-statement\" id=\"irv12754-sec-0017\"><title>CONFLICT OF INTEREST</title><p>The authors declare that they have no conflict of interest.</p></sec><sec id=\"irv12754-sec-0018\"><title>AUTHOR CONTRIBUTIONS</title><p>\n<bold>Yulin Zhang:</bold> Data curation (Equal); Formal analysis (Lead); Funding acquisition (Supporting); Investigation (Equal); Methodology (Equal); Project administration (Equal); Validation (Equal); Visualization (Equal); Writing (original) draft (Lead). <bold>Yeming Wang:</bold> Data curation (Equal). <bold>Jiankang Zhao:</bold> Investigation (Equal); Resources (Equal); Software (Equal). <bold>Zhujia Xiong:</bold> Methodology (Equal), Resources (Equal); Software (Equal); Validation (Equal). <bold>Yanyan Fan:</bold> Resources (Equal); Visualization(Equal). <bold>Wang Zhang:</bold> Resources (Equal). <bold>Xiaohui Zou:</bold> Resources (Equal). <bold>Chunlei Wang:</bold> Data curation (Equal); Resources (Equal). <bold>Jiajing Han:</bold> Resources (Equal). <bold>Binbin Li:</bold> Resources (Equal). <bold>Binghuai Lu:</bold> Project administration (Equal); Supervision (Lead); Validation (Equal). <bold>Bin Cao:</bold> Conceptualization (Lead); Funding acquisition (Lead).</p></sec></body><back><ack id=\"irv12754-sec-0016\"><title>ACKNOWLEDGEMENTS</title><p>The authors are grateful to Dr Stephen Toovey (Pegasus Research Basel, Switzerland) and Pelle Stolt 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"iso-abbrev\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"doi\">10.1111/(ISSN)1750-2659</journal-id><journal-id journal-id-type=\"publisher-id\">IRV</journal-id><journal-title-group><journal-title>Influenza and Other Respiratory Viruses</journal-title></journal-title-group><issn pub-type=\"ppub\">1750-2640</issn><issn pub-type=\"epub\">1750-2659</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32530142</article-id><article-id pub-id-type=\"pmc\">PMC7431650</article-id><article-id pub-id-type=\"doi\">10.1111/irv.12759</article-id><article-id pub-id-type=\"publisher-id\">IRV12759</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Original Article</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Original Articles</subject></subj-group></article-categories><title-group><article-title>The association between influenza infections in primary care and intensive care admissions for severe acute respiratory infection (SARI): A modelling approach</article-title><alt-title alt-title-type=\"left-running-head\">van ASTEN et al.</alt-title></title-group><contrib-group><contrib id=\"irv12759-cr-0001\" contrib-type=\"author\" corresp=\"yes\"><name><surname>van Asten</surname><given-names>Liselotte</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-4123-7595</contrib-id><xref ref-type=\"aff\" rid=\"irv12759-aff-0001\">\n<sup>1</sup>\n</xref><address><email>Liselotte.van.asten@rivm.nl</email></address></contrib><contrib id=\"irv12759-cr-0002\" contrib-type=\"author\"><name><surname>Luna Pinzon</surname><given-names>Angie</given-names></name><xref ref-type=\"aff\" rid=\"irv12759-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12759-cr-0003\" contrib-type=\"author\"><name><surname>van de Kassteele</surname><given-names>Jan</given-names></name><xref ref-type=\"aff\" rid=\"irv12759-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12759-cr-0004\" contrib-type=\"author\"><name><surname>Donker</surname><given-names>Gé</given-names></name><xref ref-type=\"aff\" rid=\"irv12759-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"irv12759-cr-0005\" contrib-type=\"author\"><name><surname>de Lange</surname><given-names>Dylan W.</given-names></name><xref ref-type=\"aff\" rid=\"irv12759-aff-0003\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12759-aff-0004\">\n<sup>4</sup>\n</xref></contrib><contrib id=\"irv12759-cr-0006\" contrib-type=\"author\"><name><surname>Dongelmans</surname><given-names>Dave A.</given-names></name><xref ref-type=\"aff\" rid=\"irv12759-aff-0003\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12759-aff-0005\">\n<sup>5</sup>\n</xref></contrib><contrib id=\"irv12759-cr-0007\" contrib-type=\"author\"><name><surname>de Keizer</surname><given-names>Nicolette F.</given-names></name><xref ref-type=\"aff\" rid=\"irv12759-aff-0003\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"irv12759-aff-0006\">\n<sup>6</sup>\n</xref></contrib><contrib id=\"irv12759-cr-0008\" contrib-type=\"author\"><name><surname>van der Hoek</surname><given-names>Wim</given-names></name><xref ref-type=\"aff\" rid=\"irv12759-aff-0001\">\n<sup>1</sup>\n</xref></contrib></contrib-group><aff id=\"irv12759-aff-0001\">\n<label><sup>1</sup></label>\n<named-content content-type=\"organisation-division\">Centre for Infectious Disease Control Netherlands</named-content>\n<institution>National Institute for Public Health and the Environment (RIVM)</institution>\n<city>Bilthoven</city>\n<country country=\"NL\">The Netherlands</country>\n</aff><aff id=\"irv12759-aff-0002\">\n<label><sup>2</sup></label>\n<institution>Nivel Primary Care Database – sentinel practices</institution>\n<city>Utrecht</city>\n<country country=\"NL\">the Netherlands</country>\n</aff><aff id=\"irv12759-aff-0003\">\n<label><sup>3</sup></label>\n<institution>National Intensive Care Evaluation</institution>\n<city>Amsterdam</city>\n<country country=\"NL\">the Netherlands</country>\n</aff><aff id=\"irv12759-aff-0004\">\n<label><sup>4</sup></label>\n<named-content content-type=\"organisation-division\">Department of Intensive Care Medicine</named-content>\n<named-content content-type=\"organisation-division\">University Medical Center</named-content>\n<institution>University Utrecht</institution>\n<city>Utrecht</city>\n<country country=\"NL\">the Netherlands</country>\n</aff><aff id=\"irv12759-aff-0005\">\n<label><sup>5</sup></label>\n<named-content content-type=\"organisation-division\">Department of Intensive Care Medicine</named-content>\n<named-content content-type=\"organisation-division\">Amsterdam UMC</named-content>\n<institution>Location AMC</institution>\n<city>Amsterdam</city>\n<country country=\"NL\">The Netherlands</country>\n</aff><aff id=\"irv12759-aff-0006\">\n<label><sup>6</sup></label>\n<named-content content-type=\"organisation-division\">Department of Medical Informatics</named-content>\n<named-content content-type=\"organisation-division\">Amsterdam UMC</named-content>\n<named-content content-type=\"organisation-division\">Location AMC</named-content>\n<institution>Amsterdam Public Health research institute</institution>\n<city>Amsterdam</city>\n<country country=\"NL\">The Netherlands</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label><bold>Correspondence</bold><break/>\nLiselotte van Asten, Centre for Infectious Disease Control Netherlands, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.<break/>\nEmail: <email>Liselotte.van.asten@rivm.nl</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>12</day><month>6</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>9</month><year>2020</year></pub-date><volume>14</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1111/irv.v14.5</issue-id><fpage>575</fpage><lpage>586</lpage><history><date date-type=\"received\"><day>05</day><month>11</month><year>2019</year></date><date date-type=\"rev-recd\"><day>05</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>06</day><month>5</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 John Wiley & Sons Ltd <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Influenza and Other Respiratory Viruses</italic> published by John Wiley & Sons Ltd.</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:IRV-14-575.pdf\"/><abstract id=\"irv12759-abs-0001\"><title>Abstract</title><sec id=\"irv12759-sec-0001\"><title>Background</title><p>The burden of severe influenza virus infections is poorly known, for which surveillance of severe acute respiratory infection (SARI) is encouraged. Hospitalized SARI patients are however not always tested for influenza virus infection. Thus, to estimate the impact of influenza circulation we studied how influenza in primary care relates to intensive care unit (ICU) admissions using a modelling approach.</p></sec><sec id=\"irv12759-sec-0002\"><title>Methods</title><p>We used time‐series regression modelling to estimate a) the number of SARI admissions to ICU associated with medically attended influenza infections in primary care; b) how this varies by season; and c) the time lag between SARI and influenza time series. We analysed weekly adult ICU admissions (registry data) and adult influenza incidence (primary care surveillance data) from July 2007 through June 2016.</p></sec><sec id=\"irv12759-sec-0003\"><title>Results</title><p>Depending on the year, 0% to 12% of annual SARI admissions were associated with influenza (0‐554 in absolute numbers; population rate: 0/10 000‐0.39/10 000 inhabitants), up to 27% during influenza epidemics. The average optimal fitting lag was +1 week (SARI trend preceding influenza by 1 week), varying between seasons (−1 to +4) with most seasons showing positive lags.</p></sec><sec id=\"irv12759-sec-0004\"><title>Conclusion</title><p>Up to 12% of yearly SARI admissions to adult ICU are associated with influenza, but with large year‐to‐year variation and higher during influenza epidemics. In most years, SARI increases earlier than medically attended influenza infections in the general population. SARI surveillance could thus complement influenza‐like illness surveillance by providing an indication of the season‐specific burden of severe influenza infections and potential early warning of influenza activity and severity.</p></sec></abstract><kwd-group><kwd id=\"irv12759-kwd-0001\">association</kwd><kwd id=\"irv12759-kwd-0002\">ILI</kwd><kwd id=\"irv12759-kwd-0003\">influenza</kwd><kwd id=\"irv12759-kwd-0004\">intensive care</kwd><kwd id=\"irv12759-kwd-0005\">pneumonia</kwd><kwd id=\"irv12759-kwd-0006\">regression model</kwd><kwd id=\"irv12759-kwd-0007\">SARI</kwd><kwd id=\"irv12759-kwd-0008\">seasonality</kwd><kwd id=\"irv12759-kwd-0009\">Surveillance</kwd><kwd id=\"irv12759-kwd-0010\">time series</kwd><kwd id=\"irv12759-kwd-0011\">trends</kwd></kwd-group><funding-group><award-group id=\"funding-0001\"><funding-source><institution-wrap><institution>Ministerie van Volksgezondheid, Welzijn en Sport </institution><institution-id institution-id-type=\"open-funder-registry\">10.13039/501100002999</institution-id></institution-wrap></funding-source><award-id>V/150044/19/SS</award-id></award-group></funding-group><counts><fig-count count=\"5\"/><table-count count=\"3\"/><page-count count=\"12\"/><word-count count=\"10807\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>September 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.6 mode:remove_FC converted:18.08.2020</meta-value></custom-meta></custom-meta-group></article-meta><notes><p content-type=\"self-citation\">\n<mixed-citation publication-type=\"journal\" id=\"irv12759-cit-1001\">\n<string-name>\n<surname>van Asten</surname>\n<given-names>L</given-names>\n</string-name>, <string-name>\n<surname>Luna Pinzon</surname>\n<given-names>A</given-names>\n</string-name>, <string-name>\n<surname>van de Kassteele</surname>\n<given-names>J</given-names>\n</string-name>, et al. <article-title>The association between influenza infections in primary care and intensive care admissions for severe acute respiratory infection (SARI): A modelling approach</article-title>. <source xml:lang=\"en\">Influenza Other Respi Viruses</source>. <year>2020</year>;<volume>14</volume>:<fpage>575</fpage>–<lpage>586</lpage>. <pub-id pub-id-type=\"doi\">10.1111/irv.12759</pub-id>\n</mixed-citation>\n</p><fn-group id=\"irv12759-ntgp-0001\"><fn id=\"irv12759-note-0001\"><p>The peer review history for this article is available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://publons.com/publon/10.1111/irv.12759\">https://publons.com/publon/10.1111/irv.12759</ext-link>\n</p></fn></fn-group></notes></front><body id=\"irv12759-body-0001\"><sec id=\"irv12759-sec-0005\"><label>1</label><title>INTRODUCTION</title><p>Hospital surveillance of severe acute respiratory infection (SARI)<xref rid=\"irv12759-bib-0001\" ref-type=\"ref\">\n<sup>1</sup>\n</xref> is lacking or incomplete in most Western European countries.<xref rid=\"irv12759-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref>, <xref rid=\"irv12759-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref> Some countries do monitor laboratory‐confirmed influenza hospitalizations or intensive care unit (ICU) admissions and report this to the European Influenza Surveillance Network (EISN).<xref rid=\"irv12759-bib-0004\" ref-type=\"ref\">\n<sup>4</sup>\n</xref> However, this underestimates severe influenza burden as not all hospital patients admitted with respiratory infections undergo laboratory testing for influenza.<xref rid=\"irv12759-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref> Additionally, denominator data on the number of patients with symptoms of infectious respiratory illness are generally lacking.<xref rid=\"irv12759-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref> The Netherlands, like in most other European countries, has a robust surveillance system for influenza infections in primary care, providing information on timing and duration of the seasonal epidemic.<xref rid=\"irv12759-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref> However, the number of serious complications requiring hospitalization is not available through this system.<xref rid=\"irv12759-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref>\n</p><p>In primary care (and other outpatient settings), influenza epidemics are heterogeneous from season to season.<xref rid=\"irv12759-bib-0007\" ref-type=\"ref\">\n<sup>7</sup>\n</xref>, <xref rid=\"irv12759-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> This is also reflected by SARI admissions to ICU,<xref rid=\"irv12759-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref> with some seasons showing high peak incidence, while other seasons show lower peaks but sometimes higher cumulative incidence over the season, with or without high ICU mortality.<xref rid=\"irv12759-bib-0010\" ref-type=\"ref\">\n<sup>10</sup>\n</xref> However, these extremes in ICU do not always coincide with high burden in primary care.<xref rid=\"irv12759-bib-0010\" ref-type=\"ref\">\n<sup>10</sup>\n</xref> The ratio of SARI in ICU to influenza‐like illness (ILI) in primary care is one of the influenza severity parameters proposed by the World Health Organization (WHO). It expresses the number of SARI ICU admissions per observed ILI patient in primary care.<xref rid=\"irv12759-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref> But, while ILI is the gold standard for estimating influenza activity in the general population, SARI might be less specific for influenza circulation as it could include a higher background level of respiratory disease by other infections and causes.<xref rid=\"irv12759-bib-0011\" ref-type=\"ref\">\n<sup>11</sup>\n</xref> Thus, to gain better insight into the timing and proportion of SARI ICU admissions that are associated with influenza circulation we used a regression modelling approach.<xref rid=\"irv12759-bib-0012\" ref-type=\"ref\">\n<sup>12</sup>\n</xref>, <xref rid=\"irv12759-bib-0013\" ref-type=\"ref\">\n<sup>13</sup>\n</xref> Understanding this association will further elucidate the potential of ICU data for strengthening influenza surveillance.</p></sec><sec sec-type=\"methods\" id=\"irv12759-sec-0006\"><label>2</label><title>METHODS</title><p>A long‐running robust ILI surveillance system<xref rid=\"irv12759-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref> and a comprehensive national registry of ICU admissions<xref rid=\"irv12759-bib-0014\" ref-type=\"ref\">\n<sup>14</sup>\n</xref> provided us with reliable data to make estimates of a) the number of adult SARI ICU admissions associated with medically attended influenza infections in adults in primary care, b) how this varies yearly and c) the delay or lead time between SARI and influenza time series. The study period ran from 1 July 2007 through 30 June 2016. As influenza epidemics occur in winter, we used season‐years which we defined as running from week 27 to 26 (ie approximately from July to June).</p><sec id=\"irv12759-sec-0007\"><label>2.1</label><title>Intensive care data</title><p>Hospital data on weekly admissions to the ICU were retrieved from the National Intensive Care Evaluation (NICE) registry, originally set up for monitoring quality of ICU care.<xref rid=\"irv12759-bib-0014\" ref-type=\"ref\">\n<sup>14</sup>\n</xref> As paediatric ICUs are not included in the registry, the study focuses on the adult population. A SARI admission to ICU was defined as a patient meeting all three of the following criteria: (a) the patient was admitted to the hospital less than two days before ICU admission, (b) the ICU admission was not a readmission to the ICU within the hospitalized period, and (c) the APACHE IV<xref rid=\"irv12759-bib-0015\" ref-type=\"ref\">\n<sup>15</sup>\n</xref> reason for admission was any of the 7 following respiratory codes: <italic>Sepsis, pulmonary; Pneumonia, aspiration; Pneumonia, bacterial; Pneumonia, fungal; Pneumonia, other; Pneumonia, parasitic (ie Pneumocystis pneumonia); and Pneumonia, viral</italic>. Admissions to intensive care for elective surgery or trauma were excluded, and we categorized all remaining admissions as medical admissions. We calculated the proportion of medical ICU admissions that were a SARI by dividing the number of weekly SARI by the weekly number of medical admissions. Information on influenza laboratory testing was not available in the NICE registry. ICU coverage increased during the study period from roughly 40% to near‐complete coverage of all Dutch adult ICUs in 2016.</p></sec><sec id=\"irv12759-sec-0008\"><label>2.2</label><title>Influenza‐like Illness data</title><p>Medically attended ILI incidence data were retrieved from NIVEL Primary Care Database—sentinel general practitioner (GP) practices. This system covers approximately 0.8% of the Dutch population and is nationally representative for age, sex, regional distribution and population density.<xref rid=\"irv12759-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref> Participating GPs report weekly the number and age of ILI patients. The number of patients registered in their practice was used as a denominator for ILI incidence calculation. To confirm influenza circulation, a subset of ILI patients is systematically swabbed for laboratory testing. We calculated influenza circulation as follows: ILI incidence * the proportion of swabs positive for influenza virus. Influenza epidemics are defined within this ILI surveillance as the weeks with ILI incidence exceeding 5.1/10 000 persons for minimally two consecutive weeks.</p></sec><sec id=\"irv12759-sec-0009\"><label>2.3</label><title>Statistical analyses</title><p>We used a binomial regression model to associate the number of weekly SARI in adult ICU with the weekly influenza incidence in primary care. As the influenza surveillance data contained pre‐defined age groups, we selected the influenza incidence in the 15+ age group as this was the only available age cut‐off for child to adult. The number of SARI admissions\n<mml:math id=\"nlm-math-1\"><mml:msubsup><mml:mi>N</mml:mi><mml:mi mathvariant=\"normal\">w</mml:mi><mml:mtext>SARI</mml:mtext></mml:msubsup></mml:math> in week <italic>w</italic> (<italic>w</italic> = 1, …, 470) was used as outcome variable, while the weekly total number of medical ICU admissions\n<mml:math id=\"nlm-math-2\"><mml:msubsup><mml:mi>N</mml:mi><mml:mi mathvariant=\"normal\">w</mml:mi><mml:mtext>ICU</mml:mtext></mml:msubsup></mml:math> was included in the model as a denominator to adjust for the increasing number of ICUs participating in the NICE registry (Figure <xref rid=\"irv12759-fig-0001\" ref-type=\"fig\">1</xref>):<disp-formula id=\"irv12759-disp-0001\"><mml:math id=\"nlm-math-3\"><mml:mrow><mml:msubsup><mml:mi>N</mml:mi><mml:mi mathvariant=\"normal\">w</mml:mi><mml:mtext>SARI</mml:mtext></mml:msubsup><mml:mo>∼</mml:mo><mml:mtext>Binomial</mml:mtext><mml:mfenced close=\")\" open=\"(\" separators=\"\"><mml:mrow><mml:msubsup><mml:mi>p</mml:mi><mml:mi mathvariant=\"normal\">w</mml:mi><mml:mtext>SARI</mml:mtext></mml:msubsup><mml:mo>,</mml:mo><mml:msubsup><mml:mi>N</mml:mi><mml:mi mathvariant=\"normal\">w</mml:mi><mml:mtext>ICU</mml:mtext></mml:msubsup></mml:mrow></mml:mfenced><mml:mo>.</mml:mo></mml:mrow></mml:math></disp-formula>\n</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"irv12759-fig-0001\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Number of SARI admissions and all medical admissions to adult ICUs</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"IRV-14-575-g001\"/></fig><p>We used an identity link function to relate the proportion of SARI admissions to the explanatory variables, which allows an additive interpretation of the regression coefficients as risk differences instead of odds ratios.</p><p>The model for\n<mml:math id=\"nlm-math-4\"><mml:msubsup><mml:mi>p</mml:mi><mml:mi mathvariant=\"normal\">w</mml:mi><mml:mtext>SARI</mml:mtext></mml:msubsup></mml:math> consists of two parts: a) a baseline model for possible underlying seasonal time trends (cyclical) which we assumed to describe SARI admissions associated with other factors than influenza and b) an influenza model that describes the association between the weekly influenza numbers and the number of SARI admissions: <disp-formula id=\"irv12759-disp-0002\"><mml:math id=\"nlm-math-5\"><mml:mrow><mml:msubsup><mml:mi>p</mml:mi><mml:mi mathvariant=\"normal\">w</mml:mi><mml:mtext>SARI</mml:mtext></mml:msubsup><mml:mo>=</mml:mo><mml:msubsup><mml:mi>p</mml:mi><mml:mi mathvariant=\"normal\">w</mml:mi><mml:mtext>base</mml:mtext></mml:msubsup><mml:mo>+</mml:mo><mml:msubsup><mml:mi>p</mml:mi><mml:mi mathvariant=\"normal\">w</mml:mi><mml:mrow><mml:mo movablelimits=\"true\">inf</mml:mo><mml:mtext>luenza</mml:mtext></mml:mrow></mml:msubsup><mml:mo>.</mml:mo></mml:mrow></mml:math></disp-formula>\n</p><p>First, we selected the best fitting baseline model consisting of cyclical terms. For the cyclical trend, we evaluated sine and cosine terms with a periodicity of 1, 1/2, 1/3 and 1/4 years. The terms were always included in the model as a sine and cosine pair, thus allowing flexible phase shifts. This resulted in 16 different potential baseline models, always with an intercept, but with and without sine cosine pairs of varying periodicity (ranging from inclusion of zero up to four pairs):<disp-formula id=\"irv12759-disp-0003\"><mml:math id=\"nlm-math-6\"><mml:mrow><mml:msubsup><mml:mi>p</mml:mi><mml:mi mathvariant=\"normal\">w</mml:mi><mml:mtext>base</mml:mtext></mml:msubsup><mml:mo>=</mml:mo><mml:msub><mml:mi>β</mml:mi><mml:mn>0</mml:mn></mml:msub><mml:mo>+</mml:mo><mml:munderover><mml:mo movablelimits=\"false\">∑</mml:mo><mml:mrow><mml:mi>k</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mn>4</mml:mn></mml:munderover><mml:mfenced close=\"}\" open=\"{\" separators=\"\"><mml:mrow><mml:msub><mml:mi>β</mml:mi><mml:mrow><mml:mn>1</mml:mn><mml:mi>k</mml:mi></mml:mrow></mml:msub><mml:mi>sin</mml:mi><mml:mfenced close=\")\" open=\"(\" separators=\"\"><mml:mfrac><mml:mrow><mml:mn>2</mml:mn><mml:mi>k</mml:mi><mml:mi>π</mml:mi><mml:mi>w</mml:mi></mml:mrow><mml:mn>52.17</mml:mn></mml:mfrac></mml:mfenced><mml:mo>+</mml:mo><mml:msub><mml:mi>β</mml:mi><mml:mrow><mml:mn>1</mml:mn><mml:mi>k</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mi>cos</mml:mi><mml:mfenced close=\")\" open=\"(\" separators=\"\"><mml:mfrac><mml:mrow><mml:mn>2</mml:mn><mml:mi>k</mml:mi><mml:mi>π</mml:mi><mml:mi>w</mml:mi></mml:mrow><mml:mn>52.17</mml:mn></mml:mfrac></mml:mfenced></mml:mrow></mml:mfenced><mml:mo>.</mml:mo></mml:mrow></mml:math></disp-formula>\n</p><p>The model with the lowest Akaike information criterion (AIC) was selected as baseline model. The model for\n<mml:math id=\"nlm-math-7\"><mml:msubsup><mml:mi>p</mml:mi><mml:mi mathvariant=\"normal\">w</mml:mi><mml:mtext>influenza</mml:mtext></mml:msubsup></mml:math> is a bit more complicated, because the dominant influenza strain and severity outcomes (hospitalization, mortality) can vary from season to season.<xref rid=\"irv12759-bib-0007\" ref-type=\"ref\">\n<sup>7</sup>\n</xref>, <xref rid=\"irv12759-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref>, <xref rid=\"irv12759-bib-0010\" ref-type=\"ref\">\n<sup>10</sup>\n</xref>, <xref rid=\"irv12759-bib-0012\" ref-type=\"ref\">\n<sup>12</sup>\n</xref> We therefore performed a time‐dependent analysis. This allowed the association (ie regression coefficient) to vary between seasons. For this, we entered the weekly influenza incidence numbers\n<mml:math id=\"nlm-math-8\"><mml:msubsup><mml:mtext>influenza</mml:mtext><mml:mi mathvariant=\"normal\">w</mml:mi><mml:mi mathvariant=\"normal\">s</mml:mi></mml:msubsup></mml:math> as separate variables per season‐year into the model (being zero everywhere, except for the specific season\n<mml:math id=\"nlm-math-9\"><mml:mi>s</mml:mi></mml:math>). Since we do not know whether SARI ICU admissions follow, coincide or precede the ILI trend, we also evaluated nine lagged values of ILI incidence per season\n<mml:math id=\"nlm-math-10\"><mml:msubsup><mml:mtext>ILI influenza</mml:mtext><mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">w</mml:mi><mml:mo>+</mml:mo><mml:msub><mml:mi mathvariant=\"normal\">j</mml:mi><mml:mi mathvariant=\"normal\">s</mml:mi></mml:msub></mml:mrow></mml:mrow><mml:mi mathvariant=\"normal\">s</mml:mi></mml:msubsup></mml:math> (where <italic>j<sub>s</sub></italic> = −4, …, 4, ie up to 4 weeks earlier and 4 weeks later in time relative to respiratory ICU admissions), including maximally one lag per season. So, per season separately, we added influenza incidence to the baseline model: testing each of the nine ILI lags <italic>j<sub>s</sub></italic> separately, that is added singularly to the baseline model (thus building nine models per season):<disp-formula id=\"irv12759-disp-0004\"><mml:math id=\"nlm-math-11\"><mml:mrow><mml:msubsup><mml:mi>p</mml:mi><mml:mi mathvariant=\"normal\">w</mml:mi><mml:mtext>SARI</mml:mtext></mml:msubsup><mml:mo>=</mml:mo><mml:munderover><mml:mo movablelimits=\"false\">∑</mml:mo><mml:mrow><mml:mi>s</mml:mi><mml:mo>=</mml:mo><mml:mn>2007</mml:mn></mml:mrow><mml:mn>2016</mml:mn></mml:munderover><mml:msub><mml:mi>β</mml:mi><mml:mi mathvariant=\"normal\">s</mml:mi></mml:msub><mml:msubsup><mml:mtext>influenza</mml:mtext><mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">w</mml:mi><mml:mo>+</mml:mo><mml:msub><mml:mi>j</mml:mi><mml:mi mathvariant=\"normal\">s</mml:mi></mml:msub></mml:mrow></mml:mrow><mml:mi mathvariant=\"normal\">s</mml:mi></mml:msubsup><mml:mo>.</mml:mo></mml:mrow></mml:math></disp-formula>\n</p><p>We repeated this influenza lag selection for each season and per season selected the influenza lag that showed the best fit (lowest AIC).</p><p>All analyses were performed using the statistical package R (version 3.4.0). Model selection was performed in this manner, as R would not run all the possible different model fits at once as this produced too many combinations.</p><p>We tested both positive and negative lags between influenza and SARI as the direction of this association is still poorly understood, with ICU admissions possibly being earlier.<xref rid=\"irv12759-bib-0016\" ref-type=\"ref\">\n<sup>16</sup>\n</xref> Influenza circulation may give birth to two distinct populations: vulnerable or fragile persons exposed to influenza in the community may come down with severe illness more quickly than generally healthy persons who may develop ILI symptoms more slowly and/or wait before seeing a GP.</p><p>By multiplying the ILI regression coefficients with the observed weekly influenza incidence (lagged according to the season‐specific lags), we calculated the influenza‐associated proportions of SARI (per week). Further multiplying these weekly proportions by the weekly number of medical ICU admissions produced the estimated absolute numbers of weekly SARI associated with influenza. We then cumulated these weekly SARI numbers by season‐year. As the number of ICUs participating in the NICE registry increased over time these absolute numbers were not directly comparable between seasons. Therefore, we chose 2015 as the index year (there was near‐complete national coverage of adult ICUs in NICE) and standardized all estimated numbers to the volume of medical ICU admissions observed in 2015.</p></sec></sec><sec sec-type=\"results\" id=\"irv12759-sec-0010\"><label>3</label><title>RESULTS</title><sec id=\"irv12759-sec-0011\"><label>3.1</label><title>Description of ILI and SARI time series</title><p>From July 2007 to June 2016, there were a total of 30 515 registered SARI admissions to ICUs with a weekly average of 65 admissions (standard deviation (SD) 28). To adjust for the increasing coverage of ICUs in the NICE registry, we also show the weekly number of SARI as a proportion (ie relative to weekly total number of medical ICU admissions) (Figure <xref rid=\"irv12759-fig-0001\" ref-type=\"fig\">1</xref>). The weekly SARI admissions, ILI and influenza incidence showed peaks in winter with surges roughly coinciding (Figure <xref rid=\"irv12759-fig-0002\" ref-type=\"fig\">2</xref>) but with the ILI and influenza trend being more pronounced owing to relatively higher peaks than the trend in SARI. Adult SARI admissions comprised 5% to 25% of medical ICU admissions weekly, ILI incidence varied between 0 and 14.8/10 000, and influenza incidence between 0 and 9.04 /10 000 by week (15+ age group). The correlation coefficient (Spearman's rank) between SARI (proportions) and influenza incidence was 0.60 (<italic>P</italic>‐value < .0001), slightly higher than influenza preceding SARI (0.44‐0.58; lag −4 to −1) or after SARI (0.53‐0.59; lag 1 to 4). Correlations with ILI instead of influenza were similar.</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"irv12759-fig-0002\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Weekly SARI numbers (as proportion of medical ICU admissions) and weekly ILI and influenza incidence</p></caption><graphic id=\"nlm-graphic-3\" xlink:href=\"IRV-14-575-g002\"/></fig></sec><sec id=\"irv12759-sec-0012\"><label>3.2</label><title>The modelled association between influenza circulation and SARI admissions</title><p>Observed and modelled SARI numbers are shown in Figure <xref rid=\"irv12759-fig-0003\" ref-type=\"fig\">3</xref>. The assumed seasonal baseline (green line) depicts SARI levels the model did not associate with influenza. From the model, we estimated that on average for the total study period, SARI increased with 7.30% (coefficient) with every increase in the influenza incidence of 1/1000 per week (Table <xref rid=\"irv12759-tbl-0001\" ref-type=\"table\">1</xref>). For example, if in a certain week the influenza incidence increases with 6/1000 compared to the previous week, the proportion of SARI increases, in an absolute sense, with 6*7.30% = 34.89% compared to the previous week. However, time‐dependent analyses show that this estimate varied significantly from season to season with regression coefficients varying between 0 (season 2013/2014) to 12.13(2009/2010 season) (Table <xref rid=\"irv12759-tbl-0001\" ref-type=\"table\">1</xref>).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"irv12759-fig-0003\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>Observed and predicted† weekly proportion of medical admissions due to a SARI (seasons 2007/2008 to 2015/2016). †Predicted weekly proportions were calculated using the parameter estimates from the regression model (with season‐specific estimates)</p></caption><graphic id=\"nlm-graphic-5\" xlink:href=\"IRV-14-575-g003\"/></fig><table-wrap id=\"irv12759-tbl-0001\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Association between respiratory ICU admissions and influenza incidence</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Season</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Best fitting lag<xref ref-type=\"fn\" rid=\"irv12759-note-0002\">\n<sup>a</sup>\n</xref>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Coefficient<xref ref-type=\"fn\" rid=\"irv12759-note-0003\">\n<sup>b</sup>\n</xref>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">95% CI</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>‐value</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2007/8 ‐ 2015/16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">+1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">7.30</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.38 ‐ 8.23</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><2.2e‐16</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2007/8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">+3</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">7.78</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.79 ‐ 15.05</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">.030657</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2008/9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">+4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">11.42</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.22 ‐ 14.75</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.59e‐12</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2009/10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">12.13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.54 ‐ 15.86</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.02e‐10</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2010/11</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">+2</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.37</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.36 ‐ 13.46</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.20e‐11</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2011/12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">+4</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4.06</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−1.00 ‐ 9.30</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">.123481</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2012/13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">7.06</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.62 ‐ 8.54</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><2.2e‐16</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2013/14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">‐</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2014/15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">+2</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">5.74</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.50 ‐ 7.00</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><2.2e‐16</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2015/16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10.03</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.46 ‐ 11.63</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><2.2e‐16</td></tr></tbody></table><table-wrap-foot id=\"irv12759-ntgp-0002\"><fn id=\"irv12759-note-0002\"><label><sup>a</sup></label><p>Weeks that SARI admissions are shifted forward (+lags, preceding influenza) or backward (−lags, lagging behind influenza) in time relative to influenza observations.</p></fn><fn id=\"irv12759-note-0003\"><label><sup>b</sup></label><p>Coefficients from a regression analysis representing the proportion of SARI admissions associated with a 1/1000 increase in influenza incidence (adjusted for baseline seasonal trends).</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap></sec><sec id=\"irv12759-sec-0013\"><label>3.3</label><title>Estimated numbers of SARI associated with influenza</title><p>On average, 7% of yearly SARI was associated with influenza but with large variations: 0%‐12% of SARI was estimated to be influenza‐associated depending on the season. The highest proportions coincided with the highest absolute number of influenza‐associated SARI (seasons 2012/13, 2014/15 and 2015/16). Figure <xref rid=\"irv12759-fig-0004\" ref-type=\"fig\">4</xref> shows the influenza‐associated proportion per week (depicting the proportion above the cyclical baseline that was estimated to be associated with influenza, ie the value of the red line minus the green line from Figure <xref rid=\"irv12759-fig-0003\" ref-type=\"fig\">3</xref>). The estimated absolute number of influenza‐associated SARI in adult ICU (standardized) varied between 0 and 554 for the whole country between the different season‐years (Table <xref rid=\"irv12759-tbl-0002\" ref-type=\"table\">2</xref>, Figure <xref rid=\"irv12759-fig-0005\" ref-type=\"fig\">5</xref>), on average 321 per season‐year. The 2013/2014 season showed the lowest number (0), the 2015/2016 season showed the highest, followed by the 2012/2013 and 2014/2015 season (554, 456 and 448, respectively, standardized, Table <xref rid=\"irv12759-tbl-0002\" ref-type=\"table\">2</xref>, Figure <xref rid=\"irv12759-fig-0005\" ref-type=\"fig\">5</xref>). When focusing only on influenza epidemic weeks instead of full season‐years, the percentage of influenza‐associated SARI was higher: on average 18% (0% to 27% between the different epidemics, Table <xref rid=\"irv12759-tbl-0003\" ref-type=\"table\">3</xref>). Modelling with ILI instead of influenza showed ILI‐associated SARI to be roughly twofold higher than influenza‐associated SARI in entire season‐years (on average 13% vs 7%), but much less different in influenza epidemic weeks (on average 22% vs 18%).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"irv12759-fig-0004\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><p>Weekly proportions of SARI associated with influenza. Predicted from a time‐dependent regression model giving season‐specific estimates</p></caption><graphic id=\"nlm-graphic-7\" xlink:href=\"IRV-14-575-g004\"/></fig><table-wrap id=\"irv12759-tbl-0002\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>SARI admissions and influenza‐associated SARI admissions to ICU per season‐year<xref ref-type=\"fn\" rid=\"irv12759-note-0004\">\n<sup>a</sup>\n</xref>\n</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Weekly max of influenza‐associated SARI<xref ref-type=\"fn\" rid=\"irv12759-note-0005\">\n<sup>b</sup>\n</xref>,</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Influenza‐associated SARI (unstandardized)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Influenza‐associated SARI<xref ref-type=\"fn\" rid=\"irv12759-note-0005\">\n<sup>b</sup>\n</xref>, incidence</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Observed crude SARI<xref ref-type=\"fn\" rid=\"irv12759-note-0005\">\n<sup>b</sup>\n</xref>, incidence</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Influenza‐associated SARI<xref ref-type=\"fn\" rid=\"irv12759-note-0005\">\n<sup>b</sup>\n</xref>, proportion</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Influenza‐associated SARI<xref ref-type=\"fn\" rid=\"irv12759-note-0005\">\n<sup>b</sup>\n</xref>, rate<xref ref-type=\"fn\" rid=\"irv12759-note-0006\">\n<sup>c</sup>\n</xref>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Observed SARI<xref ref-type=\"fn\" rid=\"irv12759-note-0005\">\n<sup>b</sup>\n</xref> rate<xref ref-type=\"fn\" rid=\"irv12759-note-0006\">\n<sup>c</sup>\n</xref>,</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2007/2008</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">52</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">144</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4595</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.10</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3.26</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2008/2009</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">52</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">179</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">345</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4566</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">8%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.25</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3.24</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2009/2010</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">41</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">189</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">283</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4408</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.20</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3.13</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2010/2011</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">48</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">202</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">276</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4710</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.20</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3.35</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2011/2012</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">48</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">61</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4363</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3.10</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2012/2013</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">396</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">456</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4680</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.32</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3.33</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2013/2014</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3853</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">2.74</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2014/2015</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">451</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">448</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4521</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">10%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.32</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3.21</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2015/2016</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">66</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">554</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">554</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4483</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">12%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.39</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3.19</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Yearly average</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">39</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">259</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">321</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">4464</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">7%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.23</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">3.17</td></tr></tbody></table><table-wrap-foot id=\"irv12759-ntgp-0003\"><fn id=\"irv12759-note-0004\"><label><sup>a</sup></label><p>Full years running from July to June.</p></fn><fn id=\"irv12759-note-0005\"><label><sup>b</sup></label><p>Standardized to the number of medical ICU admissions in season 2015.</p></fn><fn id=\"irv12759-note-0006\"><label><sup>c</sup></label><p>Rates were calculated with total 15+ Dutch population size in 2015 (scale is 1/10 000).</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"irv12759-fig-0005\" orientation=\"portrait\" position=\"float\"><label>Figure 5</label><caption><p>Standardized† number of SARI admissions associated with influenza and regression coefficients per season‡ (2007/8‐2015/16) in the Netherlands. †Standardized to the total number of medical ICU admissions in season 2015/2016. ‡ Each season representing the time period of July until June the next year (eg 2007 representing 2007/2008 season)</p></caption><graphic id=\"nlm-graphic-9\" xlink:href=\"IRV-14-575-g005\"/></fig><table-wrap id=\"irv12759-tbl-0003\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><p>All SARI admissions and Influenza‐associated SARI admissions during influenza epidemic weeks</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Dominant influenza strains</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Influenza vaccine match<xref ref-type=\"fn\" rid=\"irv12759-note-0007\">\n<sup>a</sup>\n</xref>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">% of target groups vaccinated<xref ref-type=\"fn\" rid=\"irv12759-note-0009\">\n<sup>c</sup>\n</xref>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Epidemic duration (weeks)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Observed crude SARI<xref ref-type=\"fn\" rid=\"irv12759-note-0008\">\n<sup>b</sup>\n</xref> incidence (during influenza epidemics)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Influenza‐associated SARI<xref ref-type=\"fn\" rid=\"irv12759-note-0008\">\n<sup>b</sup>\n</xref> incidence (during influenza epidemics)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Influenza‐associated SARI<xref ref-type=\"fn\" rid=\"irv12759-note-0008\">\n<sup>b</sup>\n</xref> proportion (during influenza epidemics)</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2007/2008</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">A(H1N1) dominance followed by B dominance</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">mismatch</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">74%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">929</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">58</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2008/2009</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">A(H3N2) dominance</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>match</italic>\n</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">72%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">731</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">88</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2009/2010</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">A(H1N1)pdm09 dominance</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">mismatch</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">70%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">806</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">178</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2010/2011</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">A(H1N1)pdm09 dominance followed by B dominance</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>match</italic>\n</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">69%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">890</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">153</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2011/2012</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">A(H3N2) dominance</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">mismatch</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">66%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2012/2013</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mixed A(H1N1)pdm09 and A(H3N2) dominance followed by B dominance</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">mismatch</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">62%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1996</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">418</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2013/2014</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mixed dominance with slightly more A(H3N2) than A(H1N1)pdm09</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">mismatch</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">60%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">544</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2014/2015</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">A(H3N2) dominance followed by B dominance</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">mismatch</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">57%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2366</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">404</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2015/2016</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">A(H1N1)pdm09 dominance followed by B dominance</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>match</italic>\n</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">56%</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1693</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">455</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2007/2016</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Yearly average</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"/><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1244</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">219</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18%</td></tr></tbody></table><table-wrap-foot id=\"irv12759-ntgp-0004\"><fn id=\"irv12759-note-0007\"><label><sup>a</sup></label><p>Vaccine match with the dominant influenza strain(s).</p></fn><fn id=\"irv12759-note-0008\"><label><sup>b</sup></label><p>Standardized to the number of medical ICU admissions in season 2015.</p></fn><fn id=\"irv12759-note-0009\"><label><sup>c</sup></label><p>Individuals aged 60 years or older and individuals with comorbidity who have an increased risk of complications or death due to influenza infection, % as reported previously (Ref <xref rid=\"irv12759-bib-0023\" ref-type=\"ref\">23</xref>, <xref rid=\"irv12759-bib-0024\" ref-type=\"ref\">24</xref>).</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>Using Dutch population size numbers of 2015 (as the associated numbers were standardized to year 2015), the absolute numbers translated to the following range of influenza‐associated SARI incidence rates per season‐year: 0/10 000 ‐ 0.39/10 000 (Table <xref rid=\"irv12759-tbl-0002\" ref-type=\"table\">2</xref>). Overall raw SARI incidence rates were eight‐ to 78‐fold higher (at 2.7 ‐ 3.4/10 000) than the estimated influenza‐associated SARI rate (Table <xref rid=\"irv12759-tbl-0002\" ref-type=\"table\">2</xref>). The peak also varied by season and ranged from a maximum of 0 (2013/2014) to a maximum of 66 (2015/2016) influenza‐associated SARI in one week (standardized, Table <xref rid=\"irv12759-tbl-0002\" ref-type=\"table\">2</xref>).</p><p>Seasons with higher model coefficients for influenza did not correspond with higher cumulated influenza‐associated SARI (Figure <xref rid=\"irv12759-fig-0005\" ref-type=\"fig\">5</xref>) (Spearman's rank <italic>R</italic>\n<sup>2</sup> .12 <italic>P</italic> = .78). Notably, the 2012/2013 and 2014/2015 seasons had an average coefficient size but high total number of influenza‐associated SARI while the 2009/2010 and 2010/2011 season had a high coefficient but an average number of influenza‐associated SARI.</p></sec><sec id=\"irv12759-sec-0014\"><label>3.4</label><title>Time lag between SARI and influenza or ILI trends</title><p>The overall best fitting influenza lag was on average +1 week for the total study period (ie SARI preceding influenza in the general community by one week showed the best fit). However, the optimal lag varied largely from season to season from −1 to +4 weeks, almost always with positive lags (influenza lagging behind SARI) (Table <xref rid=\"irv12759-tbl-0001\" ref-type=\"table\">1</xref>): the best fitting model was achieved when influenza coincided with SARI (lag 0 weeks) in two seasons, lagged behind SARI admissions in five seasons (lag 1 to 4 weeks) and preceded SARI admissions in one season (2012/2013). For the positive lags (SARI preceding influenza), there is no apparent association between the value of the lag and the number of influenza‐associated SARI. When assessing ILI, instead of influenza, the overall best fitting lag was also +1 week (SARI preceding ILI). Four of nine seasons showed similar lags (as between influenza and SARI); however, it was the 2015/2016 season which was the only one in which ILI preceded SARI (lag −2 weeks). This was also the season with the largest proportion of influenza‐associated SARI under study (554, 12%).</p></sec></sec><sec sec-type=\"discussion\" id=\"irv12759-sec-0015\"><label>4</label><title>DISCUSSION</title><p>This study shows how increases in influenza in primary care relate to increased SARI admissions to adult ICU in time. Varying strongly by season, 0%‐12% (0 to 554) of yearly SARI admissions to ICU are associated with medically attended influenza in the total adult Dutch population, probably reflecting the seasonal variation of circulating influenza strains. In most seasons, increases in ICU admissions occurred 1‐4 weeks earlier than increase of influenza incidence in primary care.</p><p>Over the past 10 years, there has been a concerted effort by WHO and ECDC to fill the knowledge gap in our understanding of severe influenza complications requiring hospitalization.<xref rid=\"irv12759-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref> However, a comprehensive SARI surveillance is still lacking in most Western European countries, and how SARI occurrence is associated with influenza circulation is not yet entirely clear. Our study estimates that of all adult SARI admissions to ICU, an overall 7% was associated with influenza as measured by medically attended influenza in the general adult Dutch population. This varied considerably by season‐year (0%‐12%), and was roughly twofold higher when modelling with ILI instead of influenza. The three season‐years with the highest numbers of SARI associated with influenza coincided with the seasons which had the longest influenza epidemics (lasting 12‐20 weeks in 2012/2013, 2014/2015 and 2015/2016).<xref rid=\"irv12759-bib-0017\" ref-type=\"ref\">\n<sup>17</sup>\n</xref> The percentage of influenza‐associated SARI was higher during influenza epidemic weeks with an average of 18% (0% to 27% between the different epidemics). This is lower than what is found in the extensive sentinel SARI surveillance in Belgium where in 5 influenza epidemics (2013/2014 to 2017/2018) between 31% and 46% of SARI cases were positive for influenza viruses.<xref rid=\"irv12759-bib-0001\" ref-type=\"ref\">\n<sup>1</sup>\n</xref>, <xref rid=\"irv12759-bib-0011\" ref-type=\"ref\">\n<sup>11</sup>\n</xref>, <xref rid=\"irv12759-bib-0018\" ref-type=\"ref\">\n<sup>18</sup>\n</xref> However, direct comparison is difficult as the Belgian estimates are based on all hospitalizations instead of only ICU admissions, with a different SARI case definition than ours, and with a different healthcare system. Few Western European countries other than Belgium have SARI surveillance, although some countries monitor more specifically the number of laboratory‐confirmed influenza hospitalizations and ICU admissions; this narrower case definition shows lower incidences than reported for SARI.<xref rid=\"irv12759-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref>, <xref rid=\"irv12759-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref>, <xref rid=\"irv12759-bib-0020\" ref-type=\"ref\">\n<sup>20</sup>\n</xref>, <xref rid=\"irv12759-bib-0021\" ref-type=\"ref\">\n<sup>21</sup>\n</xref>\n</p><p>A strength of our study is that we used data from primary care ILI surveillance which is the gold standard for influenza surveillance in the Netherlands and most other Western European countries. Therefore, we assume the model to give the best possible estimate of adult SARI admissions to ICU that are associated with influenza circulation in the general adult population. However, we lacked data to differentiate between influenza cases caused by different types, sub‐types and lineages of circulating influenza viruses. Thus, our estimates provide an average effect in those seasons that multiple influenza viruses played an important role in influenza circulation. There appeared to be no clear association between numbers of influenza‐associated SARI and the dominant circulating influenza virus(es) (Table <xref rid=\"irv12759-tbl-0003\" ref-type=\"table\">3</xref>). The associated numbers per season also do not show a straightforward link with seasonal vaccine match or mismatch. Only in three of nine seasons did vaccine match the dominant strain(s), but in those seasons both high (2015/2016) and low (2008/2009) influenza‐associated numbers were apparent (Table <xref rid=\"irv12759-tbl-0003\" ref-type=\"table\">3</xref>).<xref rid=\"irv12759-bib-0017\" ref-type=\"ref\">\n<sup>17</sup>\n</xref>, <xref rid=\"irv12759-bib-0022\" ref-type=\"ref\">\n<sup>22</sup>\n</xref>, <xref rid=\"irv12759-bib-0023\" ref-type=\"ref\">\n<sup>23</sup>\n</xref> As influenza vaccination uptake by risk groups (with comorbidities and/or the 60 + age group) has progressively decreased during the study period (from 74% to 56%),<xref rid=\"irv12759-bib-0024\" ref-type=\"ref\">\n<sup>24</sup>\n</xref>, <xref rid=\"irv12759-bib-0025\" ref-type=\"ref\">\n<sup>25</sup>\n</xref> the number of SARI associated with influenza has roughly increased over time with two exceptions (2011/2012: no influenza epidemic and 2013/2014: no significant association between influenza and SARI) (table <xref rid=\"irv12759-tbl-0003\" ref-type=\"table\">3</xref>).</p><p>This study is a population‐level (ecological) study comparing two trends. Although this is currently the best available approach due to a lack of structural laboratory testing of SARI patients for influenza virus, a pitfall in such time‐series analyses is finding associations that may be due to other underlying time trends. To counter this, in the model we included a seasonal baseline assuming that any associations between SARI and other seasonal aspects (for instance climatic factors and other seasonal respiratory pathogens) are accounted for by this baseline. These cyclical terms compete with the influenza variable in the model, which also exhibits a roughly cyclical pattern and the cyclical terms could potentially over‐adjust, leading to an underestimation of the association of interest: between SARI and influenza. Despite this risk of underestimation, we assume the model more valid than when leaving out baseline seasonality because other respiratory pathogens that circulate in autumn and winter can cause severe respiratory symptoms too.<xref rid=\"irv12759-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> Another risk is posed by potential misclassification of the SARI syndrome, which could also affect the estimation of the number of SARI related to influenza. Excluded diagnoses may lead to underestimation, as in the case of acute exacerbation of chronic obstructive pulmonary disease, since respiratory viruses including influenza viruses are frequently detected in upper and lower respiratory tract samples of such patients.<xref rid=\"irv12759-bib-0026\" ref-type=\"ref\">\n<sup>26</sup>\n</xref> A final issue warranting further investigation is potential differences by narrower adult age bands. The numbers of ILI patients also swabbed for laboratory influenza diagnosing are small; thus, we analysed the adult population as one whole.</p><p>Our data reflect that pressure on ICU is not only defined by the magnitude of the modelled coefficient (the number of SARI expected for every observed influenza) but by the combination with the cumulated incidence of influenza. This is reflected by the 2014/2015 season (with a long 20‐week influenza epidemic) which had an average association with influenza incidence but a high total number of influenza‐associated SARI (448, 10% of SARI), while the 2010/2011 season had a relatively high coefficient but a roughly average number of influenza‐associated SARI. This confirms that multiple measures are required to understand influenza severity and burden in secondary care<xref rid=\"irv12759-bib-0010\" ref-type=\"ref\">\n<sup>10</sup>\n</xref> and that SARI surveillance would complement ILI surveillance.</p><p>Population‐level data as used in this study can provide insight in the timing of trends of SARI admissions relative to medically attended influenza in primary care. While at an individual level a patient is not expected to be admitted to an ICU for an influenza‐associated SARI and thereafter to visit his family physician for ILI, at the population level, trends in severe illness do not necessarily follow trends in mild illness. Literature shedding light on the timing of these trends relative to each other is sparse but reports sometimes suggest that ICU admissions for SARI might actually provide an early indication of severe influenza cases.<xref rid=\"irv12759-bib-0016\" ref-type=\"ref\">\n<sup>16</sup>\n</xref> In our study, SARI surveillance based on ICU admissions, covering the entire Dutch population, could be a more sensitive measure in detecting influenza epidemic activity than ILI incidence in primary care, which has a more limited population coverage and is highly dependent on health‐seeking behaviour. We previously reported that ILI and SARI data are associated with each other at multiple lag times.<xref rid=\"irv12759-bib-0027\" ref-type=\"ref\">\n<sup>27</sup>\n</xref> In the current study, we took this further and determined the optimal lead or lag time per season. We saw that timing of the two trends differs greatly from season to season, sometimes coinciding, but more often the SARI trend shows a lead over influenza and ILI trends of 1 to 4 weeks. Only one season (2015/2016, a season of 59% influenza A(H1N1)pdm09 and 39% influenza B in influenza‐positive samples of ILI patients and with reported vaccine match), showed the opposite with SARI lagging 2 weeks behind ILI. This suggests that while SARI in ICU can coincide with ILI trends, ICU admissions more often precede increases in ILI and rarely occur <italic>later</italic> than ILI in primary care, thus being a potentially early indicator for influenza virus activity. SARI surveillance could allow preventive measures and preparedness for increasing pressure on secondary care. This is similar to the finding from a pilot in three Dutch hospitals showing that SARI admissions to hospital (not only in ICU) peaks before ILI (personal communication, S. Marbus, <italic>Hospitals peak first</italic>, submitted 2019). Perhaps vulnerable individuals progress to severe illness more rapidly, or other epidemiological characteristics (eg R0, generation time) may differ from those in the healthier general community. Whether the direction of the time shift is also informative of influenza severity is not clear as only one season in our data showed SARI in ICU lagging behind ILI (by two weeks in 2015/2016, data not shown). That was incidentally also the season with the highest influenza‐associated number and percentage of SARI (or only 2012/2013 in the SARI‐influenza analyses, and second highest). This might suggest that in the other years, when influenza burden on ICU was lower, ICU admittance may have been more accessible leading to earlier admissions and thus explaining the lead times of ICU over primary care. However, of the two seasons with coinciding ILI and SARI trends, one had high numbers (2014/2015) and the other had low numbers (2009/2010) of SARI associated with influenza.</p><p>The NICE registry was set up for benchmarking and improving ICU quality. It provides a wealth of data that have potential for additional use. Our results show that it could have additional value for understanding the severity of influenza epidemics. On retrospective data, end‐of‐season estimates can be made as we have done in our current study. Would the data flow be transferred to a more real‐time system—which the registry is aiming to do—it could be used for prospective monitoring of SARI. This would complement the weekly ILI surveillance in primary care and help fill our current knowledge gap on severe influenza complications. Such knowledge is crucial for prevention and response and for estimating the burden and societal cost of influenza epidemics or a pandemic.</p></sec><sec sec-type=\"COI-statement\" id=\"irv12759-sec-0017\"><title>CONFLICT OF INTEREST</title><p>L van Asten, AL Luna Pinzon, J. van de Kassteele, G. Donker, DW de Lange, and W. van der Hoek: None to declare. DA Dongelmans is the chairman of the National Intensive Care Evaluation (NICE) registry. NF de Keizer is a board member of the National Intensive Care Evaluation (NICE) registry. NF de Keizer is an employee of the department of medical informatics of the Amsterdam University Medical Center; this department is responsible for processing, maintaining and analysing data of the NICE registry.</p></sec><sec id=\"irv12759-sec-0018\"><title>AUTHOR CONTRIBUTION</title><p>\n<bold>Liselotte van Asten:</bold> Conceptualization (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Writing‐original draft (lead). <bold>Angie Luna Pinzon:</bold> Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Writing‐review & editing (equal). <bold>Jan van de Kassteele:</bold> Methodology (equal); Writing‐review & editing (equal). <bold>Gé Donker:</bold> Writing‐review & editing (equal). <bold>Dylan de Lange:</bold> Writing‐review & editing (equal). <bold>Dave A. Dongelmans:</bold> Writing‐review & editing (equal). <bold>Nicolette F. de Keizer:</bold> Writing‐review & editing (equal). <bold>Wim van der Hoek:</bold> Conceptualization (equal); Writing‐review & editing (equal).</p></sec></body><back><ack id=\"irv12759-sec-0016\"><title>ACKNOWLEDGEMENTS</title><p>We thank Eric van der Zwan for preparing the aggregated NICE data set and for support with data management, and Jeroen Alblas for support with data management. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"iso-abbrev\">Influenza Other Respir Viruses</journal-id><journal-id journal-id-type=\"doi\">10.1111/(ISSN)1750-2659</journal-id><journal-id journal-id-type=\"publisher-id\">IRV</journal-id><journal-title-group><journal-title>Influenza and Other Respiratory Viruses</journal-title></journal-title-group><issn pub-type=\"ppub\">1750-2640</issn><issn pub-type=\"epub\">1750-2659</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32374511</article-id><article-id pub-id-type=\"pmc\">PMC7431651</article-id><article-id pub-id-type=\"doi\">10.1111/irv.12750</article-id><article-id pub-id-type=\"publisher-id\">IRV12750</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Original Article</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Original Articles</subject></subj-group></article-categories><title-group><article-title>Are healthcare workers more likely than the general population to consult in primary care for an influenza‐like illness? Results from a case‐control study</article-title><alt-title alt-title-type=\"left-running-head\">PEYTREMANN et al.</alt-title></title-group><contrib-group><contrib id=\"irv12750-cr-0001\" contrib-type=\"author\"><name><surname>Peytremann</surname><given-names>Arnaud</given-names></name><xref ref-type=\"aff\" rid=\"irv12750-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12750-cr-0002\" contrib-type=\"author\"><name><surname>Senn</surname><given-names>Nicolas</given-names></name><xref ref-type=\"aff\" rid=\"irv12750-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"irv12750-cr-0003\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Mueller</surname><given-names>Yolanda</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-8861-4538</contrib-id><xref ref-type=\"aff\" rid=\"irv12750-aff-0001\">\n<sup>1</sup>\n</xref><address><email>Yolanda.mueller@unisante.ch</email></address></contrib></contrib-group><aff id=\"irv12750-aff-0001\">\n<label><sup>1</sup></label>\n<named-content content-type=\"organisation-division\">Center for Primary Care and Public Health (Unisanté)</named-content>\n<institution>University of Lausanne</institution>\n<city>Lausanne</city>\n<country country=\"CH\">Switzerland</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label><bold>Correspondence</bold><break/>\nYolanda Mueller, Center for Primary Care and Public Health (Unisanté), University of Lausanne, Switzerland.<break/>\nEmail: <email>Yolanda.mueller@unisante.ch</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>06</day><month>5</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>9</month><year>2020</year></pub-date><volume>14</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1111/irv.v14.5</issue-id><fpage>524</fpage><lpage>529</lpage><history><date date-type=\"received\"><day>24</day><month>2</month><year>2020</year></date><date date-type=\"rev-recd\"><day>14</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>16</day><month>4</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 John Wiley & Sons Ltd <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Influenza and Other Respiratory Viruses</italic> Published by John Wiley & Sons Ltd.</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:IRV-14-524.pdf\"/><abstract id=\"irv12750-abs-0001\"><title>Abstract</title><sec id=\"irv12750-sec-0001\"><title>Background</title><p>Healthcare workers are at increased risk of contracting influenza. However, existing studies do not differentiate professional categories or domains of the healthcare system that are most at risk.</p></sec><sec id=\"irv12750-sec-0002\"><title>Methods</title><p>This case‐control study compared proportions of patients with professional activity in the healthcare system between cases consulting their primary care physician for an influenza‐like illness (ILI) and controls from the general patient population of the same practices of the Swiss sentinel network. Influenza was confirmed by rRT‐PCR in a subset of practices. Analysis used a mixed logistic regression model, including age and sex as potential confounders.</p></sec><sec id=\"irv12750-sec-0003\"><title>Results</title><p>During the 2018/2019 influenza surveillance season, out of 4287 ILI cases and 28 561 controls reported in 168 practices, 235 (5.5%), respectively 872 (3.1%), were active in the healthcare system. After adjustment, being active in health care increased the odds of consulting for an ILI (OR 1.66, 95% CI 1.40‐1.97). The association was strongest for physicians and nursing aides. In terms of work setting, odds of consulting for ILI were increased for professionals of almost all healthcare settings except home‐based care.</p></sec><sec id=\"irv12750-sec-0004\"><title>Conclusion</title><p>Individuals active in the healthcare system were more likely to consult their primary care physician for an influenza‐like illness than for another reason, compared with individuals not active in the healthcare system. These results warrant further efforts to understand influenza transmission in the healthcare system at large.</p></sec></abstract><kwd-group><kwd id=\"irv12750-kwd-0001\">epidemiology</kwd><kwd id=\"irv12750-kwd-0002\">human</kwd><kwd id=\"irv12750-kwd-0003\">influenza</kwd><kwd id=\"irv12750-kwd-0004\">occupations</kwd><kwd id=\"irv12750-kwd-0005\">prevention and control</kwd><kwd id=\"irv12750-kwd-0006\">primary health care</kwd></kwd-group><funding-group><award-group id=\"funding-0001\"><funding-source>Swiss Society of General Internal Medicine</funding-source></award-group></funding-group><counts><fig-count count=\"0\"/><table-count count=\"3\"/><page-count count=\"6\"/><word-count count=\"4805\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>September 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.6 mode:remove_FC converted:18.08.2020</meta-value></custom-meta></custom-meta-group></article-meta><notes><p content-type=\"self-citation\">\n<mixed-citation publication-type=\"journal\" id=\"irv12750-cit-1001\">\n<string-name>\n<surname>Peytremann</surname>\n<given-names>A</given-names>\n</string-name>, <string-name>\n<surname>Senn</surname>\n<given-names>N</given-names>\n</string-name>, <string-name>\n<surname>Mueller</surname>\n<given-names>Y</given-names>\n</string-name>. <article-title>Are healthcare workers more likely than the general population to consult in primary care for an influenza‐like illness? Results from a case‐control study</article-title>. <source xml:lang=\"en\">Influenza Other Respi Viruses</source>. <year>2020</year>;<volume>14</volume>:<fpage>524</fpage>–<lpage>529</lpage>. <pub-id pub-id-type=\"doi\">10.1111/irv.12750</pub-id>\n</mixed-citation>\n</p><fn-group id=\"irv12750-ntgp-0001\"><fn id=\"irv12750-note-0001\"><p>The peer review history for this article is available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://publons.com/publon/10.1111/irv.12750\">https://publons.com/publon/10.1111/irv.12750</ext-link>\n</p></fn></fn-group></notes></front><body id=\"irv12750-body-0001\"><sec id=\"irv12750-sec-0005\"><label>1</label><title>INTRODUCTION</title><p>Healthcare workers are at increased risk of influenza infection compared to non‐HCW.<xref rid=\"irv12750-bib-0001\" ref-type=\"ref\">\n<sup>1</sup>\n</xref>, <xref rid=\"irv12750-bib-0002\" ref-type=\"ref\">\n<sup>2</sup>\n</xref>, <xref rid=\"irv12750-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref> For example, influenza‐like illness (ILI) among Italian medical residents peaks earlier compared to the general population.<xref rid=\"irv12750-bib-0003\" ref-type=\"ref\">\n<sup>3</sup>\n</xref> General practitioners (GPs) in particular have been shown to have high levels of basic immunity to influenza, probably resulting from frequent contacts with influenza viruses in the past.<xref rid=\"irv12750-bib-0004\" ref-type=\"ref\">\n<sup>4</sup>\n</xref>\n</p><p>Already during the 1918 influenza pandemic, social class based on occupation had an impact on mortality.<xref rid=\"irv12750-bib-0005\" ref-type=\"ref\">\n<sup>5</sup>\n</xref> Occupation of influenza cases has been explored in more details during the 2009 H1N1 pandemic. In a study conducted in four American states, the proportion of healthcare workers was three times higher among laboratory‐confirmed influenza cases compared to its proportion in the general workforce.<xref rid=\"irv12750-bib-0006\" ref-type=\"ref\">\n<sup>6</sup>\n</xref> In a Spanish matched case‐control study, being a healthcare worker was associated with consulting as an outpatient for influenza.<xref rid=\"irv12750-bib-0007\" ref-type=\"ref\">\n<sup>7</sup>\n</xref>\n</p><p>However, existing studies of influenza risk based on occupation do not differentiate between the different settings of the healthcare system, such as hospitals, residential homes, physician practices. Direct transmission from healthcare workers has been documented,<xref rid=\"irv12750-bib-0008\" ref-type=\"ref\">\n<sup>8</sup>\n</xref> but whether patients acquire influenza mostly from other patients or from healthcare workers is still debated.<xref rid=\"irv12750-bib-0009\" ref-type=\"ref\">\n<sup>9</sup>\n</xref>, <xref rid=\"irv12750-bib-0010\" ref-type=\"ref\">\n<sup>10</sup>\n</xref>\n</p><p>Most of the work on healthcare‐associated influenza has been conducted in hospitals<xref rid=\"irv12750-bib-0011\" ref-type=\"ref\">\n<sup>11</sup>\n</xref> or long‐term care institutions. In hospitals, a significant proportion of influenza infections is acquired during admission.<xref rid=\"irv12750-bib-0012\" ref-type=\"ref\">\n<sup>12</sup>\n</xref> Patients visiting the emergency department for another reason than influenza during the influenza season have an increased risk of contracting influenza compared with community controls.<xref rid=\"irv12750-bib-0013\" ref-type=\"ref\">\n<sup>13</sup>\n</xref> In an outpatient setting, one retrospective cohort study among children aged two to five years old reported an increased risk of 36% (incidence rate ratio 1.36; 95% CI 1.22‐1.52) of presenting for an ILI visit in the 8 days after a non‐ILI visit to a pediatric clinic.<xref rid=\"irv12750-bib-0014\" ref-type=\"ref\">\n<sup>14</sup>\n</xref>\n</p><p>Our research question was whether being professionally active in the healthcare system (exposure) increases the risk of influenza infection, assessed by consulting a primary care practitioner for influenza‐like illness (outcome). We assumed that healthcare workers would mostly consult their primary care practitioner in case of influenza‐like illness. Therefore, we estimated the association between seeking consultation for an influenza‐like illness or having confirmed influenza, and being professionally active in the healthcare system, differentiating by type of profession and work setting.</p></sec><sec sec-type=\"materials-and-methods\" id=\"irv12750-sec-0006\"><label>2</label><title>MATERIALS AND METHODS</title><p>This unmatched case‐control study was conducted within the Swiss national sentinel surveillance system (Sentinella) during the 2018‐2019 influenza surveillance season. Sentinella is a network of approximately 165 primary care physicians (general internal medicine specialist or pediatricians), maintained by the Swiss Federal Office of Public Health (SFOPH) since 1986 for the purpose of influenza surveillance. During the influenza surveillance season (epidemiological week 40 to 16), participating physicians declare on a weekly basis every case of influenza‐like illness, defined as a history of fever (>38°C), generally of abrupt onset, and presence of either sore throat or cough. Nasopharyngeal swabs are performed in a subset of practices, allowing identification of circulating strains in Switzerland by the National Reference Center of Influenza. Confirmed influenza cases are defined as ILI cases with positive nasopharyngeal swabs by rRT‐PCR. In order to obtain a denominator for ILI incidence, physicians report the daily number of patient contacts and, twice a year for a duration of two weeks, detailed patient‐contact information with documentation of age and sex. ILI incidence by number of inhabitants is extrapolated by triangulating the proportion of ILI per number of patient contacts with the number of consultation per individual, obtained from national statistics such as the Swiss Health Survey.</p><p>We used two different sets of cases in our study. First, cases were defined as all ILI cases, reported to Sentinella during the influenza surveillance period (October 2018 [week 40] to April 2019 [week 16]). In a second analysis, we restricted cases to confirmed influenza cases by rRT‐PCR. As controls, we used the patient contacts reported by physicians during week 11 and 12, 2019, minus ILI cases (patients with same sex and year of birth declared both as case and control within same week in same practice). Both for cases and controls, we added to the existing data collection a question about professional activity in the healthcare system, understood as the part of the health system providing health care to patients. Professional activity corresponded to the International Labour Office definition of occupied labor force. If professionally active, we further enquired about type of profession and work setting. Type of profession was categorized based on the International standard classification of occupations (ISCO version 08), simplified in eight categories relevant for the healthcare system, and based on the type of contact with patients: (1) physicians; (2) nurses; (3) nursing aides/personal care workers; (4) medical assistant or paramedics; (5) physical, occupational, or psycho‐therapist; (6) laboratory or radiology technician, pharmacy assistant; (7) pharmacist or dentist; (8) administrative personal; (9) other; and (10) unknown. Work setting was categorized as: (1) private practices; (2) hospital; (3) pharmacy; (4) at‐home care; (5) nursing home; (6) reeducation center; (7) dentist or therapist practices; (8) radiology or laboratory center; (9) office space; (10) other; and (11) unknown. In case of missing information about professional activity, data of people born before 1954 and after 2003 were recoded as “not active,” and the remaining “missing” recoded as unknown.</p><p>For both cases and controls, the following variables were obtained from the routinely collected Sentinella: week, age, sex. In addition, for ILI cases we collected whether the swab was sent to the reference laboratory, and rRT‐PCR result. At practice level, we obtained the region and total number patient‐physician contacts during influenza surveillance season. The project made full use of the quality assurance system of Sentinella. Declaring GPs received instructions about data collection, with main messages reinforced by regular Newsletters. Predefined checks in electronic data entry diminished the risk of data entry errors. The Sentinella program Commission, consisting of regional representatives of declaring physicians, Swiss family medicine institutes, and the SFOPH, reviewed the study protocol and data collection forms.</p><p>Analysis of this case‐control study was based on a mixed logistic regression model, taking into account the clustering by practice by including a random intercept. We considered age and sex as potential confounders, because age was associated with both types of profession and ILI incidence, and sex was associated with types of profession, as well as possibly associated with ILI incidence and health‐seeking behavior in case of ILI. Profession and work setting of patients active in the healthcare system were compared to those not active, excluding those with unknown or missing activity information (complete case analysis). If active, other professions with <5% of total and unknown profession were regrouped into a single category. If active, but profession, respectively, work setting, was missing, it was recoded as unknown. For confirmed influenza cases, the dataset was restricted to practices where swabs were performed. Separate models were used for activity in the healthcare system in general, categories of professional activity if active in the healthcare system, and categories of work settings, because of collinearity between these variables. In a sensitivity analysis, we repeated the model for activity in the healthcare system, setting all missing data to “inactive,” To examine possible over‐ or underrepresentation of some professions among controls, we compared the proportion of individuals active in each professional category among subjects aged 15‐64 years old with national occupational statistics.<xref rid=\"irv12750-bib-0015\" ref-type=\"ref\">\n<sup>15</sup>\n</xref> We used the Stata 15 software for all analyses.</p><p>The investigators had access only to anonymized data. Neither additional health‐related data nor biological material was collected specifically for the study. As such, the project was not under the scope of the Swiss human research law (LRH) and did not require formal ethical review.</p></sec><sec sec-type=\"results\" id=\"irv12750-sec-0007\"><label>3</label><title>RESULTS</title><p>During the 2018/2019 influenza surveillance season, there were 4287 ILI cases reported from 168 practices, out of which 346 were confirmed for influenza from the 79 practices swabbing ILI cases. During weeks 11 and 12, 28 561 controls were recorded, reduced to 15 463 after restricting the dataset to practices doing swabs.</p><p>The median age for the ILI cases was 33 (12‐52, 95% CI), compared with 52 (27‐71, 95% CI) for controls (Table <xref rid=\"irv12750-tbl-0001\" ref-type=\"table\">1</xref>). There were slightly more females among controls than among ILI cases (52.7% vs 50.2%, <italic>P</italic> = .001). Of the total, ILI cases 235 (5.5%) were working in the healthcare system, compared to 872 (3.1%) for controls. Professional activity was unknown for 546 (12.7%) ILI cases and 2865 (10.0%) of controls.</p><table-wrap id=\"irv12750-tbl-0001\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Sample characteristics of influenza‐like illness (ILI), respectively, rRT‐PCR‐confirmed influenza cases, and controls representing the general patient population of primary care practices of the Swiss sentinel network Sentinella, 2018‐2019 influenza surveillance season</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"> </th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cases (ILI)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Controls</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cases (confirmed influenza)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Controls</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">N observation</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">N = 4287</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">N = 28 561</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">N = 346</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">N = 15 463</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Median age in years (IQR)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">33 (12‐52)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">52 (26‐71)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">35 (15‐55)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">54 (25‐72)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">N female (%)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2147 (50.1)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">15 047 (52.7)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">173 (50.0)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">8174 (52.9)</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Active in the healthcare system<xref ref-type=\"fn\" rid=\"irv12750-note-0500\">*</xref>\n</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">235 (5.5)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">872 (3.1)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">23 (6.7)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">434 (2.8)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3506 (81.8)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">24 824 (86.9)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">298 (86.1)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">13 478 (87.2)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Unknown</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">546 (12.7)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2865 (10.0)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">25 (7.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 551 (10.0)</td></tr></tbody></table><table-wrap-foot id=\"irv12750-ntgp-0500\"><title>Note</title><fn id=\"irv12750-note-0500\"><label>*</label><p>Missing activity and born before 1954 and after 2003 recoded as “not active”; otherwise recoded as unknown.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>Being active in the healthcare system was associated with increased odds of consulting for an ILI (crude OR 1.91, 95% CI 1.65‐2.21; Table <xref rid=\"irv12750-tbl-0002\" ref-type=\"table\">2</xref>). The associations persisted after adjustment for age, sex, and inclusion of a random intercept for practice (Adj OR 1.66, 95% CI 1.40‐1.97). The association was strongest for the physicians (Adj OR 2.85, 95% CI 1.47‐5.53) and nursing aides (Adj OR 2.01, 95% CI 1.42‐2.85). Odds were also increased for administrative staff and for other or unknown profession. After adjustment, we found no increased odds for nurses nor for medical assistant and paramedical staff.</p><table-wrap id=\"irv12750-tbl-0002\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Association between being active in the healthcare system and consulting for an influenza‐like illness (ILI)</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"2\" valign=\"top\" colspan=\"1\"> </th><th align=\"left\" style=\"border-bottom:solid 1px #000000\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Cases (ILI)</p>\n<p>N = 3741</p>\n</th><th align=\"left\" style=\"border-bottom:solid 1px #000000\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Controls (ILI)</p>\n<p>N = 25 696</p>\n</th><th align=\"left\" style=\"border-bottom:solid 1px #000000\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Crude OR</p>\n<p>(95% CI)</p>\n</th><th align=\"left\" style=\"border-bottom:solid 1px #000000\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Adjusted OR</p>\n<p>(95% CI)</p>\n</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n (%)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n (%)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"> </th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"> </th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Not active in the healthcare system</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3506 (93.7)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">24 824 (96.6)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Active in the healthcare system</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">235 (6.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">872 (3.4)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.91 (1.65‐2.21)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.66 (1.40‐1.97)</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Profession if active in the healthcare system</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Nurse</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">61 (1.6)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">259 (1.0)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.67 (1.26‐2.21)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.28 (0.95‐1.74)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Nursing aide</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">54 (1.4)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">156 (0.6)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.45 (1.79‐3.35)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.01 (1.42‐2.85)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Medical assistants/ paramedics</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">24 (0.6)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">66 (0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.57 (1.61‐4.11)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.46 (0.88‐2.44)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Administrative staff</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">17 (0.5)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">65 (0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.85 (1.08‐3.16)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.84 (1.02‐3.30)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Physician</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">14 (0.4)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">42 (0.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.36 (1.29‐4.33)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.85 (1.47‐5.53)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Occupational, physical therapy, dietitian</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7 (0.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">52 (0.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.95 (0.43‐2.10)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0.96 (0.41‐2.24)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Laboratory and radiology technicians, pharmacy assistants</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">8 (0.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">14 (0.1)</td><td align=\"char\" char=\"(\" rowspan=\"4\" colspan=\"1\">1.77 (1.32‐2.36)</td><td align=\"char\" char=\"(\" rowspan=\"4\" colspan=\"1\">1.95 (1.40‐2.72)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Pharmacist, dentist</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (0.1)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">14 (0.1)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Other</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">31 (0.8)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">101 (0.4)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Unknown</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">17 (0.5)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">74 (0.3)</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Work setting if active in the healthcare system</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Nursing home</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">76 (2.0)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">198 (0.8)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.72 (2.08‐3.55)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.06 (1.53‐2.78)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Hospital</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">51 (1.4)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">187 (0.7)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.93 (1.41‐2.64)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.66 (1.18‐2.32)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Private practice</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">31 (0.8)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">80 (0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.74 (1.81‐4.16)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.26 (1.43‐3.58)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Home‐based care</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">13 (0.4)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">56 (0.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.64 (0.90‐3.01)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.53 (0.79‐2.94)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Administration</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7 (0.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">8 (0.0)</td><td align=\"char\" char=\"(\" rowspan=\"7\" colspan=\"1\">1.29 (0.99‐1.69)</td><td align=\"char\" char=\"(\" rowspan=\"7\" colspan=\"1\">1.24 (0.92‐1.67)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Pharmacy</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5 (0.1)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">18 (0.1)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Dentist, physical, occupational therapy</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5 (0.1)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">31 (0.1)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Radiology, laboratory</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (0.1)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">18 (0.1)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Rehabilitation</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (0.0)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">19 (0.1)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Other</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">15 (0.4)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">90 (0.4)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Unknown</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">29 (0.8)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">167 (0.6)</td></tr></tbody></table><table-wrap-foot id=\"irv12750-ntgp-0002\"><title>Note</title><fn id=\"irv12750-note-0002\"><p>Missing activity excluded. Model adjusted for age (linear and quadratic), sex and cluster effect by practice. Unknown or missing activity excluded.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>In terms of work setting, we found increased odds of consulting for ILI for professionals of almost all healthcare settings except home‐based care. The association was strongest for those working in private practices (Adj OR 2.26, 95% CI 1.43‐3.58) and nursing homes (Adj OR 2.06, 1.53‐2.78). It was also increased, to a lesser degree, for professionals working in hospitals. It was not significantly increased for workers in home‐based care and other healthcare settings.</p><p>Results for PCR‐confirmed influenza, although based on a limited number of cases, were consistent with results obtained for ILI overall (Table <xref rid=\"irv12750-tbl-0003\" ref-type=\"table\">3</xref>). The odds of consulting for a confirmed influenza were particularly high among physicians (Adj OR 6.83, 95% CI 1.78‐36.1) and nursing aides (Adj OR 2.32, 95% CI 1.02‐5.29), and for staff active in private practices (Adj OR 4.53, 95% CI 1.65‐12.41), hospitals (Adj OR 2.56, 95% CI 1.05‐6.23), and nursing homes (Adj OR 2.44, 95% CI 1.08‐5.53). No significant associations were found between confirmed influenza and being an administrative staff or a staff active in another or unknown profession.</p><table-wrap id=\"irv12750-tbl-0003\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><p>Association between being active in the healthcare system and consulting for PCR‐confirmed influenza</p></caption><table frame=\"hsides\" rules=\"groups\"><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><col style=\"border-right:solid 1px #000000\" span=\"1\"/><thead valign=\"top\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"2\" valign=\"top\" colspan=\"1\"> </th><th align=\"left\" style=\"border-bottom:solid 1px #000000\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Cases (confirmed influenza)</p>\n<p>N = 321</p>\n</th><th align=\"left\" style=\"border-bottom:solid 1px #000000\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Controls (confirmed influenza)</p>\n<p>N = 13 912</p>\n</th><th align=\"left\" style=\"border-bottom:solid 1px #000000\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Crude OR</p>\n<p>(95% CI)</p>\n</th><th align=\"left\" style=\"border-bottom:solid 1px #000000\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Adjusted OR</p>\n<p>(95% CI)</p>\n</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n (%)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n (%)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"> </th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"> </th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Not active in the healthcare system</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">298 (92.8)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">13 478 (96.9)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Active in the healthcare system</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">23 (7.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">434 (3.1)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.40 (1.55‐3.70)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.81 (1.13‐2.90)</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Profession if active in the healthcare system</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Nurse</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4 (1.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">124 (0.9)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.46 (0.54‐3.97)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.15 (0.41‐3.23)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Nursing aide</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7 (2.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">100 (0.7)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.17 (1.46‐6.87)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.32 (1.02‐5.29)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Medical assistants/paramedics</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2 (0.6)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">37 (0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.44 (0.59‐10.19)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.40 (0.32‐6.24)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Administrative staff</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">40 (0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.13 (10.15‐8.25)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1.24 (0.16‐9.55)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Physician</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3 (0.9)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">20 (0.1)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6.78 (2.00‐23.0)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6.83 (1.78‐36.1)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Occupational, physical therapy, dietician</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">29 (0.2)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Laboratory and radiology technicians, pharmacy assistants</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">22 (0.2)</td><td align=\"char\" char=\"(\" rowspan=\"4\" colspan=\"1\">3.23 (1.40‐7.45)</td><td align=\"char\" char=\"(\" rowspan=\"4\" colspan=\"1\">0.93 (0.93‐5.46)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Pharmacist, dentist</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6 (0.0)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Other</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5 (1.6)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">39 (0.3)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Unknown</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">17 (0.1)</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Work setting if active in the healthcare system</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Nursing home</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7 (2.2)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">114 (0.8)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.78 (1.28‐6.01)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.44 (1.08‐5.53)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Hospital</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6 (1.9)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">88 (0.6)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">3.08 (1.34‐7.11)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">2.56 (1.05‐6.23)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Private practice</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">5 (1.6)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">46 (0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4.92 (1.94‐12.5)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">4.53 (1.65‐12.41)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Home‐based care</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">32 (0.2)</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"char\" char=\".\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Administration</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7 (0.1)</td><td align=\"char\" char=\"(\" rowspan=\"7\" colspan=\"1\">1.47 (0.60‐3.60)</td><td align=\"char\" char=\"(\" rowspan=\"7\" colspan=\"1\">0.89 (0.35‐2.25)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Pharmacy</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">6 (0.0)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Dentist, physical, occupational therapy</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">17 (0.1)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Radiology, laboratory</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">7 (0.1)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Rehabilitation</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">10 (0.1)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Other</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">41 (0.3)</td></tr><tr><td align=\"left\" style=\"padding-left:10%\" rowspan=\"1\" colspan=\"1\">Unknown</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">1 (0.3)</td><td align=\"char\" char=\"(\" rowspan=\"1\" colspan=\"1\">66 (0.5)</td></tr></tbody></table><table-wrap-foot id=\"irv12750-ntgp-0003\"><title>Note</title><fn id=\"irv12750-note-0003\"><p>Missing activity excluded. Model adjusted for age (linear and quadratic), sex and cluster effect by practice. Unknown or missing activity excluded.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><p>In sensitivity analyses, we considered all individuals with unknown or missing activity in the healthcare system as not active instead of excluding them from the logistic regression models (Table <xref rid=\"irv12750-sup-0001\" ref-type=\"supplementary-material\">S1</xref>). All associations found in the main analysis were confirmed. Associations were also consistent when restricting the data to cases and controls to individuals aged 15‐64 years old (Table <xref rid=\"irv12750-sup-0001\" ref-type=\"supplementary-material\">S2</xref>). Finally, to get a sense of the healthy worker bias present in our data, we compared the proportions of individuals working in different categories or work settings among our control population with available national statistics (Table <xref rid=\"irv12750-sup-0001\" ref-type=\"supplementary-material\">S3</xref>). With the exception of nurses, all professional categories were rather underrepresented among controls. Comparing disease severity of ILI between healthcare workers (HCW) and non‐healthcare workers, there were 1.8% (4/218) clinical pneumonia among HCW, compared with 3.9% among non‐HCW (126/3149), a difference that was not significant even after adjustment for risk of complication and age in a logistic regression model (Adj OR for pneumonia among HCW 0.56, 95% CI 0.20‐1.54).</p></sec><sec sec-type=\"discussion\" id=\"irv12750-sec-0008\"><label>4</label><title>DISCUSSION</title><p>In this study, individuals active in the healthcare sector were more likely to consult their primary care physician for an influenza‐like illness, respectively, confirmed influenza, than for another reason. In terms of professional categories, the association was particularly strong for physicians and nursing aides. Surprisingly, being active either as an administrative staff or as any other or unknown profession in the healthcare system was also associated with an increased risk of consulting for an ILI. This could be due both to a higher risk of infection and to more sensitization in healthcare settings to abstain from work in case of ILI symptoms. In terms of work settings, private practices and nursing home particularly stood out, followed by hospitals.</p><p>The main limitation of this work is that health‐seeking behavior of health professional in case of ILI may differ from the general patient population. However, we have few reasons to believe that health professionals would consult more frequently for ILI, a rather mild illness in the active population, than for other health issues, which would have led to overestimation of the association. On the contrary, previous studies have shown that health professionals tend to minimize ILI symptoms and continue to work despite recommendations against this.<xref rid=\"irv12750-bib-0016\" ref-type=\"ref\">\n<sup>16</sup>\n</xref>, <xref rid=\"irv12750-bib-0017\" ref-type=\"ref\">\n<sup>17</sup>\n</xref>, <xref rid=\"irv12750-bib-0018\" ref-type=\"ref\">\n<sup>18</sup>\n</xref> There were not significantly less patients presenting with clinical pneumonia among healthcare staff. In addition, we recognize that it would have been preferable to sample controls from the patient population over the same time‐period as the cases, but this was not considered feasible within the sentinel set‐up, and would have probably resulted in many more missing data. By contrast with other professional categories, we found no association between being active as a nurse and consulting for ILI. However, nurses were also more represented among controls than other healthcare worker categories, which could have biased the result toward the null.</p><p>This is the first study to explore the question of healthcare setting‐associated influenza transmission from a primary care standpoint. Individuals active in the healthcare system appear to be overrepresented both among ILI and among confirmed influenza cases. The observed differences between professions and work settings could reflect different contact intensity between professionals and influenza‐infected patients, as well as differences in adhesion to infection prevention and control measures.</p><p>However, our results suggest that other professionals working in health care, for example administrative staff, may also be at increased risk of influenza. One could argue that individuals not in direct contact with patients do not pose a particular hazard for vulnerable patients. However, they may contribute to the overall burden of circulating viruses. Besides, these professionals may also in contact with patients, for example when working at reception desks. Decreasing circulation of influenza viruses in healthcare settings is likely to be beneficial to patients. Also, for their individual health, staff should be informed of their increased risk of influenza if this finding is confirmed.</p><p>Currently, apart from influenza vaccination, most specific influenza control measures such as mask wearing focus on droplet transmission. More attention to standard precautions, including hand hygiene, surface disinfection, and ventilation, may be required to prevent influenza in the healthcare workforce at large. Our results suggest that private practices and nursing homes could constitute weak spots of infection control. While efforts to increase staff vaccination coverage should be sustained, specific infection control recommendations targeting these settings should be developed, taking into account their specificities. To guide such recommendations, further studies on transmission modes and evidence on effective interventions should be directly generated in the relevant settings, and not extrapolated from hospitals. For example, a prospective cohort study among staff of primary care practices should be conducted to estimate infection rates without being confounded by differences in health‐seeking behavior.</p><p>While sentinel practices do not constitute a representative sample of all primary care practices, we have no reason to believe that Sentinella practices would be more or less likely to have health professionals among their patients than other private practices. Also, the Swiss sentinel network covers all six regions of the country, and the demographic structure of the adult patient population is overall similar to the one of Swiss practices.<xref rid=\"irv12750-bib-0019\" ref-type=\"ref\">\n<sup>19</sup>\n</xref> While these results cannot be used to extrapolate the proportions of professionals working in the healthcare system, we believe that the reported associations are valid. Still, we cannot exclude the possibility that health professionals were more likely to consult their physician for ILI, knowing that their physician was part of Sentinella. Overall, these findings certainly justify further attention to prevention of influenza transmission in the health system, particularly outside hospitals.</p></sec><sec sec-type=\"supplementary-material\"><title>Supporting information</title><supplementary-material content-type=\"local-data\" id=\"irv12750-sup-0001\"><caption><p>Table S1‐S3</p></caption><media xlink:href=\"IRV-14-524-s001.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"irv12750-sec-0009\"><title>ACKNOWLEDGEMENTS</title><p>We acknowledge the contributions of Damir Perisa and Raphael Rytz from the Federal Office of Public Health in communicating the study information to the Sentinella members and transmitting the surveillance data to the investigators. We thank the members of the Sentinella Program Commission for reviewing the study protocol, and physicians and staff of the Sentinella network for collecting the data. 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pub-id-type=\"doi\">10.1029/2020GH000247</article-id><article-id pub-id-type=\"publisher-id\">GH2178</article-id><article-id pub-id-type=\"other\">2020GH000247</article-id><article-categories><subj-group subj-group-type=\"article-subject-classification\"><subject>Atmospheric Composition and Structure</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Pollution: Urban and Regional</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Aerosols and Particles</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Biogeosciences</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Pollution: Urban, Regional and Global</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Geochemistry</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Instruments and Techniques</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Composition of Aerosols and Dust Particles</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Oceanography: General</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Marine Pollution</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Natural Hazards</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Megacities and Urban Environment</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Oceanography: Biological and Chemical</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Aerosols</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Paleoceanography</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Aerosols</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Geographic Location</subject></subj-group><subj-group subj-group-type=\"article-subject-classification\"><subject>Africa</subject></subj-group><subj-group subj-group-type=\"overline\"><subject>Research Article</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Research Articles</subject></subj-group></article-categories><title-group><article-title>Air Quality Impacts at an E‐Waste Site in Ghana Using Flexible, Moderate‐Cost and Quality‐Assured Measurements</article-title><alt-title alt-title-type=\"left-running-head\">Kwarteng et al.</alt-title></title-group><contrib-group><contrib id=\"gh2178-cr-0001\" contrib-type=\"author\"><name><surname>Kwarteng</surname><given-names>Lawrencia</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-3954-8757</contrib-id><xref ref-type=\"aff\" rid=\"gh2178-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"gh2178-cr-0002\" contrib-type=\"author\"><name><surname>Baiden</surname><given-names>Emmanuel Acquah</given-names></name><xref ref-type=\"aff\" rid=\"gh2178-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"gh2178-cr-0003\" contrib-type=\"author\"><name><surname>Fobil</surname><given-names>Julius</given-names></name><xref ref-type=\"aff\" rid=\"gh2178-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"gh2178-cr-0004\" contrib-type=\"author\"><name><surname>Arko‐Mensah</surname><given-names>John</given-names></name><xref ref-type=\"aff\" rid=\"gh2178-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"gh2178-cr-0005\" contrib-type=\"author\"><name><surname>Robins</surname><given-names>Thomas</given-names></name><xref ref-type=\"aff\" rid=\"gh2178-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"gh2178-cr-0006\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Batterman</surname><given-names>Stuart</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-9894-5325</contrib-id><xref ref-type=\"aff\" rid=\"gh2178-aff-0002\">\n<sup>2</sup>\n</xref><address><email>stuartb@umich.edu</email></address></contrib></contrib-group><aff id=\"gh2178-aff-0001\">\n<label><sup>1</sup></label>\n<named-content content-type=\"organisation-division\">Department of Biological, Environmental and Occupational Health Sciences</named-content>\n<institution>University of Ghana</institution>\n<city>Accra</city>\n<country country=\"GH\">Ghana</country>\n</aff><aff id=\"gh2178-aff-0002\">\n<label><sup>2</sup></label>\n<named-content content-type=\"organisation-division\">Environmental Health Sciences</named-content>\n<institution>University of Michigan</institution>\n<city>Ann Arbor</city>\n<named-content content-type=\"country-part\">Michigan</named-content>\n<country country=\"US\">USA</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label><bold>Correspondence to:</bold><break/>\nS. Batterman,<break/><email>stuartb@umich.edu</email><break/></corresp></author-notes><pub-date pub-type=\"collection\"><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>01</day><month>8</month><year>2020</year></pub-date><volume>4</volume><issue>8</issue><issue-id pub-id-type=\"doi\">10.1002/gh2.v4.8</issue-id><elocation-id>e2020GH000247</elocation-id><history><date date-type=\"received\"><day>20</day><month>3</month><year>2020</year></date><date date-type=\"rev-recd\"><day>19</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>23</day><month>6</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> ©2020. American Geophysical Union. All Rights Reserved. <copyright-statement>--><copyright-statement content-type=\"article-copyright\">©2020. The Authors.</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:GH2-4-e2020GH000247.pdf\"/><abstract><title>Abstract</title><p>Air quality information is scarce in low‐ and middle‐income countries. This study describes the application of moderate cost approaches that can provide spatial and temporal information on concentrations of particulate matter (PM) needed to assess community and occupational exposures. We evaluated PM levels at the Agbogbloshie e‐waste and scrap yard site in Accra, Ghana, and at upwind and downwind locations, obtaining both optical and gravimetric measurements, local meteorological data and satellite aerosol optical depth. Due to overload issues, the gravimetric 24‐hr samplers were modified for periodic sampling and some optical data were screened for quality assurance. Exceptionally high concentrations (e.g., 1‐hr average PM<sub>10</sub> exceeding 2000 μg/m<sup>3</sup>) were sometimes encountered near combustion sources, including open fires at the e‐waste site and spoil piles. 24‐hr PM<sub>2.5</sub> levels averaged 31, 88 and 57 μg/m<sup>3</sup> at upwind, e‐waste and downwind sites, respectively, and PM<sub>10</sub> averaged 145, 214 and 190 μg/m<sup>3</sup>, considerably exceeding air quality standards. Upwind levels likely reflected biomass burning that is prevalent in the surrounding informal settlements; levels at the e‐waste and downwind sites also reflected contributions from biomass combustion and traffic. The highest PM levels occurred in evenings, influenced by diurnal changes in emission rates, atmospheric dispersion and wind direction shifts. We demonstrate that moderate cost instrumentation, with some modifications, appropriate data cleaning protocols, and attention to understanding local sources and background levels, can be used to characterize spatial and temporal variation in PM levels in urban and industrial areas.</p></abstract><abstract abstract-type=\"short\"><title>Key Points</title><p>\n<list list-type=\"bullet\" id=\"gh2178-list-0001\"><list-item id=\"gh2178-li-0001\"><p>Ambient particulate matter was monitored onsite, upwind and downwind of an e‐waste site in Ghana using gravimetric and optical measurements</p></list-item><list-item id=\"gh2178-li-0002\"><p>E‐waste site emissions increased 24‐hr PM<sub>2.5</sub> levels by 57 μg/m<sup>3</sup> over upwind levels of 31 μg/m<sup>3</sup>, and some exceptionally high levels were measured</p></list-item><list-item id=\"gh2178-li-0003\"><p>Moderate cost methods can measure air quality and source impacts given attention to study design, sampler performance and local influences</p></list-item></list>\n</p></abstract><kwd-group kwd-group-type=\"author-generated\"><kwd id=\"gh2178-kwd-0001\">Air pollution</kwd><kwd id=\"gh2178-kwd-0002\">particulate matter</kwd><kwd id=\"gh2178-kwd-0003\">e‐waste</kwd><kwd id=\"gh2178-kwd-0004\">fires</kwd><kwd id=\"gh2178-kwd-0005\">monitoring</kwd></kwd-group><funding-group><award-group id=\"funding-0001\"><funding-source>US NIH</funding-source><award-id>1U2RTW010110‐01</award-id><award-id>5U01TW010101</award-id><award-id>P30ES017885</award-id></award-group></funding-group><counts><fig-count count=\"6\"/><table-count count=\"1\"/><page-count count=\"17\"/><word-count count=\"8907\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>August 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.6 mode:remove_FC converted:18.08.2020</meta-value></custom-meta></custom-meta-group></article-meta><notes><p content-type=\"self-citation\">\n<mixed-citation publication-type=\"journal\" id=\"gh2178-cit-9001\">\n<string-name>\n<surname>Kwarteng</surname>, <given-names>L.</given-names>\n</string-name>, <string-name>\n<surname>Baiden</surname>, <given-names>E. A.</given-names>\n</string-name>, <string-name>\n<surname>Fobil</surname>, <given-names>J.</given-names>\n</string-name>, <string-name>\n<surname>Arko‐Mensah</surname>, <given-names>J.</given-names>\n</string-name>, <string-name>\n<surname>Robins</surname>, <given-names>T.</given-names>\n</string-name>, & <string-name>\n<surname>Batterman</surname>, <given-names>S.</given-names>\n</string-name> (<year>2020</year>). <article-title>Air quality impacts at an E‐waste site in Ghana using flexible, moderate‐cost and quality‐assured measurements</article-title>. <source xml:lang=\"en\">GeoHealth</source>, <volume>4</volume>, <elocation-id>e2020GH000247</elocation-id>\n<pub-id pub-id-type=\"doi\">10.1029/2020GH000247</pub-id>\n</mixed-citation>\n</p></notes></front><body id=\"gh2178-body-0001\"><sec id=\"gh2178-sec-0001\"><label>1</label><title>Introduction</title><p>Electronic waste (e‐waste) recycling activities include the transport, dismantling, burning, and smelting of electrical and electronic equipment for the purpose of recovering valuable metals, particularly copper and gold. These activities can pose environmental and occupational health and safety concerns, particularly in low and middle income countries (LMICs) and informal settings where controls and inspections are lax or absent (Ackah, <xref rid=\"gh2178-bib-0002\" ref-type=\"ref\">2017</xref>; Gangwar et al., <xref rid=\"gh2178-bib-0022\" ref-type=\"ref\">2019</xref>; Ohajinwa et al., <xref rid=\"gh2178-bib-0037\" ref-type=\"ref\">2018</xref>; Sthiannopkao & Wong, <xref rid=\"gh2178-bib-0044\" ref-type=\"ref\">2013</xref>). Recycling activities, particularly burning and smelting, can release significant emissions of airborne pollutants that expose both on‐site workers and the nearby community. Such emissions are highly site‐ and activity‐specific and vary over time, thus, air quality monitoring is needed to assess concentrations and exposures and to determine the specific sources that should be targeted for mitigation. Despite concentrations that can greatly exceed guidelines from WHO and national standards (Djossou et al., <xref rid=\"gh2178-bib-0019\" ref-type=\"ref\">2018</xref>; Naidja et al., <xref rid=\"gh2178-bib-0033\" ref-type=\"ref\">2018</xref>), air quality monitoring in Africa is scarce, e.g., only 6 of 47 sub‐Saharan countries report PM levels (WHO, <xref rid=\"gh2178-bib-0050\" ref-type=\"ref\">2014</xref>). In consequence, the temporal and spatial variation in pollutant concentrations and exposures is poorly characterized. Monitoring plays an essential role in air quality management by documenting exposures and compliance with standards, identifying culpable emission sources, and evaluating the effectiveness of control measures.</p><p>Agbogbloshie in central Accra, Ghana has been a hub for large scale e‐waste, automobile and scrap recycling for nearly 15 years. Currently, 4,500–6,000 workers at the site use rudimentary techniques to recover valuable materials (Agyei‐Mensah & Oteng‐Ababio, <xref rid=\"gh2178-bib-0003\" ref-type=\"ref\">2012</xref>; Daum et al., <xref rid=\"gh2178-bib-0014\" ref-type=\"ref\">2017</xref>). Site activities include delivery and receipt of waste from trucks and carts; sorting and transport of waste to distinct areas for tires, refrigerators, air conditioners, starter motors, televisions, etc.; manual dismantling of some waste types, e.g., hammering‐off aluminum heat exchanger fins, stripping insulation from larger cables using machetes to obtain copper, breaking cathode ray tubes on older televisions to obtain the yoke and its copper windings; open burning of smaller insulated wires, cables and circuit boards to obtain copper; collecting and weighing the recovered metals and other valuables; and transport of products off‐site. Black plumes are frequently seen over the site, primarily originating from two waste burning areas ~350 m apart; fire accelerants used include Styrofoam insulation recovered from refrigerators, tires, and other materials (Amoyaw‐Osei et al., <xref rid=\"gh2178-bib-0005\" ref-type=\"ref\">2011</xref>). Workers typically carry out their tasks in groups of two or more without personal protective equipment in small sheds or in the open air (Oteng‐Ababio, <xref rid=\"gh2178-bib-0039\" ref-type=\"ref\">2012</xref>). Despite being a signatory to the 1989 Basel Convention that limits transboundary movement of waste and national legislation on e‐waste, Ghana produced an estimated 150,000 tons and imported 215,000 tons of e‐waste in 2009 (Ackah, <xref rid=\"gh2178-bib-0002\" ref-type=\"ref\">2017</xref>).</p><p>Ambient air quality monitoring at Agbogbloshie and the region has been limited. In addition to monthly and annual PM<sub>10</sub> concentrations reported by the Ghanaian Environmental Protection Agency (EPA) (Ghana EPA, <xref rid=\"gh2178-bib-0020\" ref-type=\"ref\">2016</xref>), we identified seven studies that reported ambient PM concentrations in Ghana over the last 20 years (Aboh et al., <xref rid=\"gh2178-bib-0001\" ref-type=\"ref\">2009</xref>; Ahiamadjie, <xref rid=\"gh2178-bib-0004\" ref-type=\"ref\">2017</xref>; Arku et al., <xref rid=\"gh2178-bib-0006\" ref-type=\"ref\">2015</xref>; Dionisio et al., <xref rid=\"gh2178-bib-0018\" ref-type=\"ref\">2010</xref>; Laskaris et al., <xref rid=\"gh2178-bib-0028\" ref-type=\"ref\">2019</xref>; Ofosu et al., <xref rid=\"gh2178-bib-0036\" ref-type=\"ref\">2012</xref>; Sulemana et al., <xref rid=\"gh2178-bib-0045\" ref-type=\"ref\">2018</xref>). While suggesting that PM concentrations can be high, the temporal and spatial coverage of these studies is limited, and diurnal patterns and the impact of e‐waste emissions on community exposure have not been evaluated. The objective of this study is to characterize PM concentrations at the Agbogbloshie e‐waste site and the nearby community, and to present moderate cost sampling methods that enable quality‐assured results. This monitoring forms part of the West Africa‐Michigan Charter II for GEOHealth cohort study, which is analyzing occupational exposures and health risks at this site. Our findings are intended to broaden knowledge on PM levels and exposures at the site, and to provide guidance for air pollution monitoring programs, particularly in LMICs.</p></sec><sec id=\"gh2178-sec-0002\"><label>2</label><title>Methods</title><sec id=\"gh2178-sec-0003\"><label>2.1</label><title>Site Description</title><p>Agbogbloshie is the informal name for an area about 1 km from central Accra and adjacent to the South Industrial Area that contains the Agbogbloshie e‐waste site and scrap yard. The site is bounded by the Abossey‐Okai Road, the Odaw River, Cemetery Drain, and the Ring Road West. This 0.365 km<sup>2</sup> area (excluding a large church and recreational fields to the west) is shown in Figure <xref rid=\"gh2178-fig-0001\" ref-type=\"fig\">1</xref>. Agbogbloshie is in the Asiedu Keteke sub‐metropolitan area, a commercial hub with ~144,000 residents in a 16 km<sup>2</sup> area, which lies within Accra, Ghana’s capital, with 2.27 million inhabitants. In addition to the scrap yard and recycling activities, the site contains a health clinic, a technical training center for e‐waste and scrap workers, a football field, mosques, extensive informal housing, workshops for motorcycle, car and electronics repair, commercial cooking, and metal fabrication (e.g., traditional charcoal pots, aluminum cooking pots).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"gh2178-fig-0001\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Area map showing e‐waste and air quality monitoring sites.</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"GH2-4-e2020GH000247-g001\"/></fig><p>The e‐waste site is adjacent to densely populated residential and commercial areas, including a large market, informal housing to the south, commercial offices and industrial areas to the N and E, vegetable markets to the E, and food markets to the NE. Markets (food, clothing, medicine, furniture, appliances, etc.), commercial cooking, metal working, and banking services are adjacent to the e‐waste site.</p><p>The present form of Agbogbloshie is relatively recent. Prior to 2002, an oxbow of the Odaw River cut into the site, the western portion was a waste dump, areas near roads had some informal businesses, and the remainder was largely vacant, based on new reports, historical imagery (Google Earth) and government reports (Awiah, <xref rid=\"gh2178-bib-0009\" ref-type=\"ref\">2017</xref>; Ministry of Works and Housing, <xref rid=\"gh2178-bib-0029\" ref-type=\"ref\">2019</xref>; Oirere, <xref rid=\"gh2178-bib-0038\" ref-type=\"ref\">2019</xref>). For flood control, the river was dredged and channelized and the oxbow drained and filled, and by 2008 the area was established as a hub for large scale e‐waste, automobile and scrap recycling. Informal housing grew throughout the site and gradually reached the southernmost portion where the Odaw River and Cemetery Drain converge. Severe floods in June 2015 led to dredging of the Odaw River and the Korle Lagoon later that year. The river choked again by September 2017 and was subsequently dredged. Another dredging cycle was completed in February 2019, removing over 1 million m<sup>3</sup> of material; one report suggested that 40,000 m<sup>3</sup> of silt was deposited annually in the basin (Gambeta, <xref rid=\"gh2178-bib-0021\" ref-type=\"ref\">2019</xref>).</p></sec><sec id=\"gh2178-sec-0004\"><label>2.2</label><title>Monitoring Approach and Site Description</title><p>We utilized “area” or fixed site monitoring with goals of measuring upwind, on‐site and downwind concentrations to understand impacts at the waste site adjusted for upwind or “background” levels, and to assess impacts at downwind locations. Our goal was to collect 24‐hr samples at the three sites simultaneously every sixth day to characterize 1‐hr, 24‐hr and long term PM levels, and to evaluate spatial differences and temporal patterns.</p><p>Monitoring site locations were selected by considering e‐waste site activities (especially burning), distance from the e‐waste site, prevailing wind directions, and the ability to obtain electrical power, rain shelter, site security and access, and permissions from property owners and operators. The siting process involved multiple site visits and consultations with leaders of the e‐waste workers, commercial and governmental facilities, and others. We obtained electrical power at two sites and installed a 12 V 80 A‐hr photovoltaic system (LCPC80–12, Jiangsu Oliter Energy Technology Co. Ltd, China) at the upwind site. Site locations are mapped in Figure <xref rid=\"gh2178-fig-0001\" ref-type=\"fig\">1</xref>; the supplemental information (SI) provides descriptions, photos and maps of each site (Figures <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S1</xref>‐<xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S3</xref>).</p><p>At the e‐waste site (site 2), sampling equipment was placed near the ceiling (~2 m height) of a 2‐wall metal shed that served as a meeting area of e‐waste leaders (Figure <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S1</xref>). This shed was ~300 m NNE of burning area 1 and ~300 m E of burning area 2 (Figure <xref rid=\"gh2178-fig-0001\" ref-type=\"fig\">1</xref>). This sometimes crowded area had many other sheds and small structures used for weighing, dismantling and storage, and a mosque (within 30 m). We frequently observed individuals cooking, eating, resting, sleeping and selling/buying food, beverages and medicine in this area. The site was 40 m from the busy 2‐lane Abossey‐Okai Road, which fronted considerable commercial activity.</p><p>The upwind site (site 1) was 1.35 km SSE of site 2, located at an inoperable pump station/sand filter at the Korle Lagoon (Figure <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S2</xref>). The pump station is a large concrete structure in an open field. Sampling equipment was placed on a concrete shelf on the third level open motor room (~6.5 m above ground level). Nearby land uses include a teaching hospital and mortuary, a dense residential area E of the lagoon, and the 4‐lane Ring Road West, which had intermittent traffic. Fields immediately W of the site had occasional vehicular movement and a few grazing cattle and goats; fields 100–300 m NW were used to transfer rubbish collected by tricycles to trucks for disposal elsewhere, occasional open burning of waste, and football. After establishing the site, we noticed intermittent waste burning at a mortuary 400 m to the W, hidden behind trees lining Ring Road West. In late 2017, the Korle Lagoon and Odaw River adjacent to the site were dredged and spoils (excavated materials) and municipal waste were placed in windrows 3–5 m high in fields immediately SE and W of the pump station. These materials sometimes burned and smoldered; as described later, this resulted in extremely high PM measurements. (After this study, in January 2019, a waste recycling and composting facility for tires, plastic water bags and bottles, metal cans, and organic waste was constructed SW of the pump station.) While site 1 was upwind of the e‐waste site, local activities significantly affected PM levels, and measurements after October 2017 could not be regarded to reflect background levels.</p><p>The downwind site (site 3) was 0.48 km NE of the e‐waste site. Monitoring instruments were mounted on a third floor balcony wall (~6 m above the ground) of a 3‐story building (Letap Jewelries Limited Building) used for pharmaceutical production (first two floors) and apartments (third floor; Figures <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S3</xref>–4). This area has considerable commercial activity, a brewery, warehouses, car and truck repair facilities, and it is near a twice‐daily used railway crossing and bridge, the busy 4‐lane Graphic Road, and the normally sluggish and polluted Odaw River channel. In addition to traffic and street merchants on nearby roads, we observed nearby building construction, e‐waste sorting, and occasional open fires on the river’s west bank from the Letap building to the e‐waste site.</p></sec><sec id=\"gh2178-sec-0005\"><label>2.3</label><title>PM Instrumentation, Modifications, and Procedures</title><p>We monitored PM, temperature and humidity using portable instruments placed in custom fabricated metal “cages” that were open on four sides and locked for safety and security (Figure <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S4</xref>). Integrated 24‐hr PM<sub>2.5</sub> samples were collected using 47 mm dia pore size 2 μm Teflon filters (SKC, PA, USA), size selective samplers (Impact Sampler, SKC), and personal sampling pumps (Leland Legacy, SKC) operating at 10 L/min. Although the pumps had internal batteries, external power packs were used to allow 24‐h sampling. Near‐continuous measurements (every 1 min) were obtained using 5‐channel optical particle counters (OPCs; Aerocet 831, Met One Instruments, Inc, Oregon, USA) operating at 2.83 L/min. External power also was used with these samplers. Temperature and relative humidity were measured every 1 min using logging instrumentation (UX100–003 data logger; Onset Corporation, Bourne, MA, USA).</p><p>Pumps were programmed to sample for a 24‐hr period, and deployment and retrieval of instruments mostly occurred from 10:00 to 16:00. Prior to sampling, new filters were installed, PM<sub>2.5</sub> pumps were set to 10 L/min using a flowmeter (VFB‐67, Dwyer Instrument Inc, IN, USA) connected to a HEPA capsule filter (Pall Gelman Science, Ann Arbor, MI, USA), and instrument clocks were synchronized. OPC instrument flows were checked and adjusted to 2.83 L/min. After sampling, flow rates were measured again, filters were removed from the sampler cassette using clean forceps, folded in half (exposed side closed), and placed in individual poly bags until weighing. All filters were stored in a clean, sealed container until gravimetric analysis. In the laboratory, filters were weighed before and after sampling using a microbalance (ME‐5, Sartorius, New York, USA) after 48‐hr conditioning at 25 ± 1 C, RH = 33 ± 2%, and deionizing for 30 min. Newly weighed filters were placed in individual labeled poly bags.</p><p>Pilot deployments showed that the integrated samplers were significantly overloaded, which was observed visually (Figure <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S5</xref>); overload could result in particle bounce and other issues that would alter sampler performance and the PM size cut. We considered several methods to resolve this problem: a lower flow rate would reduce accumulation, but also change the sampler’s size cut and thus was unacceptable for the selected inlet, and lower flow rates also can increase weighing errors; a shorter sampling period would reduce overload, however, 24‐hr periods were desired to capture both day and night periods; and an elutriator, cyclone or other device upstream of the sampler would exclude coarse PM, but would pose size, pressure drop and cost issues. Instead, we opted to use periodic sampling with a custom‐designed and fabricated system consisting of valves and a programmable cycle timer configured to sample for a 5‐min period and then bypass the sampler for 10 min. This cycle was repeated throughout the 24‐hr sampling period, thus reducing the air volume sampled by two‐thirds. (The system also enabled collection of a second sample on the 10‐min arm, with a volume reduced by one‐third for the cycling described.) Some systems allow automatic cycling of the sampling pump, which also reduces pump wear, but this was not possible with the selected pump. (We will make available the design of the cycle timer upon request to the corresponding author.)</p></sec><sec id=\"gh2178-sec-0006\"><label>2.4</label><title>Meteorological and Satellite Data</title><p>Local meteorological variables were measured using a weather station (Vantage Pro 2 Precision, Davis Instruments, Hayward, California) placed immediately SSW of site 1 at 4.5 m above ground level (36 m msl), 2.5 m above a ~ 2 m concrete wall that enclosed the sand filter. This site is 1.2 km from the coast (Gulf of Guinea). The surrounding area was largely flat and free of obstructions other than the filter enclosure and dredge spoils heaped in adjacent fields starting late 2017. 1‐min data from the upwind site were collected from 7/30/17 to 12/31/18, although the wind vane sensor failed on 8/28/18. We also obtained 2017–2018 hourly surface observations from Kotoka International Airport (latitude, longitude: 5.605, −0.167; elevation 62.5 m) from NOAA (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncdc.noaa.gov\">https://www.ncdc.noaa.gov</ext-link>). The airport site is 12.1 km from the coast and 10.2 km NE of the e‐waste site. Terrain within 900 m of the airport is open; apartments and some midrise commercial buildings are within 1 km.</p><p>To help indicate the possible presence of Harmattan dusts, we examined daily maps of aerosol optical depth (AOD) over the region from November 2017 to February 2017 using both the MODIS dark target algorithm and the combined value‐added AOD (CVA‐AOD) (<ext-link ext-link-type=\"uri\" xlink:href=\"https://earthdata.nasa.gov/earth-observation-data/near-real-time/download-nrt-data/modis-nrt\">https://earthdata.nasa.gov/earth-observation-data/near-real-time/download-nrt-data/modis-nrt</ext-link>), as described in the SI.</p></sec><sec id=\"gh2178-sec-0007\"><label>2.5</label><title>Quality Assurance</title><p>Quality assurance (QA) activities included colocation of sampling instruments, use of standard datasheets, and flow checks before and after deployment. For gravimetric samples, we used filter blanks, a minimum of two replicates of gravimetric measurements with an acceptance criterion of 10 μg, confirmation of flow volumes using the pump’s totalizer within 10%, and exclusion of PM masses over 600 mg that indicated an overloaded sampler. Due to power interruptions, sampler problems and field logistics, some sampling periods did not reach 24‐hr; we excluded periods shorter than 75% of the goal (<18 hr). Filters were handled using forceps and powder‐free gloves; different forceps were used for the oiled impactor substrate disc to avoid cross‐contamination. Filter blanks were subjected to same analyses as samples. To assess weighing accuracy, certified 200 mg standards were weighed at beginning and end of each weighing session, and after every 12<sup>th</sup> filter.</p><p>For optical measurements, daily flow and zero checks were performed using a flowmeter connected to a HEPA capsule filter. Colocation tests showed average agreement within 6% for 1‐hr averages of PM<sub>10</sub> and within 2% for PM<sub>2.5</sub>. While these instruments have a large dynamic range, very high concentrations can produce coincidence error that biases measurements, e.g., multiple small particles appear as a single larger particle that then overestimates mass in larger size channels. There is no specific threshold where coincident error becomes critical (the manufacturer did not provide guidance); our experience with the selected instrument suggests that biases start around 2000 μg/m<sup>3</sup>. Very high humidity also can bias results (as discussed later). Considering the 144,579 1‐min PM<sub>10</sub> measurements collected, the top 10 values ranged from 4,926 to 11,084 μg/m<sup>3</sup>, 86 measurements (0.06%) exceeded 3,000 μg/m<sup>3</sup>, and 365 (0.25%) exceeded 2000 μg/m<sup>3</sup>. We considered OPC data as potentially biased if PM<sub>10</sub> exceeded 2000 μg/m<sup>3</sup>. When aggregated to 1‐hr averages, this omitted 7 (0.3%) of the 2,327 hourly averages and dropped the maximum 24‐hr PM<sub>2.5</sub> average from 865 to 522 μg/m<sup>3</sup>. As discussed later, these exclusions affected only the highest PM measurements. We also checked agreement with the gravimetric measurements, and did not apply correction factors to the optical measurements.</p></sec><sec id=\"gh2178-sec-0008\"><label>2.6</label><title>Data Analysis</title><p>Hourly concentrations were calculated from OPC data if at least 80% of the 1‐min observations were available and valid, and 24‐hr averages were calculated if at least 80% of hourly averages were available. Analysis focused on PM<sub>2.5</sub> and PM<sub>10</sub>; the coarse fraction (PM<sub>2.5–10</sub>) was also determined. Optical and gravimetric PM data were compared for the same periods using correlations and scatterplots; again, at least 80% overlap of hourly data were required in these comparisons. We calculated descriptive statistics for the 1‐hr and 24‐hr data and used probability, trend plots with third order polynomial curves to fit the data, and pollution roses to assess distributions, site differences, wind direction, and time‐of‐day patterns. To provide a single “best” estimate of concentration increments over background (site 1) levels, we combined 24‐hr averages from gravimetric and optical instruments, and estimated standard deviations using Gaussian quadrature. Meteorological data was converted to 1‐hr averages, and after ensuring comparability between the sites, the 2‐site average of hourly wind speed and direction was used to obtain a nearly complete meteorological record. Data were converted to the SCRAM format to generate wind roses using WRPLOT (Lakes Environmental, Waterloo, Ontario, Canada) to summarize wind speed and direction statistics.</p></sec></sec><sec id=\"gh2178-sec-0009\"><label>3</label><title>Results</title><sec id=\"gh2178-sec-0010\"><label>3.1</label><title>Meteorology</title><p>Most of the year, this coastal area receives moist maritime air originating over the Atlantic Ocean with little variation in daily temperatures, although cloud cover and rainfall varies. As discussed later, high pressure systems above the Sahara Desert can give rise to dusty Harmattan winds from November to February (Nicholson, <xref rid=\"gh2178-bib-0034\" ref-type=\"ref\">2013</xref>). Based on 1‐hr data collected at 2017–8 Kotoka International Airport data, temperatures averaged (± standard deviation) 27.4 ± 2.2 C, relative humidity (RH) averaged 82.2 ± 11.2%, mixing heights averaged 1989 ± 2,701 m (median = 297 m), and precipitation occurred on 166 hours per year (1.9% of hours); precipitation may be underestimated since the record was incomplete (27% of hourly data was missing). Precipitation amounts and trends were similar at the two sites, although hourly and sometimes daily timing of precipitation varied (Figures <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S6</xref>‐<xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S7</xref>). The SI discusses daily and monthly trends. Shifts between day (8:00–20:00) and night (20:00–6:00) periods were modest for temperature (28.6–26.2 C), RH (83.8–87.7%), and mixing height (1989–1877 m).</p><p>Figure <xref rid=\"gh2178-fig-0002\" ref-type=\"fig\">2</xref> shows wind roses for the Kotoka International Airport and the upwind monitoring site; Figures <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S8</xref>‐<xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S9</xref> show diurnal and seasonal roses. Surface winds at the sites are similar, although velocities at the upwind site were slightly lower (3.6 ± 2.3 m/s) and directions more variable, expected given the upwind site’s greater roughness and lower elevation. Winds are predominantly SSW and W; winds from other directions are rare. The airport wind speed (10 m above the ground) averaged 4.4 ± 1.7 m/s and ranged from 1.5–8.2 m/s for the 1<sup>st</sup> and 99<sup>th</sup> percentiles. Westerly winds tended to be lighter. Calms (<1 m/s) were rare (<0.6%). The wind field rotates 75‐90<sup>o</sup> during the day: from midnight to noon, winds are westerly at lower speeds (averaging 3.45 and 4.23 m/s for 0:00–5:00 and 6:00–11:00 periods, respectively); in the afternoon (12:00–17:00), velocity increases (5.62 m/s) and direction transitions to southerly; and in the evening (18:00–23:00), winds return from the SSW and velocity decreases (4.58 m/s). This pattern is highly consistent except from June through August when winds are southwesterly with little diurnal variation and speeds increase (5.04 m/s), due in part to upwelling and cooler water in the Gulf of Ghana that increase the sea‐land temperature differential, and the northernmost movement of the low pressure intertropical convergence zone (ITCZ), which alters the NE trade winds.</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"gh2178-fig-0002\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Wind rose at Kotoka airport using hourly data from 2017 and 2018.</p></caption><graphic id=\"nlm-graphic-3\" xlink:href=\"GH2-4-e2020GH000247-g002\"/></fig><p>In Accra, the consistent wind patterns suggest that plumes from the e‐waste site will disperse N during the afternoon, NE from evening to midnight, and E from midnight to noon. Higher concentrations can occur at night with greater atmospheric stability that decreases dispersion. Later, we show that PM levels at the downwind site (NE of the e‐waste site) increase in the evening, suggesting contributions from the e‐waste site.</p></sec><sec id=\"gh2178-sec-0011\"><label>3.2</label><title>Comparison of Gravimetric and Optical Measurements</title><p>Of the 99 gravimetric measurements of PM<sub>2.5</sub> collected, 61 passed QA checks and 43 had corresponding and valid OPC measurements. These 24‐hr average measurements showed reasonable agreement with the optical measurements, e.g., R<sup>2</sup> = 0.47 (<italic>N</italic> = 43; Figure <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S10</xref>). Previously, the same gravimetric and optical instrumentation (but without periodic sampling) used for 4‐hr shift samples at Agbogbloshie and a local comparison site showed better agreement (R<sup>2</sup> = 0.75, <italic>N</italic> = 142), probably due to the ability to maintain flows, avoid filter overload with shorter sampling periods, and avoid periods with very high RH (only daytime sampling was used). Calculating the bias between gravimetric and optical measurements as 100% [1 – C<sub>opt</sub>/C<sub>grav</sub>], where C<sub>opt</sub> and C<sub>grav</sub> are the average of paired optical and gravimetric measurements, respectively (Williams et al., <xref rid=\"gh2178-bib-0051\" ref-type=\"ref\">2014</xref>), optical measurements were 21% low relative to gravimetric measurements, considered as the reference measurement. Importantly, the bias did not change significantly between sites 1–3, which might happen if the optical properties of PM varied at the sites. Because the bias was small and within the expected error range of the gravimetric measurements (due to combined variation in flows, weight determinations, impactor performance, etc.), no correction factor was used for the optical measurements.</p><p>High RH may bias optical measurements, particularly for hygroscopic particles, which can demonstrate an exponential increase in hygroscopic growth at high RH, e.g., >85% (Crilley et al., <xref rid=\"gh2178-bib-0013\" ref-type=\"ref\">2018</xref>; Di Antonio et al., <xref rid=\"gh2178-bib-0017\" ref-type=\"ref\">2018</xref>). OPC responses and light scattering are affected by particle mass, size distribution and composition, including water content (Holstius et al., <xref rid=\"gh2178-bib-0025\" ref-type=\"ref\">2014</xref>). Most studies examining RH effects have measured aerosols in industrialized countries where much of the RH artifact is due to rapid growth of the inorganic fraction of the aerosol, e.g., sodium chloride, ammonium nitrate and ammonium sulfate. Among simultaneous 24‐hr gravimetric and optical measurements, we found few divergent cases that could be attributed to high RH; the outstanding case involved concentrations of 120 and 180 μg/m<sup>3</sup> (gravimetric and optical measurements, respectively) during a 24‐hr period when the RH averaged 92%. Otherwise, screening by humidity did not change agreement between optical and gravimetric measurements, though it is seen in some of the 1‐hr data (discussed below). Potentially a large fraction of PM at Agbogbloshie is organic, suggested by the black color on filters and impaction substrates, the poorly controlled combustion sources, and source apportionments in the literature (described later), and thus is relatively hydrophobic. Our ability to investigate RH biases was constrained since we compared 24‐h PM measurements, and RH stayed in a fairly narrow band.</p></sec><sec id=\"gh2178-sec-0012\"><label>3.3</label><title>Highest PM Levels</title><p>The highest PM levels in the study occurred at the upwind site for a 12‐hour period starting 23:00 on Jan. 14, 2018 when 1‐hr (optical) PM<sub>2.5</sub> levels reached 523 μg/m<sup>3</sup> (average: 369 ± 114 μg/m<sup>3</sup>) and 1‐hr PM<sub>10</sub> reached 908 μg/m<sup>3</sup> (665 ± 203 μg/m<sup>3</sup>). These statistics excluded 5 hours with 1‐min PM levels over 2000 μg/m<sup>3</sup>. Including these (potentially biased) data would have increased 1‐hr PM<sub>2.5</sub> levels to 865 μg/m<sup>3</sup> (average: 545 ± 223 μg/m<sup>3</sup>), and 1‐hr PM<sub>10</sub> levels to 2,138 μg/m<sup>3</sup> (average: 1109 ± 569 μg/m<sup>3</sup>). During this period, QA checks were not met for the corresponding filter‐based measurement (sample U0026). At site 2 during this period, optical measurements were unavailable and the gravimetric measurement (M0043) had flow discrepancies and an overloaded filter. At site 3, levels were fairly typical, e.g., 1‐hr PM<sub>2.5</sub> levels reached 143 μg/m<sup>3</sup> (average: 108 ± 15 μg/m<sup>3</sup>) and PM<sub>10</sub> reached 418 μg/m<sup>3</sup> (average: 345 ± 34 μg/m<sup>3</sup>), and the gravimetric measurement (L0043) was excluded due to (minor) flow discrepancies; if accepted, the concentration would have been 88 μg/m<sup>3</sup>. The meteorology during this period was not unusual; winds shifted from the SW to the W and wind speeds were low (1.5–3 m/s) during the 5 hours with the highest PM levels, however, RH averaged 89% and reached 94%. Other days with very high PM levels at site 1 showed similar patterns, e.g., on Feb. 8, 2017 from 1:00 to 8:00, 1‐hr PM<sub>2.5</sub> levels averaged 423 ± 72 μg/m<sup>3</sup> at site 1; 74 ± 21 at site 2, and 36 ± 11 μg/m<sup>3</sup> at site 3, and again, winds were light and easterly, and RH was high (average 89% and up to 94%). As discussed later, these high measurements did not occur during Harmattan dust events.</p><p>The pattern of nighttime, high and isolated PM events at site 1 with slight E winds is strong evidence of impacts from nearby fires in the spoil piles: most piles were W of the site, fires were sometimes very close (<10 m) to the sampling site, and pile heights approached the sampling height. Time lapse photography at this unoccupied site could have confirmed these emissions, as shown elsewhere (Laskaris et al., <xref rid=\"gh2178-bib-0028\" ref-type=\"ref\">2019</xref>). In addition, RH was high during portions of these periods, and site 1 was closest to the coast and possibly experienced higher RH than the airport measurements, suggesting an RH artifact at this site that biased optical measurements upwards.</p></sec><sec id=\"gh2178-sec-0013\"><label>3.4</label><title>Hourly and Daily PM Concentrations and Site Differences</title><p>Statistics of the 24‐hr data are presented in Table <xref rid=\"gh2178-tbl-0001\" ref-type=\"table\">1</xref> and trends are plotted in Figure <xref rid=\"gh2178-fig-0003\" ref-type=\"fig\">3</xref>. (Table <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S1</xref> lists statistics of the 1‐hr data.) Across the study, 24‐hr PM<sub>2.5</sub> levels averaged 63 ± 31 μg/m<sup>3</sup> (<italic>N</italic> = 61) for the gravimetric data and 79 ± 33 μg/m<sup>3</sup> (<italic>N</italic> = 81) for the optical data; PM<sub>10</sub> levels (optical) averaged 210 ± 91 μg/m<sup>3</sup> (N = 81). Concentrations varied widely, e.g., 1‐hr PM<sub>2.5</sub> ranged from 7 to 523 μg/m<sup>3</sup>, and 24‐hr levels from 23–192 μg/m<sup>3</sup> (N = 81). These levels are well over 24‐hr and annual average WHO and Ghanaian standards and guidelines. Probability plots of 1‐hr PM<sub>2.5</sub> and PM<sub>10</sub> concentrations suggest lognormal distributions for PM<sub>2.5</sub> and PM<sub>10</sub> at sites 2 and 3 (Figure <xref rid=\"gh2178-fig-0004\" ref-type=\"fig\">4</xref>). However, at site 1, the distribution’s upper tail is elevated with levels about twice that expected. Up to the 92<sup>nd</sup> percentile, PM<sub>2.5</sub> concentrations were lowest at site 1 (upwind) and highest at site 2 (e‐waste); the PM<sub>10</sub> plot shows that concentrations were similar at the three sites up to the ~90<sup>th</sup> percentile. While the distributional analysis uses unmatched data and sites have varying number of measurements, it shows the high concentration events that occurred at site 1 after October 2017.</p><table-wrap id=\"gh2178-tbl-0001\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Summary of 24‐hr average PM<sub>2.5</sub> and PM<sub>10</sub> data at the three sites. Matched data shows statistics for days when all three sites have valid data</p></caption><table frame=\"hsides\" rules=\"groups\"><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><thead valign=\"bottom\"><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">PM</th><th align=\"center\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Sample</th><th align=\"center\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\"/><th style=\"border-bottom:solid 1px #000000\" align=\"center\" colspan=\"4\" valign=\"bottom\" rowspan=\"1\">2/18/17 to 2/26/18</th><th style=\"border-bottom:solid 1px #000000\" align=\"center\" colspan=\"4\" valign=\"bottom\" rowspan=\"1\">2/18/17 to 10/27/17</th><th style=\"border-bottom:solid 1px #000000\" align=\"center\" colspan=\"4\" valign=\"bottom\" rowspan=\"1\">10/28/17–2/26/18</th></tr></thead><tbody valign=\"top\"><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Size</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Type</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Statistic</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Site1</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Site2</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Site3</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">All</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Site1</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Site2</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Site3</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">All</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Site1</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Site2</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Site3</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">All</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<bold>PM2.5</bold>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Filter‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Average</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">38</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">85</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">53</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">63</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">88</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">46</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">59</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">61</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">78</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">74</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">71</td></tr><tr><td rowspan=\"14\" align=\"left\" valign=\"top\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">based</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">St. Dev.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">19</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">19</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">24</td></tr><tr><td rowspan=\"3\" align=\"left\" valign=\"top\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Min</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">38</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">42</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">38</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Max</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">95</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">120</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">122</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">122</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">120</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">81</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">120</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">95</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">103</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">122</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">122</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">NOBs</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">24</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">61</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">19</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">18</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Optical‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Average</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">74</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">90</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">69</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">79</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">88</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">68</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">74</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">95</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">93</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">69</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">88</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">based</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">St. Dev.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">41</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">24</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">38</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">36</td></tr><tr><td rowspan=\"3\" align=\"left\" valign=\"top\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Min</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">32</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">59</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">32</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">44</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">28</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Max</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">186</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">191</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">122</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">191</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">85</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">171</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">122</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">171</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">186</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">191</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">102</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">191</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">NOBs</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">81</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">31</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Combined</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Average</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">88</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">61</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">72</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">88</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">57</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">67</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">84</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">88</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">71</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">81</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Estimate</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">St. Dev.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">32</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">32</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">34</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">32</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">NOBs</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">32</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">60</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">142</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">93</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">49</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Concen.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Average</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">57</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">−12</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Increment</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">St. Dev.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<bold>PM10</bold>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Optical‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Average</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">222</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">216</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">191</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">210</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">145</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">214</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">190</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">195</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">273</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">220</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">192</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">233</td></tr><tr><td rowspan=\"6\" align=\"left\" valign=\"top\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">based</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">St. Dev.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">129</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">77</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">72</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">91</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">66</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">53</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">63</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">62</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">137</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">118</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">94</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">122</td></tr><tr><td rowspan=\"3\" align=\"left\" valign=\"top\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Min</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">84</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">90</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">84</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">135</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">90</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">84</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">122</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">92</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">43</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Max</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">543</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">527</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">359</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">543</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">289</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">364</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">265</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">364</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">543</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">527</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">359</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">543</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">NOBs</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">81</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">50</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">31</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Concen.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Average</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">−6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">−31</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">69</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">−53</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">−81</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Increment</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">St. Dev.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">99</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">101</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">56</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">64</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">129</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">122</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">‐</td></tr></tbody></table></table-wrap><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"gh2178-fig-0003\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>Trends of 24‐hr PM<sub>2.5</sub> (optical and gravimetric) and PM<sub>10</sub> (optical) concentrations at the three sites. Curves use 3<sup>rd</sup> order polynomial. PM<sub>10</sub> data is based on optical measurements.</p></caption><graphic id=\"nlm-graphic-5\" xlink:href=\"GH2-4-e2020GH000247-g003\"/></fig><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"gh2178-fig-0004\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><p>Probability plots of 1‐hr PM<sub>2.5</sub> and PM<sub>10</sub> concentrations.</p></caption><graphic id=\"nlm-graphic-7\" xlink:href=\"GH2-4-e2020GH000247-g004\"/></fig><p>The trend plots (Figure <xref rid=\"gh2178-fig-0003\" ref-type=\"fig\">3</xref>) show striking and fairly consistent differences in PM<sub>2.5</sub> levels between the three sites for the period prior to October 26, 2017 when concentrations averaged 31, 88 and 57 μg/m<sup>3</sup> at sites 1, 2 and 3, respectively (<italic>N</italic> = 14, 43, 36; combined optical/gravimetric estimate; Table <xref rid=\"gh2178-tbl-0001\" ref-type=\"table\">1</xref>). Considering upwind site as “background,” the increment over background for PM<sub>2.5</sub> was 57 ± 21 μg/m<sup>3</sup> at the e‐waste site (site 2) and 26 ± 21 μg/m<sup>3</sup> at the downwind site (site 3). PM<sub>2.5–10</sub> increments were small, 16–18 μg/m<sup>3</sup>, thus differences between sites for PM<sub>10</sub> were mainly due to PM<sub>2.5</sub>. Increments based on medians or matched data (when all three sites had measurements) were similar. Following October 26, 2017, site 1 levels generally exceeded those at the other two sites, likely reflecting dredging and spoil pile burning discussed earlier. For this later period, modest or negative increments were estimated.</p><p>Monitoring height can affect concentration measurements, particularly if monitoring sites are near sources and the atmospheric is stable, which tends to limit vertical mixing. Such effects may be predicted using dispersion models such as AERSCREEN (U.S. EPA, <xref rid=\"gh2178-bib-0020\" ref-type=\"ref\">2016</xref>) that represent plume spread which is governed by the vertical dispersion coefficient σ<sub>SC,X</sub> for stability class SC and downwind distance X. A height change of Δ<sub>H</sub> (m) changes the concentration by a factor of 1 ‐ exp{− (Δ<sub>H</sub>)<sup>2</sup>/(σ<sub>SC,X</sub>)<sup>2</sup>}. For example, for a distance of 100 m and stable conditions (class F), σ<sub>F,100</sub> is 2.3 and 7.5 m for rural and urban terrain, respectively; under more common neutral stability (class D), σ<sub>D,100</sub> is 4.7 and 13.8 m (determined using AERSCREEN). The terrain at Agbogbloshie is urban, thus, the latter dispersion coefficients apply. Monitoring site heights were from 2 to 6.5 m above ground level. A height change of 4 m under stable conditions yields a concentration change of 25% for a source that is 100 m distant (σ<sub>F,100</sub> = 7.5 m) and 4% for a source that is 300 m distant (σ<sub>F,300</sub> = 19.9 m). Under neutral stability, changes are 8 and 1%, respectively, for sources 100 and 300 m distant (σ<sub>D,100</sub> = 13.8 m and σ<sub>D,300</sub> = 40.2 m). Atmospheric conditions change hourly, and conditions are often unstable and well mixed during the day, which would reduce differences. Site‐specific conditions will determine impacts. At site 1 (upwind), most sources were 100–300 m distant or further, except in late 2017 when smoldering and burning windrows of dredged materials were proximate and nearly at monitoring height (6.5 m) when some of the highest concentrations were observed. At site 2 (e‐waste site), the monitor was 2 m above ground (near breathing height). This site is surrounded by e‐waste activity, e.g., dismantling occurred within 50 m and burning within 300 m; this site would (as was intended) capture high concentrations. Site 3 was ~0.5 km from (most) e‐waste activities, but near a busy road. The monitoring height of 6 m would not significantly decrease concentrations attributable to the e‐waste site, but would likely lower contributions from nearby traffic.</p><p>Overall, results indicate that the upwind site functioned as a background site up to October 2017 but not afterwards, that PM<sub>2.5</sub> at the e‐waste site was highly elevated over background, and that PM<sub>2.5</sub> at the downwind site was moderately elevated over background. Site differences for PM<sub>10</sub> were smaller on a relative basis and largely attributable to PM<sub>2.5</sub>.</p></sec><sec id=\"gh2178-sec-0014\"><label>3.5</label><title>Diurnal and Wind Sector Variation in PM Levels</title><p>Figure <xref rid=\"gh2178-fig-0005\" ref-type=\"fig\">5</xref> displays the diurnal variation of PM levels at each site, using the median hourly concentration to reduce effects of potential outliers. While the sites have some similarities, there are important differences. At site 1, the diurnal pattern was bimodal with a short early morning peak (5:00–7:00) and a prolonged evening peak (17:00–00:00). After October 2017, PM<sub>10</sub> levels at site 1 were elevated during midday, possibly reflecting dredging activities (Figure <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S11</xref>). At site 2, PM levels peaked in the evening (19:00–22:00) when PM<sub>2.5</sub> reached nearly 150 μg/m<sup>3</sup>; then gradually declined to 50 μg/m<sup>3</sup> by mid‐to‐late morning; PM<sub>10</sub> levels increased somewhat in the early morning (5:00–7:00). As discussed later, prevailing winds shift from the south in the afternoon to the southwest in the evening, which can bring plumes from e‐waste burning in area 1 (which often continues in the evening) and also from cooking using biomass fuels in the extensive informal settlements located south and west of site 2 (Figure <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S1</xref>); in addition, concentrations will increase as the boundary layer height and dispersion are reduced in the evening. At site 3, PM levels rose sharply throughout the evening (17:00–00:00) and a second peak occurred in early morning (5:00–7:00).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"gh2178-fig-0005\" orientation=\"portrait\" position=\"float\"><label>Figure 5</label><caption><p>PM<sub>2.5</sub> and PM<sub>10</sub> concentrations by time of day. Shows median concentration at each hour. (N = 1,069 at each site.)</p></caption><graphic id=\"nlm-graphic-9\" xlink:href=\"GH2-4-e2020GH000247-g005\"/></fig><p>“Pollutant roses” plotting median and 90<sup>th</sup> percentile 1‐hr concentrations by wind direction are shown in Figure <xref rid=\"gh2178-fig-0006\" ref-type=\"fig\">6</xref>. Because N and E winds were very uncommon, rose “petals” in these directions are unlikely to be representative and should be discounted. At site 1, high concentrations from the NNW suggest e‐waste site emissions, and the high 90<sup>th</sup> percentile concentrations from the W suggest fires at the adjacent spoil piles. At site 2, median concentrations were approximately uniformly distributed, suggesting emission sources in all directions; 90<sup>th</sup> percentile levels were highest from the SW (disregarding PM<sub>10</sub> levels from the NE and E), suggesting burn area 1 or other local activities. At site 3, both median and 90<sup>th</sup> percentile levels (again neglecting NE and E arms for 90<sup>th</sup> percentile PM<sub>10</sub>) were highest from the SW, also suggesting e‐waste emissions.</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"gh2178-fig-0006\" orientation=\"portrait\" position=\"float\"><label>Figure 6</label><caption><p>PM<sub>2.5</sub>, PM<sub>10</sub>, and PM<sub>2.5–10</sub> (coarse fraction) concentrations by wind sector at three sites. Left panel shows median concentration by wind sector. Right panel shows 90<sup>th</sup> percentile concentrations. Concentration scales differ for each row and set. Scale numbering (0 to 30) is arbitrary.</p></caption><graphic id=\"nlm-graphic-11\" xlink:href=\"GH2-4-e2020GH000247-g006\"/></fig></sec><sec id=\"gh2178-sec-0015\"><label>3.6</label><title>Satellite Data and Harmattan Dusts</title><p>The MODIS AOD and CVA‐AOD data are summarized in Table <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S2</xref>, and daily AOD maps are displayed in Figures <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S12</xref>–<xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">15</xref>. In November and December, 2017, no Harmattan impact is suggested by the satellite or EPA data (Table <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S2</xref>). In 2018, the AOD maps suggests several prolonged and widespread Harmattan dusts episodes over Accra, including Jan. 1–6, possibly on Jan. 8–14, and on Jan. 16–18, Jan. 22–24, Jan. 27–28, Jan. 31‐Feb 2, and Feb. 7–8. We obtained PM measurements on only 3 of these days, and while some levels were high (24‐hr PM<sub>10</sub> ranged from 92–544 μg/m<sup>3</sup>), Harmattan dusts were not indicated for several reasons: few hours of N to E winds occurred on these days; PM levels or increases across sites were not comparable (Figure <xref rid=\"gh2178-fig-0003\" ref-type=\"fig\">3</xref>) as would be expected for regional events; and the highest levels occurred at sites 1 and 2 in short periods that suggest local burning. Possibly, PM levels attributable to Harmattan dusts were too small to discern given the magnitude of local sources near our monitoring sites. As discussed below, earlier studies have shown elevated PM levels during the Harmattan season.</p></sec></sec><sec id=\"gh2178-sec-0016\"><label>4</label><title>Discussion</title><sec id=\"gh2178-sec-0017\"><label>4.1</label><title>PM Monitoring and Sources</title><p>The Ghanaian EPA started monitoring in Accra around 2011 and by 2015, 24‐hr PM<sub>10</sub> samples were being collected every 6<sup>th</sup> day at 4–5 “permanent” and 10–11 roadside sites; several sites also measure PM<sub>2.5</sub>. Based on annual reports (Ghana EPA, <xref rid=\"gh2178-bib-0020\" ref-type=\"ref\">2016</xref>) and EPA data (E. Apoh, personal communication, Jan. 20, 2020), we list 2015–7 data in Tables <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S3</xref>‐<xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S5</xref>. Only monthly summaries were available, and most PM<sub>2.5</sub> data was for 2015. We first consider the March through November data, i.e., the non‐Harmattan season. In 2015, PM<sub>2.5</sub> levels at nearby sites averaged 82 μg/m<sup>3</sup> at the South Industrial Area (SIA) site, located ~1 km N of site 2, and 93 μg/m<sup>3</sup> at Graphic Road, a roadside site ~460 m ESE of site 3. In 2017, PM<sub>10</sub> averaged 137 μg/m<sup>3</sup> at SIA and 237 and 188 μg/m<sup>3</sup> at Graphic Road for 2015 and 2017, respectively. We obtained comparable levels at sites 2 and 3 (88 and 57 μg/m<sup>3</sup> for PM<sub>2.5</sub>; 214 and 190 μg/m<sup>3</sup> for PM<sub>10</sub>), although site 3 was ~62 m upwind (SW) and sheltered from traffic on Graphic Road by a large building, while the EPA site was in the middle of this 4‐lane road (Figure <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S16</xref>). Figure <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S17</xref> maps 2017 PM<sub>10</sub> data, and shows some of the highest levels in central Accra and at the e‐waste site. The EPA data also shows higher PM levels during the Harmattan season (December–February) and the highest levels near busy roads, e.g., Weija Junction averaged 155 and 245–295 μg/m<sup>3</sup> for PM<sub>2.5</sub> and PM<sub>10</sub>, respectively.</p><p>Seven studies were identified that reported PM concentrations in or near Accra; several others examined household pollution from cook stoves (Delapena et al., <xref rid=\"gh2178-bib-0016\" ref-type=\"ref\">2018</xref>; Wylie, <xref rid=\"gh2178-bib-0052\" ref-type=\"ref\">2017</xref>; Zhou et al., <xref rid=\"gh2178-bib-0054\" ref-type=\"ref\">2011</xref>). From April to August 2007, optical PM<sub>2.5</sub> and PM<sub>10</sub> measurements collected daily while walking 8–9 km paths over 1 week periods in four neighborhoods had geometric means of 35 and 86 μg/m<sup>3</sup> in Asylum Down and 21 and 49 μg/m<sup>3</sup> in Jamestown, the closest neighborhoods to Agbogbloshie; levels were higher along large roads and near roadside biomass fires (trash and stoves) (Dionisio et al., <xref rid=\"gh2178-bib-0018\" ref-type=\"ref\">2010</xref>). From January to August 2008, 56 students carrying backpack samplers in the same neighborhoods had 24‐hr geometric mean PM<sub>2.5</sub> levels from 37–58 μg/m<sup>3</sup>; household fuel use and school location were important determinants (Arku et al., <xref rid=\"gh2178-bib-0006\" ref-type=\"ref\">2015</xref>). Ambient levels in these studies may be underestimated due to the lack of evening and nighttime outdoor monitoring. From October to November 2013, 24‐hr PM<sub>10</sub> (and metal) measurements collected every 6<sup>th</sup> day at four roadside locations (Weija, Mallam, Kaneshie First Light, Graphic Road, also used by EPA) averaged 167 (Kaneshie) to 228 (Weija) μg/m<sup>3</sup>; the Graphic Road site (along the same road but ~5 km W of our downwind site 3) averaged 182 μg/m<sup>3</sup> (Sulemana et al., <xref rid=\"gh2178-bib-0045\" ref-type=\"ref\">2018</xref>). Lastly, from March 2017 to April 2018, midday 4‐hr shift samples collected from 142 e‐waste workers using backpack monitors and the same optical sensors as in the present study had PM<sub>2.5</sub> levels averaged 81 μg/m<sup>3</sup> (Laskaris et al., <xref rid=\"gh2178-bib-0028\" ref-type=\"ref\">2019</xref>), similar to our site 2 average of 88 μg/m<sup>3</sup>.</p><p>Three studies used receptor modeling to apportion PM sources in Ghana. For a 1‐yr period starting February 2006, gravimetric samples were collected and analyzed for PM, metals and black carbon at Kwabenya, an outlying suburb located ~20 km NE of Accra’s center (Aboh et al., <xref rid=\"gh2178-bib-0001\" ref-type=\"ref\">2009</xref>). PM<sub>2.5</sub> averaged 41 ± 54 μg/m<sup>3</sup> (median = 26; <italic>N</italic> = 171) and PM<sub>2.5–10</sub> averaged 138 ± 245 μg/m<sup>3</sup> (median = 62; N = 171); during Harmattan conditions (defined by Si levels >10 μg/m<sup>3</sup>), PM<sub>2.5</sub> increased to 97 ± 89 μg/m<sup>3</sup> (median = 53; <italic>N</italic> = 44) and PM<sub>2.5–10</sub> to 389 ± 395 μg/m<sup>3</sup> (median = 182; N = 44); a principal components analysis showed few differences between Harmattan and non‐Harmattan periods, and apportioned PM<sub>2.5</sub> to crustal (38–39%), vehicles/biomass (24–38%), and industrial (16–33%) sources, while PM<sub>2.5–10</sub> was apportioned to crustal (41–45%), vehicles/biomass (18–20%), industrial (15–17%), sea spray/other (6–13%), and sand (7%, Harmattan period only) sources. From February 2008 to March 2009, 24‐hr (12‐hr during the Harmattan due to filter overload) samples were collected and analyzed for mass, carbon and metals at Ashaiman, a city (population of 220,000) located ~30 km E of Accra and ~10 km from the industrial port city of Tema; PM<sub>2.5</sub> averaged 22 μg/m<sup>3</sup> (range: 6–73; N = 44), and sources identified using positive matrix factorization (PMF) were diesel (18%), soil/dust (18%), gasoline (16%), fresh and aged sea salt (16 and 6%), industry (11%), biomass (9.5%), and 2‐stroke engines (5%) (Ofosu et al., <xref rid=\"gh2178-bib-0036\" ref-type=\"ref\">2012</xref>). In dissertation research, PM and metals were measured from May 2010 to April 2011 at a commercial site (trucking depot) ~ 0.5 km NNE of Agbogbloshie (Ahiamadjie, <xref rid=\"gh2178-bib-0004\" ref-type=\"ref\">2017</xref>). Median PM<sub>2.5</sub> and PM<sub>10</sub> levels were 59 and 107 μg/m<sup>3</sup> (<italic>N</italic> = 145), respectively; averages were 89 and 138 μg/m<sup>3</sup>; the highest levels occurred during NE Harmattan winds; and sources determined using PMF were Harmattan dusts, e‐waste burning, resuspended soil and dust, industry, vehicles, sea spray, biomass burning and oil burning.</p><p>Overall, these studies suggest that average concentrations in urban, industrial or roadside areas in Accra range from 40–106 μg/m<sup>3</sup> for PM<sub>2.5</sub> and 91–228 μg/m<sup>3</sup> for PM<sub>10</sub> excluding the Harmattan season. These levels are in the range of measurements reported elsewhere in sub‐Saharan Africa, e.g., a 2‐year study at three urban sites in Abidjan, Ivory Coast (423 km W of Accra) and one site in Cotonou, Benin (300 km ENE) using weekly gravimetric sampling at traffic and urban sites reported lower PM<sub>2.5</sub> levels, averaging 28–32 μg/m<sup>3</sup>, but higher levels, 145 μg/m<sup>3</sup> in an Abidjan market with ~25 wood‐burning fireplaces that smoked meat and fish or roasted peanuts (Djossou et al., <xref rid=\"gh2178-bib-0019\" ref-type=\"ref\">2018</xref>). Such results demonstrate the importance of nearby sources in interpreting monitoring results.</p><p>The main PM sources in Accra, as elsewhere in the region, are biomass burning, traffic, industry/energy, and Saharan dust (Aboh et al., <xref rid=\"gh2178-bib-0001\" ref-type=\"ref\">2009</xref>; Ahiamadjie, <xref rid=\"gh2178-bib-0004\" ref-type=\"ref\">2017</xref>; Naidja et al., <xref rid=\"gh2178-bib-0033\" ref-type=\"ref\">2018</xref>; Ofosu et al., <xref rid=\"gh2178-bib-0036\" ref-type=\"ref\">2012</xref>; WHO, <xref rid=\"gh2178-bib-0049\" ref-type=\"ref\">2006</xref>). Traffic emissions include vehicle exhaust, secondary aerosol formation, and entrained dust from paved and unpaved roads. Additional PM sources include construction activity and sea spray from the Gulf of Guinea. Mechanically‐generated PM, e.g., entrained dust, is predominantly large particles (Pakbin et al., <xref rid=\"gh2178-bib-0040\" ref-type=\"ref\">2010</xref>; Rezaei et al., <xref rid=\"gh2178-bib-0042\" ref-type=\"ref\">2018</xref>). Importantly, outside of the Harmattan season, combustion sources are responsible for most of the PM<sub>2.5</sub> (Ahiamadjie, <xref rid=\"gh2178-bib-0004\" ref-type=\"ref\">2017</xref>; Ofosu et al., <xref rid=\"gh2178-bib-0036\" ref-type=\"ref\">2012</xref>). Biomass burning of waste and wood for cooking is widespread and was observed near each monitoring site, e.g., site 1 had nearby refuse and spoil pile fires (after October 2017); site 2 had nearby and extensive commercial cooking, and biomass fuel use is widespread in the nearby informal housing areas; and site 3 was downwind of occasional fires on the river’s banks. Biomass burning and vehicle exhaust emissions likely form most of the “urban background” found across Accra, including in residential areas. The lowest long‐term PM<sub>2.5</sub> level in Accra (excluding data from the Harmattan season) was 48 μg/m<sup>3</sup> measured in a residential area (Danosoman) by EPA in 2015; we measured comparable levels, 44 ± 23 μg/m<sup>3</sup>, at the upwind site. Of note, 24‐hr PM<sub>2.5</sub> levels at even these sites exceeded the WHO and Ghanaian EPA guidelines (25 and 30 μg/m<sup>3</sup>, respectively). Local impacts due to e‐waste, biomass burning, industry, traffic and Harmattan dusts can be sizeable and add to these levels, further degrading air quality.</p></sec><sec id=\"gh2178-sec-0018\"><label>4.2</label><title>PM Attributable to e‐Waste Sites</title><p>Several studies have reported PM levels at e‐waste sites. In Moradabad City, India, which has extensive and illegal waste recycling activities, ambient PM<sub>10</sub> determined gravimetrically during 3 winter months at 3 sites averaged 193–243 μg/m<sup>3</sup> (Gangwar et al., <xref rid=\"gh2178-bib-0022\" ref-type=\"ref\">2019</xref>). In Taizhou, China where a large (1 km<sup>2</sup>) industrial zone recycles ~2 million tons of e‐waste annually, PM<sub>2.5</sub> measured using high‐volume sampling at two sites 400–500 m distant averaged 38–49 μg/m<sup>3</sup> (<italic>N</italic> = 7 per site) in summer and 109–154 μg/m<sup>3</sup> (<italic>N</italic> = 6) in winter (Gu et al., <xref rid=\"gh2178-bib-0024\" ref-type=\"ref\">2010</xref>). In Guiyu, China, a community with extensive e‐waste processing, PM<sub>2.5</sub> samples collected over a 1‐year period averaged 50 μg/m<sup>3</sup> (<italic>N</italic> = 133), higher than at a reference site (38 μg/m<sup>3</sup>; <italic>N</italic> = 33) (Zheng et al., <xref rid=\"gh2178-bib-0053\" ref-type=\"ref\">2016</xref>). Worker exposure estimates based on personal sampling have shown higher concentrations. At Agbogbloshie, the median PM<sub>2.5</sub> level in shift sampling was 61 μg/m<sup>3</sup>, but tasks like burning resulted in far higher levels (Laskaris et al., <xref rid=\"gh2178-bib-0028\" ref-type=\"ref\">2019</xref>). Extremely high levels were reported for workers burning e‐waste in Thailand where PM<sub>2.5</sub> and PM<sub>10</sub> levels averaged 2,774 ± 4,173 and 3,215 ± 4,858 μg/m<sup>3</sup> (N = 33) (Bungadaeng et al., <xref rid=\"gh2178-bib-0012\" ref-type=\"ref\">2019</xref>). Indoor PM<sub>10</sub> levels measured in several e‐waste recycling plants averaged 247–651 μg/m<sup>3</sup> (Papaoikonomou et al., <xref rid=\"gh2178-bib-0041\" ref-type=\"ref\">2018</xref>). These studies note the importance of combustion (especially open burning of waste, circuit boards, wires/cables, Styrofoam, etc.) for fine fraction PM emissions (e.g., PM<sub>2.5</sub>), and the contribution of mechanical processes (dismantling, sorting, shredding, transportation) for coarse PM emissions (e.g., PM<sub>2.5–10</sub>).</p><p>The spatial and temporal variability in PM levels is governed by multiple factors. First, some sources have large and regular diurnal changes in emission rates, e.g., traffic associated emissions (vehicle exhaust and road dust) contribute to morning and late afternoon concentration peaks associated with rush‐hour, and cooking using biomass fuels increases in the evening. Other sources have intermittent or irregular emissions, e.g., open burning emissions depend on the fuel, fire stage, wind speed, accelerant, etc. Second, while highly variable, atmospheric dispersion is generally much reduced after sunset as the ground cools (reducing the boundary layer height and mixing), which can greatly elevate concentrations from nearby ground‐level sources (Roy et al., <xref rid=\"gh2178-bib-0043\" ref-type=\"ref\">2012</xref>). This was seen at sites 1 and 3, and may have contributed to increases at site 2; however, the diurnal pattern at site 2 (Figure <xref rid=\"gh2178-fig-0005\" ref-type=\"fig\">5</xref>) suggest additional factors, e.g., emissions associated with waste handling and possibly cooking. Third, prevailing winds determine impacts downwind of emission sources (Dehghanpour et al., <xref rid=\"gh2178-bib-0015\" ref-type=\"ref\">2014</xref>); the regular southerly winds in the afternoon likely decreased PM levels at sites 1 and 3 since few sources were upwind, but increased levels at site 2 due to emissions from burning area 1; evening southwesterly winds brought plumes from burning area 1 to site 3 as seen by a prolonged PM peak; and midnight to noon westerly winds transported emissions from burn area 2 to site 3, and possibly emissions from the mortuary burn area to site 1 (Figure <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S8</xref>). These patterns are consistent and suggest the impact of e‐waste site emissions at site 3. Additional evidence of impacts from Agbogbloshie, up to a distance of up to 2 km (several times longer than to site 3), was suggested by elevated metal concentrations (e.g., Mn, Zn, Cu, Pb) in surface dusts measured along downwind transects (Mudge et al., <xref rid=\"gh2178-bib-0031\" ref-type=\"ref\">2019</xref>). We estimate that PM<sub>2.5</sub> levels at site 3 exceed background levels (at site 1) by 26 μg/m<sup>3</sup> (Table <xref rid=\"gh2178-tbl-0001\" ref-type=\"table\">1</xref>). Much of this increment can be attributed to e‐waste emissions; other sources include traffic on Graphic Road and nearby but infrequent fires. Fourth, background sources, including the regional background and dusty Harmattan winds, discussed next, can affect levels in the region.</p></sec><sec id=\"gh2178-sec-0019\"><label>4.3</label><title>PM Attributable to the Harmattan</title><p>The Harmattan season is highly variable from year to year and characterized by dry and hazy conditions and little rainfall, which allows widespread entrainment and dispersal of fine and coarse fraction dust from the Sahara Desert across western Africa (Awadzi & Breuning‐Madsen, <xref rid=\"gh2178-bib-0007\" ref-type=\"ref\">2009</xref>; Lanzerstorfer, <xref rid=\"gh2178-bib-0027\" ref-type=\"ref\">2017</xref>; Toure et al., <xref rid=\"gh2178-bib-0047\" ref-type=\"ref\">2019</xref>). In Ghana, these dusts usually originate in intense dust storms (“haboobs”) caused by the Bodélé depression occurring between Tibesti and Lake Chad (K. Sunnu et al., <xref rid=\"gh2178-bib-0046\" ref-type=\"ref\">2018</xref>; Naidja et al., <xref rid=\"gh2178-bib-0033\" ref-type=\"ref\">2018</xref>). NE Harmattan winds produce white and hazy skies and elevated PM levels in Ghana, typically from December to February. In northern Ghana, the season is dominated by the NE Harmattan winds, and by SW monsoon winds in southern Ghana, although instabilities in the ITCZ can lead to NE Harmattan winds in the south (Awadzi & Breuning‐Madsen, <xref rid=\"gh2178-bib-0007\" ref-type=\"ref\">2009</xref>). While only surface PM measurements are directly relevant to community exposures, satellite estimates of AOD and other optical properties, and surface sun photometer estimates of AOD can indicate the potential presence of Harmattan dusts (Toure et al., <xref rid=\"gh2178-bib-0047\" ref-type=\"ref\">2019</xref>). These indicators have limitations: the column integrated measures are estimated only during daytime and when clouds do not obscure the sun, MODIS satellite coverage is incomplete at the equator (often the study area is excluded), and most importantly, the relationship between AOD and surface PM concentrations is not direct. While our sampling did not discern impacts of Harmattan events, these events can greatly elevate PM levels (Aboh et al., <xref rid=\"gh2178-bib-0001\" ref-type=\"ref\">2009</xref>). For example, 24‐hr PM<sub>2.5</sub> and PM<sub>10</sub> levels in Accra reached 350 and 449 μg/m<sup>3</sup>, respectively, in the midst of a severe Harmattan dust event (January 11, 2011) and the PM<sub>2.5</sub>/PM<sub>10</sub> fraction reached ~80%, compared to 50–60% in non‐Harmattan season (Ahiamadjie, <xref rid=\"gh2178-bib-0004\" ref-type=\"ref\">2017</xref>). As noted earlier, EPA data (Tables <xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S3</xref>‐<xref rid=\"gh2178-supitem-0001\" ref-type=\"supplementary-material\">S5</xref>) also showed higher PM levels during the Harmattan season.</p></sec><sec id=\"gh2178-sec-0020\"><label>4.4</label><title>Air Quality Assessment, and Study Strengths and Limitations</title><p>This study combined integrated sampling using gravimetric measurements, which are usually required in regulatory and compliance applications, with continuous optical measurements, which are increasingly used in environmental and other applications (Kumar & Gurjar, <xref rid=\"gh2178-bib-0026\" ref-type=\"ref\">2019</xref>; Morawska et al., <xref rid=\"gh2178-bib-0030\" ref-type=\"ref\">2018</xref>; Mukherjee et al., <xref rid=\"gh2178-bib-0032\" ref-type=\"ref\">2017</xref>). The accuracy, precision and reliability of the optical sensors depend on the instrument, PM characteristics (e.g., shape, density and reflectivity) and meteorology (Belosi et al., <xref rid=\"gh2178-bib-0010\" ref-type=\"ref\">2013</xref>; Mukherjee et al., <xref rid=\"gh2178-bib-0032\" ref-type=\"ref\">2017</xref>; Njalsson & Novosselov, <xref rid=\"gh2178-bib-0035\" ref-type=\"ref\">2018</xref>), thus, performance and the need for site‐specific calibrations should be evaluated (Binnig et al., <xref rid=\"gh2178-bib-0011\" ref-type=\"ref\">2007</xref>; Walser et al., <xref rid=\"gh2178-bib-0048\" ref-type=\"ref\">2017</xref>). As shown by the diurnal and pollution rose plots, the continuous measurements provided by sensors can be highly informative.</p><p>Regardless of the instrumentation used, this study shows the importance of an appropriate study design and quality assurance procedures. Our design included: use of both gravimetric and optical sensors to assess the need for correction factors (the gravimetric samples also collected PM for future chemical analyses); use of upwind sites to assess background, e‐waste and downwind impacts; documentation of “microinventories” around monitoring sites that might affect PM levels and interpretations; examination of regional sources and seasonal variation by obtaining sufficient observations and by examining other monitoring and satellite data; collection and analysis of local meteorological information to select monitoring sites and understand collected measurements; and analyses that applied rigorous QA checks, provided appropriate averaging, identified potential outliers, examined temporal, spatial and directional patterns, and used robust statistics.</p><p>We recognize limitations of the study. Due to logistical issues, equipment failures, QA checks, and other reasons, our data record had gaps and coverage was incomplete, particularly during the Harmattan season. Upwind, on‐site and downwind levels cannot be fully captured using only three monitoring sites for such a complex environment as Agbogbloshie. The difficulties in establishing appropriate sites should not be underestimated. Continuous monitoring would enable other types of analyses. (The closest known site with continuous hourly or daily PM measurements is in Bamako, Senegal at the US Embassy; the U.S. Embassy in Accra is to start monitoring in 2020). RH measurements at each site might aid detection of RH‐induced artifacts. While perhaps a minor issue in Accra given the very consistent wind patterns, analysis during precipitation events, the Harmattan season, and other periods could be informative. Chemical analysis of the PM, and collection of additional pollutants, would aid source identification.</p></sec></sec><sec id=\"gh2178-sec-0021\"><label>5</label><title>Conclusions</title><p>We characterized ambient PM levels at the Agbogbloshie e‐waste recycling site and nearby communities using gravimetric and optical instruments and several analysis techniques. Levels at the site were significantly elevated over the background level, which itself was often high, mainly due to biomass burning and vehicle emissions. Very high PM levels, which sometimes overloaded the instruments, typically occurred in the evening due to nearby waste and biomass fires. Overall, we demonstrate that low to moderate cost instrumentation, with some modifications in hardware, appropriate data cleaning, and attention to understanding local sources, meteorology and background levels, can be used to characterize the spatial and temporal variation in PM levels in complex urban and industrial areas. The dearth of air quality information in low‐ and middle‐income countries (LMICs), where pollution levels often far exceed air quality standards, can be addressed using such approaches.</p></sec><sec sec-type=\"COI-statement\" id=\"gh2178-sec-0023\"><title>Conflict of Interest</title><p>The authors declare no conflicts of interest relevant to this study.</p></sec><sec sec-type=\"supplementary-material\"><title>Supporting information</title><supplementary-material content-type=\"local-data\" id=\"gh2178-supitem-0001\"><caption><p>Supporting Information S1</p></caption><media xlink:href=\"GH2-4-e2020GH000247-s001.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"gh2178-sec-0022\"><title>Acknowledgments</title><p>We acknowledge funding from the West Africa‐Michigan CHARTER in GEOHealth which is supported by the United States National Institutes of Health (NIH)/Fogarty International Center grants 1U2RTW010110‐01 and 5U01TW010101, and from Canada’s International Development Research Center grant 108121‐001. Additional support for this research was provided by grant P30ES017885 from the National Institute of Environmental Health Sciences, NIH. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We appreciate the assistance and cooperation of our field staff, the Ghana EPA, and the e‐waste leaders. The 1‐hr and 24‐hr PM data utilized in this paper is available in the Deep Blue data repository at <ext-link ext-link-type=\"uri\" xlink:href=\"https://doi.org/10.7302/fjst-6n49\">https://doi.org/10.7302/fjst-6n49</ext-link>.</p></ack><ref-list id=\"gh2178-bibl-0001\" content-type=\"cited-references\"><title>References</title><ref id=\"gh2178-bib-0001\"><mixed-citation publication-type=\"journal\" id=\"gh2178-cit-0002\">\n<string-name>\n<surname>Aboh</surname>, <given-names>I. J. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Cell Dev. Biol.</journal-id><journal-title-group><journal-title>Frontiers in Cell and Developmental Biology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-634X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850834</article-id><article-id pub-id-type=\"pmc\">PMC7431653</article-id><article-id pub-id-type=\"doi\">10.3389/fcell.2020.00715</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cell and Developmental Biology</subject><subj-group><subject>Mini Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Mechanism of Activation of Mechanistic Target of Rapamycin Complex 1 by Methionine</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Kitada</surname><given-names>Munehiro</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/148565/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Xu</surname><given-names>Jing</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/967851/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ogura</surname><given-names>Yoshio</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/703690/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Monno</surname><given-names>Itaru</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/703722/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Koya</surname><given-names>Daisuke</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/78927/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Diabetology and Endocrinology, Kanazawa Medical University</institution>, <addr-line>Uchinada</addr-line>, <country>Japan</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Division of Anticipatory Molecular Food Science and Technology, Medical Research Institute, Kanazawa Medical University</institution>, <addr-line>Uchinada</addr-line>, <country>Japan</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Giovanni Corsetti, University of Brescia, Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Michael A. Kalwat, University of Texas Southwestern Medical Center, United States; Reinhard Christoph Dechant, ETH Zürich, Switzerland</p></fn><corresp id=\"c001\">*Correspondence: Munehiro Kitada, <email>kitta@kanazawa-med.ac.jp</email></corresp><corresp id=\"c002\">Daisuke Koya, <email>koya0516@kanazawa-med.ac.jp</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Signaling, a section of the journal Frontiers in Cell and Developmental Biology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>715</elocation-id><history><date date-type=\"received\"><day>26</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>13</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Kitada, Xu, Ogura, Monno and Koya.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Kitada, Xu, Ogura, Monno and Koya</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Nutrients are closely involved in the regulation of lifespan and metabolic health. Cellular activities, such as the regulation of metabolism, growth, and aging, are mediated by a network of nutrients and nutrient-sensing pathways. Among the nutrient-sensing pathways, the mechanistic target of rapamycin complex 1 (mTORC1) acts as the central regulator of cellular functions, which include autophagy. Autophagy plays a significant role in the removal of protein aggregates and damaged or excess organelles, including mitochondria, to maintain intracellular homeostasis, which is involved in lifespan extension and cardiometabolic health. Moreover, dietary methionine restriction may have a beneficial effect on lifespan extension and metabolic health. In contrast, methionine may activate mTORC1 and suppress autophagy. As the mechanism of methionine sensing on mTORC1, SAMTOR was identified as a sensor of <italic>S</italic>-adenosyl methionine (SAM), a metabolite of methionine, in the cytoplasm. Conversely, methionine may activate the mTORC1 signaling pathway through the activation of phosphatase 2A (PP2A) because of increased methylation in response to intracellular SAM levels. In this review, we summarized the recent findings regarding the mechanism via which methionine activates mTORC1.</p></abstract><kwd-group><kwd>methionine</kwd><kwd><italic>S</italic>-adenosyl methionine</kwd><kwd>mechanistic target of rapamycin complex 1</kwd><kwd>autophagy</kwd><kwd>SAMTOR</kwd><kwd>phosphatase 2A methylation</kwd></kwd-group><counts><fig-count count=\"2\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"59\"/><page-count count=\"7\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>All organisms adapt and respond to the nutrients available in the environment. Cellular activities, including the regulation of metabolism, cell growth, and aging, are mediated by a network that comprised nutrients and nutrient-sensing pathways (<xref rid=\"B9\" ref-type=\"bibr\">Efeyan et al., 2015</xref>). Dietary interventions, such as calorie or dietary restriction and protein restriction, have been widely explored for their impact on lifespan extension or the prevention of age-related diseases through effects on cardiometabolic health. Calorie or dietary restriction without malnutrition has been demonstrated to extend the lifespan of organisms and improve their cardiometabolic health (<xref rid=\"B7\" ref-type=\"bibr\">Colman et al., 2009</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Fontana et al., 2010</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Mattison et al., 2012</xref>, <xref rid=\"B28\" ref-type=\"bibr\">2017</xref>). However, recent studies have reported that protein restriction, rather than calorie or dietary restriction, is more strongly involved in the lifespan extension and cardiometabolic health (<xref rid=\"B33\" ref-type=\"bibr\">Nakagawa et al., 2012</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Levine et al., 2014</xref>; <xref rid=\"B46\" ref-type=\"bibr\">Solon-Biet et al., 2014</xref>; <xref rid=\"B45\" ref-type=\"bibr\">Simpson et al., 2017</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Kitada et al., 2019</xref>). Moreover, accumulated evidence from experimental studies indicates that the restriction of specific amino acids, such as branched-chain amino acids (BCAAs) or methionine, promotes longevity and cardiometabolic health (<xref rid=\"B12\" ref-type=\"bibr\">Fontana et al., 2016</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Lee et al., 2016</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Cummings et al., 2018</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Kitada et al., 2019</xref>), which possibly mediates the benefits of protein restriction.</p><p>Among the nutrient-sensing pathways, the mechanistic target of rapamycin complex 1 (mTORC1) is a serine/threonine protein kinase that acts as the central regulator of cell growth and metabolism in response to the changes in nutrients or growth factors (<xref rid=\"B20\" ref-type=\"bibr\">Kim and Guan, 2019</xref>). Numerous studies on the pharmacological inhibition of mTORC1 by rapamycin have demonstrated the lifespan-extension benefit of this approach (<xref rid=\"B16\" ref-type=\"bibr\">Harrison et al., 2009</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Anisimov et al., 2011</xref>; <xref rid=\"B51\" ref-type=\"bibr\">Wilkinson et al., 2012</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Miller et al., 2014</xref>; <xref rid=\"B56\" ref-type=\"bibr\">Zhang et al., 2014</xref>), which suggest that mTORC1 is closely involved in lifespan regulation. The mechanism via which the suppression of mTORC1 leads to lifespan extension includes the induction of the autophagy (<xref rid=\"B20\" ref-type=\"bibr\">Kim and Guan, 2019</xref>). Autophagy is a lysosomal degradation pathway that plays an important role in the removal of protein aggregates and damaged or excess organelles, such as mitochondria, to maintain homeostasis and cell function (<xref rid=\"B32\" ref-type=\"bibr\">Mizushima et al., 2008</xref>). An appropriate autophagy may protect cells against various age-related stress conditions, which results in lifespan extension and cardiometabolic health (<xref rid=\"B54\" ref-type=\"bibr\">Wong et al., 2020</xref>). mTORC1 has been recognized as a crucial regulator of autophagy, and amino acids are one of the strong factors that affect mTORC1 activation (<xref rid=\"B20\" ref-type=\"bibr\">Kim and Guan, 2019</xref>). Thus, the beneficial effect of protein restriction on lifespan extension may be mediated through the induction of autophagy via the suppression of mTORC1 under amino-acid restriction. Recent findings have clarified that essential amino acids, including BCAAs or methionine, are possibly related to the regulation of the aging process, lifespan, and cardiometabolic health through multiple physiological and molecular mechanisms. In particular, the mechanisms underlying the role of methionine in the regulation of aging or lifespan have been widely investigated through dietary intervention via the application of a methionine restriction diet. Among these mechanisms, the involvement of methionine in the regulation of mTORC1 and autophagy has been elucidated based on the results of those studies. In the current review, we summarized the recent findings regarding the mechanism of mTORC1 activation by methionine.</p></sec><sec id=\"S2\"><title>Role of Rags on the Regulation of mTORC1 Activity by Amino-Acid Sensing</title><p>The mTORC1 activity is regulated by several molecules in response to changes in nutrients, including amino acids and growth factors. Moreover, the upstream component of the amino-acid–sensing pathway of mTORC1 is complicated (<xref rid=\"B20\" ref-type=\"bibr\">Kim and Guan, 2019</xref>). The regulation of mTORC1 activity by amino acids occurs through the translocation and localization of mTORC1 to lysosomes. The heterodimers of low-molecular-weight GTPases, RagA or B, and RagC, or D (<xref rid=\"B19\" ref-type=\"bibr\">Kim et al., 2008</xref>; <xref rid=\"B40\" ref-type=\"bibr\">Sancak et al., 2008</xref>; <xref rid=\"B2\" ref-type=\"bibr\">Anandapadamanaban et al., 2019</xref>), which are localized in lysosomes, play an important role in the activation of mTORC1 by amino acids. RagA and RagC exist as a dimer, and the GTP-bound form of RagA is its active form, whereas the GDP-bound form of RagC is its active form. In the presence of amino acids, these proteins function as activated GTP-RagA or GDP-RagC. In contrast, under amino-acid starvation, they function as a combination of inactivated GDP-RagA or GTP-RagC. The activated Rag dimer binds to Raptor, which is a major component of mTORC1, and participates in the translocation and localization of mTORC1 from the cytoplasm to lysosomes. Thereafter, in the lysosome, mTORC1 is activated by GTP-Rheb.</p><p>The GATOR1 and GATOR2 complexes are recognized as Rag regulators and are localized in the cytoplasm (<xref rid=\"B4\" ref-type=\"bibr\">Bar-Peled et al., 2013</xref>). GATOR1 is a complex composed of three proteins, DEPDC5, NPRL2, and NPRL3, and has RagA-binding ability and GTPase-activating protein (GAP) activity for RagA (<xref rid=\"B43\" ref-type=\"bibr\">Shen et al., 2018</xref>). DEPDC5 of GATOR1 contains a GAP domain, which binds directly to RagA, thus inactivating it. However, deletion of the GATOR1 component results in the amino-acid–independent localization and activation of mTORC1 in the lysosome, which demonstrates that GATOR1 is a negative regulator of mTORC1. In contrast, GATOR2 is a complex consisting of five proteins, Sec13, Seh1L, WDR24, WDR59, and Mios (<xref rid=\"B4\" ref-type=\"bibr\">Bar-Peled et al., 2013</xref>). GATOR2 binds to GATOR1; GATOR2 acts as the positive regulator of mTORC1 by suppressing the GAP activity of GATOR1. Leucine and arginine bind to sestrin1/2 and CASTOR1, respectively, and sestrin1/2 and CASTOR1 are also recognized as sensors of leucine or arginine (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). Amino-acid–bound sensor proteins dissociate from GATOR2, thus losing their ability to inactivate GATOR2 (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Chantranupong et al., 2014</xref>, <xref rid=\"B5\" ref-type=\"bibr\">2016</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Parmigiani et al., 2014</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Saxton et al., 2016a</xref>, <xref rid=\"B42\" ref-type=\"bibr\">b</xref>; <xref rid=\"B52\" ref-type=\"bibr\">Wolfson et al., 2016</xref>). Consequently, the activated GATOR2 triggers the activation of mTORC1 through the inactivation of GATOR1. Conversely, during leucine or arginine starvation, sestrin1/2 and CASTOR1 bind to GATOR2 and inactivate GATOR2, which results in mTORC1 inactivation via an increase in the RagA GAP activity of GATOR1 (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Sestrin1/2, CASTOR1, and SAMTOR are cytosolic sensors of leucine, arginine, and SAM for the regulation of mTORC1. <bold>(A)</bold> Sestrin1/2 and CASTOR1 sense leucine and arginine in the cytoplasm, respectively. Upon binding of leucine or arginine to sestrin1/2 and CASTOR1, these proteins dissociate from GATOR2, which releases their suppressive effect on GATOR2. GATOR2 suppresses GATOR1, which consequently activates mTORC1 via GTP-bounded RagA formation by GAP inactivation. <bold>(B)</bold> In conditions of low leucine and arginine levels, sestrin1/2 and CASTOR1 suppress GATOR2 (the negative regulator of GATOR1), which leads to the activation of GATOR1 and results in the suppression of mTORC1 via GAP activation of RagA. <bold>(C)</bold> (1) Ragulator releases GTP from RagC; (2) SLC38A9 is activated by arginine in the lysosome; (3) SLC38A9 converts RagA from the GDP- to the GTP-bound state, leading to the activation of the Rags; (4) Ragulator and SLC38A9 recruit the mTORC1 to the lysosomal surface; and (5) mTORC1 is activated. <bold>(D)</bold> SAMTOR senses SAM in the cytoplasm. Upon the binding of SAM to SAMTOR, SAMTOR dissociates from GATOR1. The disruption of the SAMTOR–GATOR1 complex leads to the inactivation of GATOR1, which results in mTORC1 activation via the inhibition of GAP activation and increased GTP binding to RagA. <bold>(E)</bold> In conditions of low levels of SAM, the SAMTOR–GATOR1 complex suppresses mTORC1 activity. SAM, <italic>S</italic>-adenosyl methionine; mTORC1, mechanistic target of rapamycin complex 1; GAP, GTPase-activating protein.</p></caption><graphic xlink:href=\"fcell-08-00715-g001\"/></fig><p>In addition to the cytosolic amino-acid–sensing branch, Shen and Sabatini reported that Ragulator and SLC38A9 are two critical regulators of the activation of mTORC1 as the lysosomal amino-acid–sensing branch (<xref rid=\"B44\" ref-type=\"bibr\">Shen and Sabatini, 2018</xref>) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref>). Ragulator tethers the Rag heterodimer to the lysosomal surface, and the SLC38A9 transmembrane protein is a lysosomal arginine sensor that stimulates mTORC1 activity through the regulation of Rags. Ragulator and SLC38A9 are guanine exchange factors that lead the Rags toward the active form (<xref rid=\"B44\" ref-type=\"bibr\">Shen and Sabatini, 2018</xref>). Ragulator triggers GTP release from RagC, thus lifting the locked inactivated state of the Rags (<xref rid=\"B44\" ref-type=\"bibr\">Shen and Sabatini, 2018</xref>). Upon arginine binding, SLC38A9 converts RagA from the GDP- to the GTP-bound state, leading to the activation of the Rags (<xref rid=\"B44\" ref-type=\"bibr\">Shen and Sabatini, 2018</xref>). Thus, Ragulator and SLC38A9 activate mTORC1 by recruiting it to the lysosomal surface via Rag activation in response to arginine levels in the lysosome. Moreover, v-ATPase interacts with Ragulator, Rags, and SLC38A9 and is involved both in amino-acid–sensing and in efflux from the lysosome (<xref rid=\"B59\" ref-type=\"bibr\">Zoncu et al., 2011</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Abu-Remaileh et al., 2017</xref>; <xref rid=\"B55\" ref-type=\"bibr\">Wyant et al., 2017</xref>). However, it remains unknown whether v-ATPase senses amino acids.</p></sec><sec id=\"S3\"><title>Methionine-Induced mTORC1 Activation and the Role of Samtor as a Sam Sensor That Provides a Link to the Methionine Metabolism</title><p>The KICSTOR complex is one of the regulators of mTORC1 and comprises kaptin (KPTN), the integrin-α FG-GAP repeat-containing protein 2 (ITFG2), C12orf66, and seizure threshold 2 (SZT2) (<xref rid=\"B53\" ref-type=\"bibr\">Wolfson et al., 2017</xref>). C7orf60 was identified as an interacting protein of GATOR1 and was subsequently renamed SAMTOR (<xref rid=\"B15\" ref-type=\"bibr\">Gu et al., 2017</xref>). The overexpression of SAMTOR suppresses mTORC1 activity, which indicates that SAMTOR is a negative regulator of mTORC1. SAM is converted from methionine, and methionine starvation reduces the concentration of SAM in the cytoplasm. When present of SAM, SAM binds to SAMTOR, which then dissociates from GATOR1 (<xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>). The disruption of the SAMTOR–GATOR1 complex leads to the inactivation of GATOR1, which then results in mTORC1 activation through the inhibition of GAP activation and increased binding of GTP to RagA. In contrast, methionine starvation reduces SAM levels below the dissociation constant of the SAM–SAMTOR complex, thus promoting SAMTOR–GATOR1 binding and, in turn, suppressing mTORC1 activity (<xref ref-type=\"fig\" rid=\"F1\">Figure 1E</xref>). However, loss of SAMTOR activates mTORC1, even in conditions of methionine starvation. In addition, SAMTOR mutants that cannot bind to SAM fail to transmit methionine sufficiently to mTORC1, therefore suppressing mTORC1. These results indicate that SAMTOR serves as a SAM sensor in the methionine-mediated mTORC1 activation.</p></sec><sec id=\"S4\"><title>Role of the Induction of the Methylation of PP2A by Sam in mTORC1 Activation</title><p>The study performed by Sutter et al. also showed that methionine regulates the mTORC1 signaling pathway and autophagy through the regulation of the methylation status of phosphatase 2A (PP2A) in yeast (<xref rid=\"B49\" ref-type=\"bibr\">Sutter et al., 2013</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Laxman et al., 2014</xref>). In the presence of high levels of intracellular SAM, Ppm1 induces the methylation of the catalytic subunit of PP2A in response to SAM concentration. PP2A is activated by its methylation; thereafter, methylated PP2A can suppress Npr2 through its dephosphorylation, which results in mTORC1 activation and the suppression of autophagy (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>). The complex consisting of Npr2, Npr3, and Iml1 (NPRL2, NPRL3, and DEPDC5 in mammals, respectively) is termed SEACIT in yeast (GATOR1 in mammals, as described above) (<xref rid=\"B37\" ref-type=\"bibr\">Panchaud et al., 2013</xref>) and functions as a negative regulator of mTORC1 via a GAP activity toward the yeast Rag orthologs, that is, Gtr1/2 (Rags family in mammals) (<xref rid=\"B14\" ref-type=\"bibr\">Gao and Kaiser, 2006</xref>). Therefore, suppression of SEACIT by the dephosphorylation of Npr2 induced by the activation of PP2A results in the activation of mTORC1. In contrast, lower SAM levels in cells reduce the methylation levels of PP2A and promote the phosphorylation of Npr2, which results in the suppression of mTORC1 activity and the induction of autophagy (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). In mammalian cells, the methylation of PP2A is catalyzed by a specific <italic>S-</italic>adenosyl methionine (SAM)–dependent methyltransferase, the leucine carboxyl methyltransferase 1 (LCMT1) (<xref rid=\"B47\" ref-type=\"bibr\">Stanevich et al., 2011</xref>). Activated PP2A possibly dephosphorylates NPRL2 and results in mTORC1 activation in mammalian cells; however, no report has shown whether PP2A is directly involved in the regulation of the phosphorylation state of NPRL2. Therefore, further studies are necessary to clarify this issue.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Regulation of mTORC1 via the methylation of PP2A in response to SAM. <bold>(A)</bold> In conditions of high levels of intracellular SAM in yeast, Ppm1 induces the methylation of the catalytic subunit of PP2A in response to SAM concentration. The activated (methylated) form of PP2A suppresses Npr2 through its dephosphorylation. The complex consisting of Npr2, Npr3, and Iml1 (SEACIT) is a negative regulator of mTORC1; therefore, the suppression of SEACIT via the dephosphorylation of Npr2 results in the activation of mTORC1. In mammalian cells, LCMT1 induces the methylation of the catalytic subunit of PP2A in response to SAM concentration, leading to the activation of mTORC1, possibly through the activation of GATOR1. Moreover, in mammalian cells, PP2A possibly regulates the phosphorylation levels of NPRL2 in response to SAM levels. <bold>(B)</bold> Lower SAM levels reduce the methylation levels of PP2A in yeast and mammalian cells and promote the activation of Npr2 via its phosphorylation, which results in the suppression of mTORC1 activity. In mammalian cells, PP2A possibly regulates the phosphorylation levels of NPRL2 in response to SAM levels. mTORC1, mechanistic target of rapamycin complex 1; PP2A, phosphatase 2A; SAM, <italic>S</italic>-adenosyl methionine; GAP, GTPase-activating protein; LCMT1, leucine carboxyl methyltransferase 1.</p></caption><graphic xlink:href=\"fcell-08-00715-g002\"/></fig><p>We also reported that a low-protein diet ameliorates diabetes-induced kidney injury and that dietary methionine abrogates the beneficial effects of a low-protein diet in diabetic kidneys (<xref rid=\"B22\" ref-type=\"bibr\">Kitada et al., 2020</xref>). More specifically, diabetic rats that were fed a low-protein + methionine diet exhibited increased expression of LCMT1 and methyl-PP2A compared with control (standard-diet–fed) and low-protein-diet–fed diabetic rats, which was accompanied by an increase in renal SAM levels. Although the expression of glycine <italic>N</italic>-methyltransferase (Gnmt), which is a SAM-converted enzyme, was decreased in diabetic rat kidneys, changes in renal SAM levels were dependent on the dietary methionine content (<xref rid=\"B22\" ref-type=\"bibr\">Kitada et al., 2020</xref>). Consistent with the alteration of LCMT1 and methyl-PP2A, mTORC1 activation and autophagy suppression were observed in standard-diet–fed and low-protein + methionine–fed diabetic rats. Furthermore, we also used cultured human kidney-2 cells to confirm that the administration of SAM-induced methylated PP2A increased the expression of methyl-PP2A and activated mTORC1 (<xref rid=\"B22\" ref-type=\"bibr\">Kitada et al., 2020</xref>). However, the involvement of SAM-induced methylated PP2A in mTORC1 activation through NPRL2 and the activation of the negative regulator of mTORC1 by its increased phosphorylation, such as that observed for Npr2 in yeast, remain unknown.</p></sec><sec id=\"S5\"><title>Methionine Activates mTORC1 Through TAS1R1/TAS1R3</title><p>Nelson et al. previously identified a mammalian amino-acid taste receptor, the taste 1 receptor member 1 (TAS1R1)/taste 1 receptor member 3 (TAS1R3) heterodimer, which is a cell-surface G-protein–coupled receptor (<xref rid=\"B34\" ref-type=\"bibr\">Nelson et al., 2002</xref>). This receptor broadly functions as an amino-acid sensor that responds to most of the 20 standard amino acids. Upon sensing amino acids, this receptor activates mTORC1 through the activation of phospholipase C, the increase in intracellular calcium, and the activation of the mitogen-activated protein kinase 1/mitogen-activated protein kinase 3 (<xref rid=\"B50\" ref-type=\"bibr\">Wauson et al., 2015</xref>). TAS1R1–TAS1R3 is required for the amino-acid–induced mTORC1 localization to the lysosome, which is a necessary step in mTORC1 activation. Several reports have demonstrated that TAS1R1–TAS1R3 may serve as a sensor of extracellular methionine and that it activates mTORC1 in cultured C2C12 cells and bovine epithelial cells (<xref rid=\"B57\" ref-type=\"bibr\">Zhou et al., 2016</xref>, <xref rid=\"B58\" ref-type=\"bibr\">2018</xref>).</p></sec><sec id=\"S6\"><title>Discussion</title><p>In this study, we described the recent findings regarding the mechanism via which methionine induces the activation of mTORC1. mTORC1 may be activated by sensing SAM rather than methionine. A previous report by Obata et al. provided evidence that SAM, rather than methionine, may be the main contributor to the aging process (<xref rid=\"B35\" ref-type=\"bibr\">Obata and Miura, 2015</xref>). Those authors showed that increasing SAM catabolism via the action of glycine <italic>N</italic>-methyltransferase (Gnmt) extends the lifespan in <italic>Drosophila</italic>. In particular, SAM is upregulated in older flies, even if the transcription of Gnmt is induced in a forkhead box O (FOXO)–dependent manner. However, overexpression of Gnmt suppresses the age-dependent increase in SAM and extends lifespan in <italic>Drosophila</italic>. In addition, metabolic impairment, such as insulin resistance in obesity, is closely involved in the aging process. A previous report demonstrated that plasma SAM concentrations were related to higher fasting insulin levels, the homeostasis model assessment of insulin resistance, and the tumor necrosis factor α in a cross-sectional study that involved subjects with metabolic syndrome (<xref rid=\"B27\" ref-type=\"bibr\">Lind et al., 2018</xref>). Another report also revealed that plasma SAM, and not methionine, is independently related to fat mass and truncal adiposity in a cross-sectional study involving elderly individuals (<xref rid=\"B11\" ref-type=\"bibr\">Elshorbagy et al., 2013</xref>); in contrast, overfeeding increases serum SAM in proportion to the fat mass gained (<xref rid=\"B10\" ref-type=\"bibr\">Elshorbagy et al., 2016</xref>). Thus, the upregulation of SAM associated with overfeeding or metabolic dysfunction may be involved in whole-body metabolic impairment. These data indicate that increased levels of SAM in the process of methionine metabolism may be related to the stimulation of aging and metabolic impairment, including insulin resistance, which is particularly associated with obesity. Previous reports have shown that dietary methionine restriction extends the lifespan or improves cardiometabolic health (<xref rid=\"B36\" ref-type=\"bibr\">Orentreich et al., 1993</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Miller et al., 2005</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Hasek et al., 2010</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Plaisance et al., 2011</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Johnson and Johnson, 2014</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Lee et al., 2014</xref>; <xref rid=\"B48\" ref-type=\"bibr\">Stone et al., 2014</xref>). The effect of methionine restriction on lifespan extension or cardiometabolic health may be exerted through multiple mechanisms, including antioxidative stress, the production of hydroxy sulfates, the downregulation of GH/insulin growth factor 1 signaling, the production of fibroblast growth factor 21, the suppression of mTORC1, and the induction of autophagy (<xref rid=\"B21\" ref-type=\"bibr\">Kitada et al., 2019</xref>). Among them, the suppression of mTORC1 is induced by decreasing SAM levels. Therefore, the regulation of SAM levels and sensing of SAM in the cytoplasm may be key factors in the mechanism of lifespan extension, which may be mediated by the regulation of mTORC1. Because the selective suppression of mTORC1 induced by SAM may be a therapeutic target for aging, metabolic impairment, or aging-related disease, further studies are necessary to address these issues.</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>MK designed the manuscript, the guarantor of this work, and wrote and edited the manuscript. JX, YO, IM, and DK contributed to the discussion. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>Boehringer Ingelheim, Mitsubishi Tanabe Pharma, Kyowa Kirin, Taisho Pharmaceutical Co., Ltd., and Ono Pharmaceutical Co., Ltd. contributed to establishing the Division of Anticipatory Molecular Food Science and Technology. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>This study was supported in part by the Japan-China Sasakawa Medical Fellowship to JX.</p></ack><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abu-Remaileh</surname><given-names>M.</given-names></name><name><surname>Wyant</surname><given-names>G. A.</given-names></name><name><surname>Kim</surname><given-names>C.</given-names></name><name><surname>Laqtom</surname><given-names>N. N.</given-names></name><name><surname>Abbasi</surname><given-names>M.</given-names></name><name><surname>Chan</surname><given-names>S. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Microbiol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Microbiol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Microbiol.</journal-id><journal-title-group><journal-title>Frontiers in Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-302X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849424</article-id><article-id pub-id-type=\"pmc\">PMC7431654</article-id><article-id pub-id-type=\"doi\">10.3389/fmicb.2020.01819</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Microbiology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Bacteria Modify Their Sensitivity to Chemerin-Derived Peptides by Hindering Peptide Association With the Cell Surface and Peptide Oxidation</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Godlewska</surname><given-names>Urszula</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/413290/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Bilska</surname><given-names>Bernadetta</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/938133/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Majewski</surname><given-names>Paweł</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Pyza</surname><given-names>Elzbieta</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/50674/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zabel</surname><given-names>Brian A.</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Cichy</surname><given-names>Joanna</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/306841/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Immunology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University</institution>, <addr-line>Kraków</addr-line>, <country>Poland</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Cell Biology and Imaging, Institute of Zoology and Biomedical Research, Jagiellonian University</institution>, <addr-line>Kraków</addr-line>, <country>Poland</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Palo Alto Veterans Institute for Research, VA Palo Alto Health Care System</institution>, <addr-line>Palo Alto, CA</addr-line>, <country>United States</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Santi M. Mandal, Indian Institute of Technology Kharagpur, India</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Piyush Baindara, University of Missouri, United States; Dennis Ken Bideshi, California Baptist University, United States</p></fn><corresp id=\"c001\">*Correspondence: Joanna Cichy, <email>Joanna.Cichy@uj.edu.pl</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1819</elocation-id><history><date date-type=\"received\"><day>04</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>10</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Godlewska, Bilska, Majewski, Pyza, Zabel and Cichy.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Godlewska, Bilska, Majewski, Pyza, Zabel and Cichy</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Chronic inflammatory skin diseases like psoriasis alter the local skin microbiome and lead to complications such as persistent infection with opportunistic/pathogenic bacteria. Disease-associated changes in microbiota may be due to downregulation of epidermal antimicrobial proteins/peptides, such as antimicrobial protein chemerin. Here, we show that chemerin and its bioactive derivatives have differential effects on the viability of different genera of cutaneous bacteria. The lethal effects of chemerin are enhanced by bacterial-derived ROS-induced chemerin peptide oxidation and suppressed by stationary phase sigma factor RpoS. Insight into the mechanisms underlying changes in the composition of cutaneous bacteria during autoreactive skin disease may provide novel ways to mobilize chemerin and its peptide derivatives for maximum antimicrobial efficacy.</p></abstract><kwd-group><kwd>chemerin</kwd><kwd>antimicrobial peptide</kwd><kwd>skin</kwd><kwd>psoriasis</kwd><kwd>sigma factor</kwd></kwd-group><counts><fig-count count=\"5\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"28\"/><page-count count=\"11\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>To prevent pathologic outcomes, the skin must continuously confront a wide and diverse array of bacterial challenges. The primary line of defense relies on keratinocytes and their ability to secrete antimicrobial peptides. Among keratinocyte-derived factors equipped with antimicrobial potential is chemerin. Chemerin is a secreted multifunctional protein that is known mainly for its properties to support immune cell infiltration to inflammatory sites and regulate differentiation of adipocytes (<xref rid=\"B23\" ref-type=\"bibr\">Wittamer et al., 2003</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Zabel et al., 2005b</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Goralski et al., 2007</xref>). Chemerin is secreted as a functionally inert precursor protein (Chem163S, with number and capital letter referring to the terminal amino acid position and single amino acid code). Chem163S can be converted to active isoforms through posttranslational carboxyl-terminal processing. Proteolytic cleavage at serine 157 in the carboxyl-terminus of Chem163S results in generation of Chem157S isoform that is effective in triggering chemotaxis of several types of immune cells (<xref rid=\"B24\" ref-type=\"bibr\">Yamaguchi et al., 2011</xref>). This isoform also exhibits much stronger growth inhibitory potential compared with Chem163S against bacteria (<xref rid=\"B16\" ref-type=\"bibr\">Kulig et al., 2011</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Godlewska et al., 2017</xref>). However, in contrast to C-terminal region responsible for chemotactic potential, antimicrobial activity is mainly associated with a domain localized in the middle of the chemerin sequence, Val<sup>66</sup>-Pro<sup>85</sup> peptide (p4), which embodies the majority of chemerin’s anti-microbial activity (<xref rid=\"B3\" ref-type=\"bibr\">Banas et al., 2013</xref>). Therefore, to exhibit potent antimicrobial activity, holoprotein Chem163S requires removal of a terminal inhibitory peptide, possibly to enable structural accessibility of its antimicrobial domain, and/or a release of its central antimicrobial peptide. Chem157S has been isolated from human biological specimens, including ascites and serum (<xref rid=\"B28\" ref-type=\"bibr\">Zabel et al., 2006</xref>). Although cutaneous chemerin isoforms remain to be identified, endogenous chemerin is largely responsible for the natural antimicrobial activity present in keratinocyte secretions (<xref rid=\"B3\" ref-type=\"bibr\">Banas et al., 2013</xref>), suggesting that chemerin isoform(s) capable of controlling bacteria growth are generated in the epidermis. Given that several chemerin receptors can retain chemerin on the cell surface (<xref rid=\"B26\" ref-type=\"bibr\">Zabel et al., 2014</xref>), structural features/changes in chemerin structure following binding to these receptors might enable the p4 domain to interact with bacteria. Since chemerin is highly susceptible to proteolytic cleavage (<xref rid=\"B26\" ref-type=\"bibr\">Zabel et al., 2014</xref>), it is also likely that p4 can be released from chemerin by endogenous (<xref rid=\"B22\" ref-type=\"bibr\">Wittamer et al., 2005</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Zabel et al., 2005a</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Schultz et al., 2013</xref>) or bacteria-derived proteases (<xref rid=\"B17\" ref-type=\"bibr\">Kulig et al., 2007</xref>) and act independently of the rest of chemerin protein. Synthetic p4 is effective in treating experimental <italic>S. aureus</italic> skin infections (<xref rid=\"B10\" ref-type=\"bibr\">Godlewska et al., 2019</xref>), suggesting that it could impact the clinical management of <italic>S. aureus</italic> and potentially other bacteria-mediated skin pathologies.</p><p>Despite our growing understanding of the local and systemic role of chemerin in immunity, the function of this protein at body barriers remains poorly understood. Chemerin is abundantly expressed by keratinocytes in healthy skin but it is markedly downregulated in the epidermis of patients suffering from the autoinflammatory skin disease psoriasis (<xref rid=\"B2\" ref-type=\"bibr\">Albanesi et al., 2009</xref>). Since the overall microbial community (microbiota) of normal and psoriatic skin can differ substantially (<xref rid=\"B9\" ref-type=\"bibr\">Gao et al., 2008</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Chang et al., 2018</xref>), active chemerin derivatives may contribute to skin pathophysiology by shaping the composition of the skin microbiota.</p><p>Here, we demonstrate differentiating potentials of chemerin isoforms and p4 in controlling cutaneous bacteria and identify novel bacteria-mediated mechanisms that influence the antimicrobial activity of chemerin peptide, which may modify the genus-, species-, and strain-level structure of the skin microbiome.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Bacterial Strains</title><p>The following laboratory or clinical reference strains were used in the study: <italic>Escherichia coli</italic> HB101, <italic>Escherichia coli</italic> DH5α, and <italic>Escherichia coli</italic> NiCo21(DE3)<italic>; Staphylococcus aureus</italic> strain 8325-4, <italic>Staphylococcus epidermidis</italic> DSM 20044, <italic>Staphylococcus hominis</italic> DSM 20328, <italic>Staphylococcus capitis</italic> DSM 20326, <italic>Staphylococcus caprae</italic> DSM 20608, <italic>Streptococcus mitis</italic> DSM 12643, <italic>Corynebacterium simulans</italic> DSM 44392, <italic>Corynebacterium tuberculostearicum</italic> DSM 44922, <italic>Cutibacterium acnes</italic> DSM 16379. The clinical reference strains were obtained from DSMZ German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig). <italic>Rhodobacter capsulatus</italic> (<italic>R. capsulatus</italic>) strains included <italic>R. capsulatus</italic> pMTS1/MTR<italic>bc</italic>1 strain overproducing cytochrome <italic>bc</italic><sub>1</sub> (WT), the MT-RBC1 knockout strain with a deletion of the operon coding for cytochrome <italic>bc</italic><sub>1</sub>, G167P strain with glycine 167 to proline mutation in the cytochrome <italic>b</italic> protein of the <italic>bc</italic><sub>1</sub>complex, and 2Ala with alanine insertion mutation in the iron-sulfur protein subunit of the <italic>bc</italic><sub>1</sub>complex (<xref rid=\"B5\" ref-type=\"bibr\">Borek et al., 2008</xref>, <xref rid=\"B4\" ref-type=\"bibr\">2015</xref>). <italic>R. capsulatus</italic> strains were kindly donated by Dr. A. Osyczka (Dept. of Molecular Biophysics, Jagiellonian University, Kraków, Poland).</p></sec><sec id=\"S2.SS2\"><title>Production of Recombinant Human Chemerin Isoforms</title><p>Recombinant human full-length chemerin variant Chem163S and chemerin variant Chem157S, lacking 6 aa at C-terminus, were produced in <italic>Escherichia coli</italic>. DNA fragments corresponding to the desired chemerin proteins were amplified by PCR and cloned into the pNIC28-Bsa4 expression vector (Addgene, LGC Standards, Teddington, United Kingdom) at a site preceded by the sequence coding for hexahistidine tag, using the overlap-extension PCR (<xref rid=\"B6\" ref-type=\"bibr\">Bryksin and Matsumura, 2010</xref>). Both constructs lacked the first 20 aa native chemerin signal peptide. The identity of the created pNIC28-Bsa4-chem157 or pNIC28-Bsa4-chem163 constructs was verified by sequencing (Genomed, Warsaw, Poland). Recombinant chemerin isoforms were expressed and purified in <italic>E. coli</italic> strain NiCo21(DE3) (New England Biolabs, MA, United States), transformed with the plasmids described above. Recombinant proteins were purified from the inclusion bodies of bacteria transformants, using Ni-Sepharose 6 Fast Flow, followed by Q Sepharose Fast Flow (both from GE Healthcare, Uppsala, Sweden). The recombinant chemerin variants were first eluted with 500 mM imidazole, and in the second purification step with 25–35 mM (Chem163S) or 35–50 mM (Chem157S) NaCl and dialyzed against PBS. The obtained proteins were routinely >90% pure as assessed by SDS-PAGE and Coomassie Blue staining.</p></sec><sec id=\"S2.SS3\"><title>Chemerin Peptide 4 (p4)</title><p>Peptide p4 was chemically synthesized by ChinaPeptide (Shanghai, China) at ≥95% purity. Biotin- or FITC-labeled p4 were synthesized by CASLO (Kongens Lyngby, Denmark) at ≥95% or ≥98% purity. Biotin was added directly at the N-terminus of p4. For FITC-labeled p4, C-terminal lysine was added to p4 and FITC was conjugated to the side chain of this C-terminal lysine. Both biotin-labeled and FITC-labeled p4 displayed similar antimicrobial activity to unmodified p4.</p></sec><sec id=\"S2.SS4\"><title>Antimicrobial Microdilution Assay (MDA)</title><p>For antimicrobial experiments all staphylococci and <italic>C. acnes</italic> were cultured in brain heart infusion broth (BHI) (Sigma-Aldrich). <italic>S. mitis</italic> and <italic>C. simulans</italic> were cultured in BHI supplemented with 0.3% yeast extract (Sigma-Aldrich), whereas <italic>C. tuberculostearicum</italic> was grown in BHI supplemented with 0.3% yeast extract and 1% Tween 80 (Sigma-Aldrich). Bacteria were grown under aerobic condition with two exceptions. <italic>C. acnes</italic> was grown in BHI in an anaerobic atmosphere using a GasPak<sup>TM</sup> EZ Anaerobe Pouch System (BD), and <italic>S. mitis</italic> was grown in a 5% CO2 atmosphere using a GasPak<sup>TM</sup> EZ CO2 Pouch System (BD). To determine the antimicrobial activity of chemerin and peptide p4 against skin-associated species, bacteria in mid-logarithmic phase were diluted to 4 × 10<sup>5</sup> CFU/ml with PBS containing a series of 2-fold dilution of p4, chemerin isoforms Chem157S and Chem163S (5 μM), or PBS (control) and incubated for 2 h. The number of viable bacteria were enumerated by colony forming units (CFU) counting. In the experiments with <italic>R. capsulatus</italic> strains, bacteria were grown protected from light in mineral-peptone-yeast extract (MPYE) at 30°C. Bacteria in mid-logarithmic phase were diluted to 4 × 10<sup>5</sup> CFU/ml with PBS containing 1% (v/v) of medium and preincubated with 2 μM antimycin (Sigma-Aldrich) for 15 min to promote ROS production by <italic>bc</italic><sub>1</sub>, before adding 2.5 μM p4. The number of viable bacteria were enumerated by CFU counting. To determine whether susceptibility to p4 is growth-dependent, <italic>E. coli</italic> WT and <italic>rpoS</italic> mutants were cultured in Luria-Bertani (LB) medium (Sigma-Aldrich), and when required supplemented with antibiotics. Bacteria in logarithmic or stationary growth phase were diluted to 4 × 10<sup>5</sup> CFU/ml with PBS containing a series of 2-fold dilution of p4 or PBS (control) and incubated for 2 h. The surviving bacteria were enumerated by CFU counting.</p></sec><sec id=\"S2.SS5\"><title>ATP Determination</title><p>Total ATP levels were determined using The BacTiter-Glo viability assay kit<sup>®</sup> (Promega), following the manufacturer’s instructions. In brief, bacteria in mid-logarithmic phase were diluted to 8 × 10<sup>6</sup> CFU/ml with PBS containing a series of 2-fold dilution of p4 or PBS (control). After incubation with p4, BacTiter-Glo reagent was added to each well (1:1 [v:v]) and incubated in darkness for 5 min at room temperature (RT). The luminescent signal was recorded using Tecan Infinite M200 Plate Reader.</p></sec><sec id=\"S2.SS6\"><title>Fluorometric Measurement of Membrane Potential</title><p>Membrane potential was measured by using a voltage-sensitive dye DiSC3(5) (Sigma-Aldrich). <italic>E. coli</italic> or <italic>S. epidermidis</italic> cultures were grown in BHI to the mid-logarithmic phase and diluted to an OD600 of 0.2. 15 mM. EDTA was added to <italic>E. coli</italic> dilution to facilitate the uptake of the DiSC3(5). DiSC3(5) was added to each well to a final concentration of 1 μM followed by 25 μM p4 to monitor the dissipation of membrane potential. Gramicidin (1 μM) (Sigma-Aldrich) was used as a positive control. The fluorescence at an excitation wavelength of 615 nm and an emission wavelength of 665 nm was measured using Tecan Infinite M200 Plate Reader.</p></sec><sec id=\"S2.SS7\"><title>Fluorescence Microscopy</title><p><italic>Escherichia coli</italic> in logarithmic or stationary phase (1 × 10<sup>8</sup> CFU) were incubated for indicated time with FITC-labeled p4. Cells were washed three times with PBS to remove the peptide, attached to slides by cytospin centrifugation, fixed in 3.7% paraformaldehyde (Sigma-Aldrich), and counterstained with 1 μg/mL Hoechst dye 33258 (Life Technologies) for 30 min at RT. Images were captured with a fluorescence microscope (Eclipse; Nikon) and analyzed using NIS-Elements (Nikon) Imaging Software AR 5.01.00. Nucleoid lengths were measured automatically. Stained nucleoids less than 42 μm in length or obtained from cell duplicates were excluded from data analysis. Quantification of FITC-p4 labeled cells was done manually using NIS-Elements software.</p></sec><sec id=\"S2.SS8\"><title>DNA Binding Assay</title><p>Total genomic DNA from <italic>E. coli</italic> was isolated using a DNA extraction kit (Thermo Fisher Scientific), according to the manufacturer’s instruction. Gel retardation experiments were performed by mixing 150 ng of the DNA and increasing amounts of p4 in buffer containing 10 mM Tris–HCl, 1 mM EDTA, pH 8.0. The mixtures were incubated at RT for 30 min, and subsequently analyzed by electrophoresis on a 0.8% agarose gel in the TBE buffer (90 mM Tris-borate, 2 mM EDTA, pH 8.3).</p></sec><sec id=\"S2.SS9\"><title>qRT-PCR</title><p>The total fraction of RNAs was isolated from the <italic>E</italic>. <italic>coli</italic> HB101 using Total RNA Zol-Out<sup>TM</sup> (A&A Biotechnology) and converted to cDNA using NxGen M-MulV reverse transcriptase (Lucigen) with random hexamers (Promega). Real time PCR was performed on the C1000 Thermal cycler (CFX96 Real Time System, Bio-Rad) using SYBR Green I containing universal PCR master mix (A&A Biotechnology) and primers specific for <italic>16S rRNA</italic>, 5′- TGTSTGCAYGGYTGTCGTCA-3′, 5′- ACGTCRTCCMCACCTTCCTC-3′; <italic>gyrA</italic>, 5′- CGAGCG CGGATATACACCTT-3′, 5′- TCCGGTATCGCCGTAGGTAT-3′ (Genomed).</p></sec><sec id=\"S2.SS10\"><title>Transmission Electron Microscopy (TEM)</title><p>5 × 10<sup>8</sup>\n<italic>E. coli, S. epidermidis</italic>, or <italic>S. aureus</italic> cells were treated with p4 or vehicle (PBS) for 2 h at 37°C. <italic>E. coli</italic> cell pellets were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) overnight at 4°C while Staphylococci pellets were washed three times in PBS for 5 min and fixed overnight in 2.5% glutaraldehyde in PBS at 4°C. <italic>E. coli</italic> was then washed in 0.1M sodium cacodylate buffer, post-fixed in 1% osmium tetroxide in 0.1M cacodylic buffer for 1 h at RT, washed again two times in the buffer and distilled water, and stained “en bloc” with 2% uranyl acetate aqueous solution for 1 h at RT. <italic>S. aureus</italic> was washed in PBS and post-fixed with 1% osmium tetroxide for 2 h at 4°C. Samples were embedded in epoxy resin (PolyBed 812; Polyscienses, Inc., Warrington, PA, United States) after dehydration in graded ethanol series (50–100%) and in propylene oxide. Ultrathin sections (65 nm) were cut using ultramicrotome (Leica EM UC7) and post-stained with uranyl acetate and lead citrate. Specimens were observed using a transmission electron microscope (JEOL JEM2100) operating at an accelerating voltage of 80 kV.</p></sec><sec id=\"S2.SS11\"><title>Immunogold Labeling</title><p><italic>Escherichia coli</italic> bacteria were treated with p4-biotin or unlabeled p4 as a control. Ultrathin sections of the bacteria on nickel grids were incubated with 4% sodium metaperiodate for 10 min, followed by 1% aqueous periodic acid for 10 min, and 1% fish skin gelatin (FSG) in PBS for 2 h. Sections were incubated with primary mouse anti-biotin Abs (clone 3D6.6) in 1% FSG overnight at 4°C followed by secondary antibodies (12 nm Colloidal Gold-Donkey anti-mouse Abs; both from Jackson ImmunoResearch) for 2 h at RT. Sections were fixed in 1% glutaraldehyde for 5 min, stained with uranyl acetate, and examined in TEM.</p></sec><sec id=\"S2.SS12\"><title>Scanning Electron Microscopy (SEM)</title><p>Preparation of the bacteria samples was conducted in the same manner as for TEM procedure. In brief, bacteria in mid-logarithmic phase were incubated with p4 or PBS for 2 h at 37°C on coverslips in 12-wells plates. After incubation bacteria were fixed for 10 min in 2.5% glutaraldehyde in PBS, washed three times with PBS, and dehydrated in gradient ethanol series (15–100%). The samples were then transferred to a mixture (1:1, v:v) of ethanol and acetone, and pure acetone for two times 10 min each. Dry specimens were then coated with gold. Examination and photography were carried out with a HITACHI S-4700 scanning electron microscope.</p></sec><sec id=\"S2.SS13\"><title>CRISPR/Cas9 Genome Editing</title><p>The plasmids, primers, and sgRNA target used to CRISPR/Cas-induced genetic modifications in <italic>E. coli</italic> are listed in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>. Gene targeting sequence was cloned into pgRNA-bacteria plasmid using Q5<sup>®</sup> Site-Directed Mutagenesis Kit (New England BioLabs). For knockout mutation, electrocompetent <italic>E. coli</italic> HB101 was transformed with pCas plasmid and isolates were selected on LB agar plates containing kanamycin (50 μg/ml) (BioShop). Bacteria harboring pCas were then grown at 30°C in LB medium with kanamycin (50 μg/ml). Arabinose (10 mM final concentration) (Sigma-Aldrich) was added to the culture at an OD600 of 0.3 to 0.4 for induction of the recombinase (λ-Red) expression. When the optical density reached 0.5 to 0.7, cells were harvested, washed three times in ice-cold 10% glycerol, and mixed with 100 ng of pgRNA-bacteria plasmid and 1 μg of donor DNA (containing stop codon and sequence for <italic>Hin</italic>dIII digestion). Electroporation was done in a pre-chilled 2-mm electroporation cuvette (Sigma-Aldrich) at 2.5 kV and 5 ms. Cells were recovered in LB medium without antibiotics for 1 h at 30°C, plated onto LB agar containing kanamycin (50 μg/ml) and ampicillin (100 μg/ml) (Sigma-Aldrich) and incubated overnight at 30°C. Transformants were verified by colony PCR, <italic>Hin</italic>dIII (Thermo Fisher Scientific) digestion, and DNA sequencing (Genomed). Experiments with silencing <italic>rpoS</italic> expression were performed as follows. The chemical competent <italic>E. coli</italic> DH5α was transformed with plasmid pdCas9-bacteria expressing catalytically “dead” dCas9. The positive clones were selected on LB agar plates containing 25 μg/ml chloramphenicol (Sigma-Aldrich) and used for additional chemical transformation with pgRNA-bacteria plasmid. For the knockdown experiments, bacteria were grown in LB supplemented with antibiotics overnight at 37°C. The following day, bacteria were diluted and incubated in LB with appropriate antibiotics for additional 6 h to OD<sub>600</sub> of 0.5–0.8. To obtain KD phenotype LB was additionally supplemented with 0.1 μM anhydrotetracycline (aTc) (Cayman Chemical).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>List of plasmids, primers, and sgRNA sequences used in the CRISPR/Cas9 experiments.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Plasmid</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Purpose</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Source</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pgRNA-bacteria</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Expression of guide RNA (gRNA)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Addgene #44251</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pCas</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Constitutive expression of cas9 and inducible expression of λ-Red</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Addgene #62225</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pdCas9-bacteria</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">aTc-inducible expression of a catalytically inactive bacterial Cas9 (dCas9)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Addgene #44249</td></tr><tr><td valign=\"top\" align=\"justify\" colspan=\"3\" rowspan=\"1\"><bold>Primers</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CCATAACGGGTTTTAGAGCTAGAAATAGC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insertion of gene targeting sequence in pgRNA-bacteria plasmid</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This study</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAATCGTGGTCACTAGTATTATACCTAGGAC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insertion of gene targeting sequence in pgRNA-bacteria plasmid</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This study</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GGCGTATCACGAGGCAGAAT</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Identification of modification of pgRNA-bacteria plasmid</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This study</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CGACTCGGTGCCACTTTTTC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Identification of modification of pgRNA-bacteria plasmid</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This study</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GTTGCGTATGTTGAGAAGCGG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Amplification of <italic>rpoS</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This study</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AACTGTTATCGCAGGGAGCC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Amplification of <italic>rpoS</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This study</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AACGCCAGCTAAAGC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Binding to modified fragment of <italic>rpoS</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This study</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AAATCGGCGGAACCA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Binding to modified fragment of <italic>rpoS</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This study</td></tr><tr><td valign=\"top\" align=\"justify\" colspan=\"3\" rowspan=\"1\"><bold>Target part of gene (+PAM sequence)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GACCACGATTGCCATAACGGCGG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Binding of dCas9</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This study</td></tr><tr><td valign=\"top\" align=\"justify\" colspan=\"3\" rowspan=\"1\"><bold>Donor DNA template</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TCTTCGATAAGGTCCAGCAACGCCAGCTAAAGCTT GCCTTAACGGCGGGCAATTTTTACCACCAGACGCA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Modification of <italic>rpoS</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This study</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TGCGTCTGGTGGTAAAAATTGCCCGCCGTTAAGGC AAGCTTTAGCTGGCGTTGCTGGACCTTATCGAAGA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Modification of <italic>rpoS</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This study</td></tr></tbody></table></table-wrap></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Chem157S and p4 Display Differential Antimicrobial Activity Against Skin Bacteria</title><p>Previous studies have identified specific alterations in the cutaneous microbiome in lesional psoriatic skin vs. healthy skin (<xref rid=\"B9\" ref-type=\"bibr\">Gao et al., 2008</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Fahlen et al., 2012</xref>). Since chemerin is substantially reduced in psoriatic (lesional and non-lesional) epidermis vs. healthy epidermis, we hypothesized that psoriatic epidermis is preferentially inhabited by bacteria that are sensitive to chemerin antimicrobial activity. To test this hypothesis, we selected a diverse set of nine common clinical bacterial isolates altered in psoriatic skin for chemerin-mediated killing activity. These include <italic>S. aureus, S. hominis, S. epidermidis, S. capitis, S caprae, Streptococcus mitis</italic> (<italic>Str. mitis</italic>), <italic>Corynebacterium simulans</italic> (<italic>C. simulans</italic>), <italic>C. tuberculostearicum</italic>, and <italic>Propionibacterium acnes</italic> recently renamed <italic>Cutibacterium acnes</italic> (<italic>C. acnes</italic>) (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). Chem157S (5 μM) restricted the growth of all <italic>Staphylococcus</italic> spp. and <italic>C. simulans</italic>, but it was ineffective against <italic>Str. mitis</italic> and <italic>C. acnes</italic>. In contrast, Chem163S had no antimicrobial activity against most tested strains with exception of <italic>S. caprae</italic> (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Chemerin peptide p4 and Chem157S shared a similar pattern of activity against cutaneous microbiota (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref> and <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>), but the effects of p4 were overall more robust than Chem157S based on the complete inhibition of growth of sensitive bacteria at concentration ≥MIC (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). Specifically, among the sensitive strains, p4 displayed best efficacy against <italic>C. tuberculostearicum, S. caprae, S. capitis</italic>, and <italic>S. epidermidis</italic> (MIC = 3.1–6.3 μM) (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). P4 was ineffective against <italic>C. acnes</italic> and <italic>Str. mitis</italic> (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). Therefore, antimicrobial chemerin derivatives exhibit selectivity against common and psoriasis-relevant cutaneous microbes.</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Antimicrobial activity of peptide p4 against skin bacteria.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Strain</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MIC [μM]</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Sensitive/Resistant [100 μM]</td><td valign=\"top\" align=\"left\" colspan=\"2\" rowspan=\"1\">Strain-level skin changes, healthy individuals vs. donors with psoriasis</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>S. aureus</italic> 8325-4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.5–25</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">S</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">↑</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B9\" ref-type=\"bibr\">Gao et al., 2008</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Chang et al., 2018</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>S. hominis</italic> DSM 20328</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.5–25</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">S</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">↑</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B9\" ref-type=\"bibr\">Gao et al., 2008</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>S. epidermidis</italic> DSM 20044</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">S</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">↑</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B9\" ref-type=\"bibr\">Gao et al., 2008</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>S. capitis</italic> DSM 20326</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.1–6.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">S</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">↑</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B9\" ref-type=\"bibr\">Gao et al., 2008</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>S. caprae</italic> DSM 20608</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">S</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">↓∼</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B9\" ref-type=\"bibr\">Gao et al., 2008</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Str. mitis</italic> DSM 12643</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">≥100</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">↓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B9\" ref-type=\"bibr\">Gao et al., 2008</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>C. simulans</italic> DSM 44392</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">S</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">↑</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B9\" ref-type=\"bibr\">Gao et al., 2008</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>C. tuberculostearicum</italic> DSM 44922</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">S</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">↑</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B9\" ref-type=\"bibr\">Gao et al., 2008</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>C. acnes</italic> DSM 16379</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">>100</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">↓</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B9\" ref-type=\"bibr\">Gao et al., 2008</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Chang et al., 2018</xref></td></tr></tbody></table><table-wrap-foot><attrib><italic>Selected cutaneous bacteria previously shown to be qualitatively altered in psoriatic vs. healthy skin were incubated with chemerin p4. The MIC was defined as the lowest concentration of p4 showing no visible growth (100% of killing). Strains were defined as sensitive (S) or resistant (R) if they were or were not completely killed by 100 μM p4, respectively, in all experiments, <italic>n</italic> = 3.</italic></attrib></table-wrap-foot></table-wrap><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Chemerin controls growth of cutaneous bacteria. Bacteria were incubated with chemerin isoforms Chem157S, Chem163S (5 μM), or PBS (control) for 2 h. Cell viability in CFU/ml, shown as the percentage of control cells, was analyzed by MDA assay. Results are expressed as the mean ± SD of at least two independent experiments, performed with technical triplicates. ***<italic>p</italic> < 0.001, **<italic>p</italic> < 0.01, *<italic>p</italic> < 0.05 by one-way ANOVA with <italic>post hoc</italic>: Tukey’s multiple comparisons test.</p></caption><graphic xlink:href=\"fmicb-11-01819-g001\"/></fig></sec><sec id=\"S3.SS2\"><title>P4 Suppresses Bacteria Growth by Directly Targeting Envelope-Dependent and Independent Pathways</title><p>To better understand strain-dependent differences in p4 efficacy, we extended our analysis into mechanisms by which p4 controls bacteria growth. We previously showed that peptide p4 rapidly destabilized membrane integrity in p4-sensitive <italic>E. coli</italic> strains (MIC = 6.3–12.5 μM). However, we noted that p4 localized to multiple bacteria cell compartments 10 min after treatment, including the cell wall and nucleoid (<xref rid=\"B10\" ref-type=\"bibr\">Godlewska et al., 2019</xref>) and <xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>, suggesting that p4 affects bacteria viability by directly interfering with several key pathways. Combined scanning and transmission electron microscopy (SEM and TEM, respectively), demonstrated massive loss of cell surface structures and/or peeled cell envelope in <italic>E. coli</italic> and/or <italic>S. epidermidis</italic> (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>), indicating ultrastructural disruption of bacterial membranes and peptidoglycan layers in both Gram + and Gram- strains. Treatment of <italic>E. coli</italic> with p4 also resulted in a tendency for, and significant depolarization of, membrane potential in <italic>E. coli</italic> and <italic>S. epidermidis</italic>, respectively (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>), as well as accompanying loss of ATP levels (<xref ref-type=\"fig\" rid=\"F2\">Figure 2D</xref>). Together, these data indicate that p4 affects bacteria growth by targeting bacteria shield components.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Chemerin peptide p4 localizes in different bacterial cell compartments and disrupts the cell envelope. <bold>(A)</bold> Localization of p4 is shown by immunogold labeling, followed by TEM. <italic>E. coli</italic> was incubated with 100 μM biotin-p4 (upper panel) or p4 as a control (lower panel) for 10 min, fixed, and stained with mouse anti-biotin Abs, followed by anti-mouse Abs conjugated to gold particles. Arrowheads and asterisks indicate interaction of p4 with cell envelope and nucleoid, respectively. <bold>(B)</bold> The indicated bacteria strains were incubated with 100 μM p4 or vehicle control (PBS) for 2 h, followed by SEM (upper and middle rows) or TEM (lower row). TEM and SEM images are from one experiment and are representative of at least three experiments. Scale bar = 300 nm (A), 1 μm (B SEM) or 100 nm (B,TEM). <bold>(C)</bold>\n<italic>E. coli</italic> and <italic>S. epidermidis</italic> were incubated with 25 μM p4, 1 μM gramicidin as a positive control, or vehicle control (PBS) for 10 min followed by measurement of membrane potential by fluorimetry. <bold>(D)</bold>\n<italic>E. coli</italic> and <italic>S. epidermidis</italic> were incubated with p4, or vehicle control (PBS) for 10 min followed by measurement of total ATP levels. Membrane potential <bold>(C)</bold> or ATP levels <bold>(D)</bold> are shown as the percentage of a vehicle-treated cells. Results are expressed as the mean ± SD of at least three independent experiments, performed in duplicates. ***<italic>p</italic> < 0.001, **<italic>p</italic> < 0.01, *<italic>p</italic> < 0.05, ns = non-significant by one-way ANOVA with <italic>post hoc</italic>: Tukey’s multiple comparisons test.</p></caption><graphic xlink:href=\"fmicb-11-01819-g002\"/></fig><p>P4 also induced condensation of bacteria chromosome in <italic>E. coli</italic> and Staphylococci (<xref ref-type=\"fig\" rid=\"F2\">Figures 2A</xref>, <xref ref-type=\"fig\" rid=\"F3\">3A</xref>). Given the rapid localization of p4 on nucleoid (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>), these data suggested that p4 binding to DNA may interfere with the chromosome structure. In <italic>E. coli</italic>, nucleoid condensation occurs naturally during the stationary phase of growth. Fluorescence microscopy revealed that, in common with <italic>E. coli</italic> in the stationary phase, bacteria incubated in the presence of 10 μM p4 but not vehicle control for 30 min significantly reduced the nucleoid length in the log phase. This was visualized as a change of shape of nucleoids from rod-like, relaxed form in the log phase of growth to more compact, lobular forms in the stationary phase, and in the log phase in response to p4 (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>). In contrast, condensation of bacteria nucleoids, already robust in stationary phase without any treatment, was much less dependent on p4. The qualitative data were corroborated by quantitative length measurement of Hoechst-labeled bacterial chromosomes by fluorescence microscopy (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>). To explore the direct DNA-interacting ability of p4, we performed gel retardation analysis to assess the electrophoretic mobility of DNA. Increasing concentrations of p4 retarded the migration of bacterial DNA (<xref ref-type=\"fig\" rid=\"F3\">Figure 3D</xref>). Maximal inhibition was noticed when DNA was exposed to p4 at concentrations above the MIC (12.5–100 μM); however, a lower concentration of p4 (that corresponds to 1 × MIC) was sufficient for partial retardation of DNA migration. The inhibitory effect was negligible for sublethal concentrations of p4 (<xref ref-type=\"fig\" rid=\"F3\">Figure 3D</xref>). To determine whether the interaction between p4 and DNA can influence overall bacterial fitness, we determined transcription levels of two house-keeping genes: <italic>16S rRNA</italic> and <italic>gyrA</italic>. Expression of these genes in response to p4 was not significantly altered in either log or stationary phase of growth by qPCR (<xref ref-type=\"fig\" rid=\"F3\">Figure 3E</xref>). These data suggest that p4 is not able to inhibit transcription of constitutively expressed genes, despite interacting with DNA and impacting nucleoid condensation.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Bacteria treatment with p4 results in robust nucleoid condensation in logarithmic phase of growth. <bold>(A)</bold> The indicated bacteria strains were incubated with 100 μM p4 or vehicle control for 2 h, followed by TEM. <bold>(B)</bold>\n<italic>E. coli</italic> in the indicated phase of growth was incubated with 10 μM FITC-labeled p4 or vehicle control for 30 min, stained with Hoechst to visualize DNA, and analyzed by fluorescence microscopy. FITC-p4 positive cells are shown as the percentage of total cells (Mean ± SD). <bold>(C)</bold> Length of nucleoids were measured in p4 or vehicle-treated <italic>E. coli</italic> images using NIS-Elements Imaging Software, <italic>n</italic> = 3. In each experiment, at least 2 or 3 different high-power fields were analyzed. Results are expressed as the mean ± SD. ***<italic>p</italic> < 0.001, *<italic>p</italic> < 0.05 by Kruskal-Wallis one-way ANOVA with <italic>Post hoc</italic> Dunn’s test. <bold>(D)</bold>\n<italic>E. coli</italic>-derived DNA was incubated with the indicated concentration of p4 and analyzed by gel-retardation assay, <italic>n</italic> = 3. <bold>(E)</bold> Expression of the indicated house-keeping genes was analyzed by qPCR in <italic>E. coli</italic> grown in the logarithmic or stationary phase and treated with 5 μM p4 or vehicle (PBS) for 30 min, <italic>n</italic> = 3. Expression is shown as the percentage of vehicle-treated cells. Images in panel <bold>(A)</bold> are from one experiment and are representative of at least three experiments. Scale bar = 500 nm for <italic>E. coli</italic> and 200 nm for <italic>S. aureus</italic>\n<bold>(A)</bold> and 2 μm <bold>(B)</bold>.</p></caption><graphic xlink:href=\"fmicb-11-01819-g003\"/></fig></sec><sec id=\"S3.SS3\"><title>Bacteria Modulate Sensitivity to p4 by Influencing p4 Oxidation and Cell Surface Localization</title><p>Since P4 is not uniformly bactericidal, it is possible that bacteria may employ countermeasures to ameliorate the damaging effects of p4. We showed that an oxidative environment can strongly augment p4 antimicrobial activity by supporting the formation of disulfide bonds leading to potent antimicrobial p4 dimers compared to barely active p4 monomers (<xref rid=\"B10\" ref-type=\"bibr\">Godlewska et al., 2019</xref>). To test whether bacteria themselves can modulate antimicrobial activity of p4 by oxidizing the peptide, we employed highly p4-sensitive Gram negative bacteria, <italic>Rhodobacterium capsulatus</italic> (<italic>R. capsulatus</italic>) (MIC = 5 μM) (<xref rid=\"B10\" ref-type=\"bibr\">Godlewska et al., 2019</xref>). In common with <italic>E. coli</italic> and <italic>staphyloccoci</italic>, p4 triggered robust morphological cell alterations, including cell layers distortion and condensation of nuclear material in <italic>R. capsulatus</italic> (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>). The p4-mediated effect on <italic>R. capsulatus</italic> also manifested in a rapid drop in ATP levels above MIC (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>). Changes in cytoplasmic ATP levels in response to p4 were at least partly dependent on electron transport chain (ETC), since <italic>R. capsulatus</italic> deficient in one of its key ETC enzymes, cytochrome <italic>bc</italic><sub>1</sub> (mutant MT-RBC1 KO), was less sensitive to sublethal and lethal p4 levels (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>). These data further support our previous findings, that p4 restricts the growth of <italic>R. capsulatus</italic> in association with cytochrome <italic>bc</italic><sub>1</sub> activity, which has an ability to facilitate the formation of antimicrobial p4 dimers via p4 oxidation (<xref rid=\"B10\" ref-type=\"bibr\">Godlewska et al., 2019</xref>).</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Bacteria capable of producing high level of ETC-dependent ROS are more sensitive to p4. <bold>(A)</bold>\n<italic>R. capsulatus</italic> was incubated with 100 μM p4 or vehicle (control) for 1 h followed by TEM. Scale bar = 200 nm. <bold>(B)</bold>\n<italic>R. capsulats</italic> (WT) or <italic>R. capsulatus</italic> deficient with cytochrome <italic>bc</italic><sub>1</sub> activity (MT-RBC1 KO) was incubated with the indicated concentration of p4 or vehicle control (PBS) for 30 min followed by measurement of total ATP levels. ATP levels are shown as the percentage of a vehicle-treated cells. Results are expressed as the mean ± SD of at least three independent experiments, performed in duplicates. ***<italic>p</italic> < 0.001, *<italic>p</italic> < 0.05 by one-way ANOVA with <italic>post hoc</italic>: Tukey’s multiple comparisons test. <bold>(C)</bold> The indicated strains of <italic>R. capsulatus</italic> were incubated with 2 μM of antimycin for 15 min followed by treatment with 2.5 μM of p4 or vehicle control for 2 h. <italic>n</italic> = 3. Results are expressed as the mean ± SD. ***<italic>p</italic> < 0.001, ns = non-significant by one-way ANOVA with <italic>post hoc</italic>: Tukey’s multiple comparisons test.</p></caption><graphic xlink:href=\"fmicb-11-01819-g004\"/></fig><p>To mimic the conditions under which ETC may enhance p4 activity, we forced the <italic>bc</italic><sub>1</sub>-dependent superoxide generation by treatment of bacteria with antimycin (<xref rid=\"B5\" ref-type=\"bibr\">Borek et al., 2008</xref>, <xref rid=\"B4\" ref-type=\"bibr\">2015</xref>). We reasoned that the sublethal antimycin administration in strains capable of producing reactive oxygen species (ROS) when treated with antimycin, would oxidize the peptide and boost p4 effect against <italic>R. capsulatus</italic>. Indeed, WT <italic>R. capsulatus</italic> and <italic>R. capsulatus</italic> G167P mutant with enhanced ability to produce superoxide in response to antimycin (<xref rid=\"B4\" ref-type=\"bibr\">Borek et al., 2015</xref>) were significantly more sensitive to p4 in the presence of antimycin. In contrast, there was no antimycin-dependent difference in antimicrobial p4 activity in two mutated <italic>R. capsulatus</italic> strains, MT-RBC1 KO and 2Ala (<xref rid=\"B5\" ref-type=\"bibr\">Borek et al., 2008</xref>, <xref rid=\"B4\" ref-type=\"bibr\">2015</xref>), with markedly diminished ability to produce antimycin-dependent ROS (<xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>). Together, these data suggest that bacteria have the endogenous ability to calibrate the activation status of p4, for example, by modulating their oxidative potential by ROS production at the cell membrane in the vicinity of p4.</p><p>Bacteria entering the stationary phase of growth modify their morphology, metabolic and transcriptional profile for protection against the harsh environment. These modifications include increasing resistance to antimicrobial factors (<xref rid=\"B14\" ref-type=\"bibr\">Jaishankar and Srivastava, 2017</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Agrawal et al., 2019</xref>). Our observation that bacteria exposed to p4 in the logarithmic phase of growth condense their chromosomes in a similar way as untreated bacteria in the stationary phase (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>) prompted us to ask whether p4 might induce the development of bacterial resistance conferred by the “stationary phase phenotype.” The susceptibility of <italic>E. coli</italic> to p4 in the logarithmic phase of growth was significantly higher than in cells in the stationary phase (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>). The most notable difference between <italic>E. coli</italic> treated with p4 in the log and stationary phase was observed at sublethal p4 concentrations (1.6 and 3.1 μM). However, even 100 μM p4 (the highest tested level), in spite of its strong bactericidal effect, did not completely kill <italic>E. coli</italic> in the stationary phase, since 7.4 ± 1.2% (mean ± SD) survived the p4 treatment (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>).</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Bacteria resistance to p4 depends on the phase of growth and involves RpoS-mediated control of p4 interaction with the cell surface. <bold>(A)</bold>\n<italic>E. coli</italic> in the logarithmic or stationary phase of growth was treated with the indicated concentration of p4, or vehicle control for 2 h. Cell viability in CFU/ml, shown as the percentage of control cells, was analyzed by MDA assay. <italic>n</italic> = 3, mean ± SD. <bold>(B)</bold>\n<italic>E. coli</italic> (WT) or <italic>E. coli</italic> deficient with <italic>rpoS</italic> gene (<italic>rpoS</italic> KO) was incubated in the stationary phase of growth with the indicated concentration of p4 or vehicle control for 2 h followed by MDA assay. <italic>n</italic> = 3, mean ± SD. <bold>(C)</bold> WT and <italic>rpoS</italic> KO <italic>E. coli</italic> strains were incubated in the logarithmic phase of growth with the indicated concentration of p4 or vehicle control for 2 h followed by MDA assay. <italic>n</italic> = 3, mean ± SD. <bold>(D)</bold> WT <italic>E. coli</italic> (upper panel) or <italic>E. coli</italic> with <italic>rpoS</italic> gene controlled by aTc (<italic>rpoS</italic> KD) (lower panel) were treated with 0.1 μM aTC for 6 h to silence <italic>rpoS</italic> expression. The bacteria were then treated with the indicated concentration of p4 followed by MDA assay. <italic>n</italic> = 3, mean ± SD. <bold>(E)</bold> The indicated <italic>E. coli</italic> strains were treated with 0.1 μM aTc for 6 h followed by incubation with 20 μM p4 or vehicle control for 10 min. The bacteria were stained with Hoechst to visualize DNA and analyzed by fluorescence microscopy. Scale bar = 2 μm. FITC-p4 positive cells are shown as the percentage of total cells (Mean ± SD). ***<italic>p</italic> < 0.001, **<italic>p</italic> < 0.01, *<italic>p</italic> < 0.05 by one-way ANOVA with <italic>post hoc</italic>: Tukey’s multiple comparisons test.</p></caption><graphic xlink:href=\"fmicb-11-01819-g005\"/></fig><p>One of the main factors involved in protection of bacteria during stress conditions is transcriptional factor σ<sup><italic>S</italic></sup> (RpoS) (<xref rid=\"B14\" ref-type=\"bibr\">Jaishankar and Srivastava, 2017</xref>). We next examined whether RpoS played a role in adaptability of bacteria to the p4-mediated threat. We used CRISPR/Cas to generate <italic>E. coli</italic> genetically deficient in RpoS (<italic>rpoS</italic> KO). During stationary phase, RpoSKO were significantly more susceptible to p4 than WT <italic>E. coli</italic> (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>). In contrast, <italic>rpoS</italic> KO bacteria maintained the p4 sensitivity of the WT strain in the logarithmic phase of growth (<xref ref-type=\"fig\" rid=\"F5\">Figure 5C</xref>), in agreement with RpoS function confined to the stationary phase. Similar results were obtained using an inducible system for silencing <italic>rpoS</italic> gene (<italic>rpoS</italic> knockdown, KD). A decreased tolerance to antimicrobial effect of p4 was correlated with the activity of <italic>rpoS</italic> gene (controlled by aTc) in <italic>rpoS</italic> knockdown <italic>E. coli</italic> stain but not in the WT strain (<xref ref-type=\"fig\" rid=\"F5\">Figure 5D</xref>).</p><p><italic>Escherichia coli</italic> in the stationary phase bound less FITC-labeled p4 than <italic>E. coli</italic> in the logarithmic phase (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>). These data suggested that RpoS renders bacteria less susceptible to p4 by promoting the spatial segregation of the peptide and bacteria at the cell surface. To test this possibility, we examined binding of p4 to <italic>E. coli</italic> with the silenced <italic>rpoS</italic>. Compared to the WT <italic>E. coli</italic>, the <italic>rpoS</italic> knockdown mutant displayed markedly higher potential for binding of FITC-labeled p4 to the cell surface (<xref ref-type=\"fig\" rid=\"F5\">Figure 5E</xref>). We conclude that the phenotypic resistance to p4 involves RpoS-dependent limiting of p4 association with the bacterial surface.</p></sec></sec><sec id=\"S4\"><title>Discussion</title><p>Chronic inflammatory skin diseases, including psoriasis, are associated with microbial cutaneous community changes that can negatively impact skin health. Although no single microbial biomarker indicative of psoriasis has been found, several microorganisms were linked to disease exacerbation, including chemerin-sensitive <italic>S. aureus</italic> (<xref rid=\"B21\" ref-type=\"bibr\">Tomi et al., 2005</xref>).</p><p>The skin defense system engages many components, including microbiota- and epidermis-derived antimicrobial factors (<xref rid=\"B18\" ref-type=\"bibr\">Kwiecien et al., 2019</xref>), suggesting that expression-level variations of these factors might play a pathogenic role in altering the cutaneous microbiome. However, in contrast to antimicrobial factors of microbial origin that can be expected to mainly affect the growth of other strains that compete for the same cutaneous niche, AMPs that are highly expressed in the epidermis such as chemerin may play a dominant role in restricting skin associated microbiota. Here we demonstrate the selectivity of antimicrobial chemerin derivatives against psoriasis-relevant cutaneous bacteria strains. Among nine tested strains, seven followed the predicted pattern of sensitivity to chemerin peptides in correlation with a diverse skin distribution in healthy (chemerin rich) and psoriasis (chemerin poor) epidermis. Among the sensitive chemerin strains were <italic>C. tuberculostearicum, S. capitis, C. simulans, S. epidermidis, S. hominis</italic>, and <italic>S. aureus.</italic> These strains were also reported to be present in higher quantities in non-lesional skin of psoriasis patients compared to healthy skin (<xref rid=\"B9\" ref-type=\"bibr\">Gao et al., 2008</xref>; <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>), when epidermal chemerin levels may start to decline. In contrast, Chem157S and p4 resistant-<italic>Str. mitis</italic>, which could be expected to be largely spared by chemerin peptides in epidermis, was on average unchanged or less abundant in skin of psoriasis individuals (<xref rid=\"B9\" ref-type=\"bibr\">Gao et al., 2008</xref>; <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). However, association with chemerin levels was not observed for chemerin-resistant <italic>C. acnes</italic>, which was markedly underrepresented in psoriasis skin, as well as chemerin-sensitive <italic>S. caprae</italic>, which remained unchanged in psoriatic vs. healthy skin (<xref rid=\"B9\" ref-type=\"bibr\">Gao et al., 2008</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Chang et al., 2018</xref>). The major determinants of skin inhabitance with these bacteria likely involve other factors. For example, <italic>C. acnes</italic> colonizes lipid-rich sebaceous skin areas. Since psoriatic skin is rather desiccated, skin dryness may be key factor in limiting <italic>C. acnes</italic> abundance in psoriatic skin. Chemerin exhibits antimicrobial potential against pathogenic microbes, such as <italic>S. aureus</italic>, as well as more benign skin strains such as <italic>S. epidermidis</italic>. Together these data suggest that chemerin does not selectively spare less harmful bacteria, but rather acts to restrict overgrowth of a variety of prevalent cutaneous genera, species, and strains. In the absence of the protective role of chemerin as an antimicrobial factor in epidermis, chemerin-controlled bacterial species might have a growth advantage in the skin undergoing pathological alterations, and thereby contribute to disease exacerbation.</p><p>The prophylactic potential of p4 against skin infection with a variety of microorganisms, including MRSA infection, and the therapeutic efficacy of p4 in treating experimental <italic>S. aureus</italic> skin infections [(<xref rid=\"B10\" ref-type=\"bibr\">Godlewska et al., 2019</xref>) and <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>] could form the basis of potential new drugs for the prevention/treatment of skin infections, independent of the endogenous abundance of p4 <italic>in vivo</italic>. Here, we show that p4 is likely to target multiple bacterial components, including the cell wall, cell membrane, and chromosome. Although targeting several independent components can be expected to limit the ability of bacteria to develop resistance to p4, our data suggests that bacteria can engage two discrete mechanisms to protect against p4 lethality. First, ETC-dependent ROS formation augments antimicrobial potency of p4 (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). Therefore, bacteria restricted in their ability to produce ROS at the cell membrane may, at least to some degree, be protected against the comparatively more lethal p4 dimers. Second, blocking accessibility of p4 to the bacterial surface and limiting p4 interactions with the cell membrane and intracellular targets, such as DNA, might account for better bacterial tolerance to p4. In accordance with these proposed mechanisms, bacteria in the stationary phase of growth that become more resistant to p4 are known to: (i) cope with oxidative stress, for example, by encoding enzymes that remove ROS, and (ii) maintain membrane integrity, for example, by introducing changes to membrane lipid composition (<xref rid=\"B19\" ref-type=\"bibr\">Mitchell et al., 2017</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Guo et al., 2019</xref>). Protective mechanisms available in the stationary phase of growth often activate RpoS-dependent pathways that both strengthen cell wall permeability barrier and activate efflux pumps (<xref rid=\"B15\" ref-type=\"bibr\">Kobayashi et al., 2006</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Guo et al., 2019</xref>). These findings provide corroborating support for the possible RpoS-mediated bacterial defensive strategy in response to the p4 threat. Given that <italic>rpoS</italic>-silenced <italic>E. coli</italic> diminish p4 integration to the cell surface layers (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>), bacteria may benefit by modifying their membrane properties in such a way to prevent p4 cell surface binding. Alternatively, resistance to p4 may involve alterations of p4 activity in the bacteria microenvironment, before or concurrent with p4 interactions with bacteria wall components. This could be a consequence of a release of ions and other modulating factors to the bacteria environment by RpoS-dependent efflux pumps.</p><p>We have thus characterized differential bactericidal activities of endogenous active chemerin (Chem157S) and p4 peptide against common types of cutaneous bacteria known to be altered in psoriasis. We have also discovered bacteria-mediated mechanisms that regulate the antimicrobial activity of chemerin. Given the potential clinical utility of chemerin and p4 in treating skin infections, insight into how skin bacteria can counteract p4 may prove useful in designing chemerin-based biologics geared to establish or restore a favorable cutaneous microbiome.</p></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p></sec><sec id=\"S6\"><title>Author Contributions</title><p>UG, BB, EP, and JC conceived and designed the experiments. UG and BB performed the experiments. PM contributed to the reagents and materials. JC and BZ wrote the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by grants from the Polish National Science Center UMO-2014/12/W/NZ6/00454 (to JC) and Polish Ministry of Science and Higher Education K/DCS/005445 (to UG).</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Agrawal</surname><given-names>A.</given-names></name><name><surname>Rangarajan</surname><given-names>N.</given-names></name><name><surname>Weisshaar</surname><given-names>J. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Cell Dev. Biol.</journal-id><journal-title-group><journal-title>Frontiers in Cell and Developmental Biology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-634X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850824</article-id><article-id pub-id-type=\"pmc\">PMC7431655</article-id><article-id pub-id-type=\"doi\">10.3389/fcell.2020.00704</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cell and Developmental Biology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Anticancer Effects of Fufang Yiliu Yin Formula on Colorectal Cancer Through Modulation of the PI3K/Akt Pathway and BCL-2 Family Proteins</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Dong</surname><given-names>Bingzi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1046154/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Yang</surname><given-names>Zhenjie</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/963265/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ju</surname><given-names>Qiang</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhu</surname><given-names>Shigao</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1046148/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Yixiu</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zou</surname><given-names>Hao</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1046062/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Sun</surname><given-names>Chuandong</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1046142/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zhu</surname><given-names>Chengzhan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/884972/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Shandong Key Laboratory of Digital Medicine and Computer Assisted Surgery, The Affiliated Hospital of Qingdao University</institution>, <addr-line>Qingdao</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Hepatobiliary and Pancreatic Surgery, The Affiliated Hospital of Qingdao University</institution>, <addr-line>Qingdao</addr-line>, <country>China</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of General Surgery, Anqiu People’s Hospital</institution>, <addr-line>Anqiu</addr-line>, <country>China</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Blood Transfusion, The Affiliated Hospital of Qingdao University</institution>, <addr-line>Qingdao</addr-line>, <country>China</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Department of General Medicine, Weifang Hospital of Traditional Chinese Medicine</institution>, <addr-line>Weifang</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Teresita Padilla-Benavides, Wesleyan University, United States</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Joae Qiong Wu, University of Massachusetts Medical School, United States; Jesus A. Olivares-Reyes, Centro de Investigación y Estudios Avanzados, Instituto Politécnico Nacional de México (CINVESTAV), Mexico</p></fn><corresp id=\"c001\">*Correspondence: Chengzhan Zhu, <email>zhuchengz@qduhospital.cn</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>These authors have contributed equally to this work</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Cell and Developmental Biology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>704</elocation-id><history><date date-type=\"received\"><day>27</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>10</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Dong, Yang, Ju, Zhu, Wang, Zou, Sun and Zhu.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Dong, Yang, Ju, Zhu, Wang, Zou, Sun and Zhu</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Colorectal cancer (CRC) is one of the most common malignant tumors in China. Fufang Yiliu Yin (FYY) is a traditional Chinese medicine formula used in clinical practice for cancer treatment, but its effectiveness and mechanism of action in human CRC are unclear. In this study, we investigated the antitumor effect of FYY on HCT116 and SW480 human CRC cell lines <italic>in vitro</italic> and evaluated the underlying molecular mechanism. A subcutaneous xenograft mouse model was used to confirm the antitumor effect <italic>in vivo</italic>. The components and targets of FYY were collected from the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) database. CRC targets were collected via the GeneCards and OMIM databases. Protein–protein interactions were explored using the STRING platform. Cytoscape was used to construct drug–disease–target networks. KEGG and GO analyses were performed to investigate common FYY and CRC targets. FYY significantly inhibited cell proliferation and induced HCT116 and SW480 cell apoptosis. Cell proliferation was blocked at the G0/G1 phase, while cell apoptosis was promoted at the early stage. According to the network pharmacological analysis, quercetin and kaempferol were the most bioactive compounds of FYY. The key targets of FYY were cyclin-D1, MAPK8, and EGFR. GO analysis showed that core targets included the apoptotic signaling pathway, response to steroid hormone, and cellular response to organic cyclic compound. The KEGG pathway analysis showed that FYY may affect CRC through the PI3K/Akt pathway. <italic>In vitro</italic>, FYY significantly inhibited tumor growth. Pathway analysis confirmed that FYY induced cell apoptosis by modulating PI3K/Akt signaling and BCL-2 family proteins. Hence, our findings indicate that FYY may be a promising adjuvant therapy for CRC.</p></abstract><kwd-group><kwd>colorectal cancer</kwd><kwd>traditional Chinese medicine</kwd><kwd>network pharmacology</kwd><kwd>Fufang Yiliu Yin</kwd><kwd>BCL-2 family proteins</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">China Postdoctoral Science Foundation<named-content content-type=\"fundref-id\">10.13039/501100002858</named-content></funding-source><award-id rid=\"cn001\">2016M602098 and 2018M640615</award-id></award-group><award-group><funding-source id=\"cn002\">Taishan Scholar Foundation of Shandong Province<named-content content-type=\"fundref-id\">10.13039/501100010029</named-content></funding-source><award-id rid=\"cn002\">2019010668</award-id></award-group></funding-group><counts><fig-count count=\"7\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"45\"/><page-count count=\"16\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Colorectal cancer (CRC) is one of the most common malignant tumors in China (<xref rid=\"B5\" ref-type=\"bibr\">Chen et al., 2016</xref>). CRC is one of the five leading causes of cancer death, and its incidence is gradually increasing owing to obesity and lifestyle changes (<xref rid=\"B9\" ref-type=\"bibr\">Du et al., 2015</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Chen et al., 2016</xref>). Postoperative treatments including chemotherapy and radiotherapy are important for longer patient survival. Traditional Chinese medicine (TCM) has become an option for preventing CRC metastasis and enhancing the effects of chemotherapy (<xref rid=\"B26\" ref-type=\"bibr\">Shi et al., 2017</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Xu et al., 2017</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Xie et al., 2019</xref>). TCM is used as an alternative or supplementary treatment in the United States and Europe and has been widely used to treat various diseases in Asia, especially in China (<xref rid=\"B32\" ref-type=\"bibr\">Wang et al., 2014</xref>). TCM has also been widely investigated in Asia for effective and low-toxicity monomer compounds to develop new drugs for cancer therapy and to counteract drug resistance (<xref rid=\"B28\" ref-type=\"bibr\">Sui et al., 2017</xref>; <xref rid=\"B44\" ref-type=\"bibr\">Zheng et al., 2018</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Xie et al., 2019</xref>).</p><p>In China, patients usually choose TCM for adjuvant therapy after curative resection (<xref rid=\"B36\" ref-type=\"bibr\">Xu et al., 2017</xref>). The effectiveness of TCM has been proven in multiple cancers including breast cancer (<xref rid=\"B16\" ref-type=\"bibr\">Lee et al., 2014</xref>), hepatocellular carcinoma (<xref rid=\"B4\" ref-type=\"bibr\">Chen et al., 2018</xref>), pancreatic cancer (<xref rid=\"B15\" ref-type=\"bibr\">Kuo et al., 2018</xref>), and CRC (<xref rid=\"B26\" ref-type=\"bibr\">Shi et al., 2017</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Xu et al., 2017</xref>). In CRC, TCM significantly improved disease-free survival in stage II and III CRC in a retrospective cohort study including 817 patients (<xref rid=\"B26\" ref-type=\"bibr\">Shi et al., 2017</xref>). In a multicenter prospective cohort study including 312 patients with stage II and III CRC, postoperative TCM treatment was associated with better disease-free survival and overall survival compared to those of the untreated group (<xref rid=\"B36\" ref-type=\"bibr\">Xu et al., 2017</xref>). Certain active ingredients in TCM herbs may have stronger activity in inhibiting cell proliferation and promoting cell apoptosis (<xref rid=\"B30\" ref-type=\"bibr\">Tan et al., 2011</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Huang and Hu, 2018</xref>). For example, bufalin, an active component of the TCM Chan Su, can reverse multidrug resistance by inhibiting the protein expression and efflux function of ABCB1 (<xref rid=\"B38\" ref-type=\"bibr\">Yuan et al., 2017</xref>). Cinobufagin, another cardiotonic steroid isolated from Chan Su, suppresses tumor neovascularization by disrupting the endothelial mTOR/HIF-1α pathway to trigger reactive oxygen species-mediated vascular endothelial cell apoptosis (<xref rid=\"B18\" ref-type=\"bibr\">Li et al., 2019</xref>). Of the frequently used TCM treatments, the most effective single herbs are Ginseng Radix (Ren Shen), <italic>Hedyotis diffusa</italic> Willd (Bai Hua She She Cao), <italic>Scutellaria barbata</italic> (Ban Zhi Lian), and Astragali Radix (Huang Qi) (<xref rid=\"B16\" ref-type=\"bibr\">Lee et al., 2014</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Wu et al., 2017</xref>). However, the underlying mechanisms of these remedies remain unknown. Network pharmacology can efficiently and quickly identify the interactions between drugs and target proteins, providing a foundation for TCM application (<xref rid=\"B40\" ref-type=\"bibr\">Zhang et al., 2019</xref>).</p><p>Fufang Yiliu Yin (FYY) is a TCM formula that has been used in clinical practice for cancer treatment. Our previous study found that FYY inhibited cell proliferation, migration, and invasion and promoted apoptosis in hepatocellular carcinoma (<xref rid=\"B37\" ref-type=\"bibr\">Yang et al., 2018</xref>). FYY contains eight herbs: Astragali Radix (Huang Qi), <italic>Ganoderma lucidum</italic> (Ling Zhi), Semen Armeniacae Amarum (Ku Xing Ren), <italic>H. diffusa</italic> Willd (Bai Hua She She Cao), Aconiti Lateralis Radix Praeparata (Fu Zi), <italic>Glycyrrhiza glabra</italic> Linne (Gan Cao), Radix Panacis Quinquefolii (Xi Yang Shen), and Platycodi Radix (Jie Geng). Of these herbs, Radix Panacis Quinquefolii (Ginseng Radix), <italic>H. diffusa</italic> Willd, and Astragali Radix are commonly used in anticancer formulas (<xref rid=\"B16\" ref-type=\"bibr\">Lee et al., 2014</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Wu et al., 2017</xref>). <italic>G. lucidum</italic> and Platycodi Radix also reportedly have anticancer effects; Radix Astragali (<xref rid=\"B13\" ref-type=\"bibr\">Jung et al., 2016</xref>), <italic>G. lucidum</italic> (<xref rid=\"B7\" ref-type=\"bibr\">Dai et al., 2014</xref>), Platycodi Radix (<xref rid=\"B21\" ref-type=\"bibr\">Park and Lee, 2014</xref>), and <italic>H. diffusa</italic> Willd (<xref rid=\"B39\" ref-type=\"bibr\">Zhang et al., 2016</xref>) inhibit cancer cell proliferation. Polysaccharides in <italic>G. lucidum</italic> inhibit the proliferation of CRC cells, upregulating the expression of P21 protein and blocking cells at the G2/M phase (<xref rid=\"B20\" ref-type=\"bibr\">Na et al., 2017</xref>).</p><p>In the current study, we investigated the anticancer effect of FYY on CRC cells <italic>in vitro</italic> and <italic>in vivo</italic>, and a network pharmacology analysis was performed to explore the potential molecular mechanisms. The information obtained in this study will aid in elucidating the previously unavailable mechanisms of action of FYY in CRC and developing FYY as an adjuvant therapy for CRC.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Preparation of FYY and Cell Culture</title><p>The components of FYY conformed to the provisions stated by the Chinese Pharmacopoeia; FYY was prepared at the Weifang Hospital of Traditional Chinese Medicine, Shandong, China (<xref rid=\"B37\" ref-type=\"bibr\">Yang et al., 2018</xref>). FYY (120 mg/ml) was stored at −20°C until use and was further diluted to the required concentrations in subsequent cell experiments. Human CRC cell lines (HCT116 and SW480) were purchased from the cell resource center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). HCT116 cells were grown in RPMI-1640 medium (RPMI-1640, HyClone, United States) and SW480 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, United States) containing 10% fetal bovine serum (FBS, Gibco BRL, United States) and 1% penicillin/streptomycin (Sigma-Aldrich; St. Louis, MO, United States) in 5% CO<sub>2</sub> at 37°C in a humidified incubator.</p></sec><sec id=\"S2.SS2\"><title>Cell Viability and Colony Formation Assays</title><p>Cells (3 × 10<sup>3</sup> per well) were seeded into 96-well plates and incubated overnight at 37°C, 5% CO<sub>2</sub> in a humidified incubator. When the cells adhered to the wall, HCT116 and SW480 cells were treated with 3, 6, 9, 12, or 15 mg/ml of FYY or PBS as a control for 24 and 48 h. Cell viability was measured using a cell counting kit-8 (CCK-8, Beyotime Institute of Biotechnology, Inc., Shanghai, China). Ten microliters of the CCK-8 solution was added to each well, and then samples were incubated at 37°C for 2 h. Finally, the absorbance value at 450 nm was determined using a Multiskan<sup>TM</sup> FC Microplate Photometer (Thermo Fisher Scientific Inc., United States).</p><p>HCT116 and SW480 cells were treated with 9, 12, or 15 mg/ml of FYY or PBS as a control for 24 h. The cells (1 × 10<sup>3</sup> per well) were then cultured in six-well plates, and the medium was changed every 3 days for 14 days. Cell colonies were fixed with 4% paraformaldehyde and then stained with 5% Giemsa (Beyotime Institute of Biotechnology, Inc., Shanghai, China) for 15 min. A colony formation assay was performed to count viable colonies (>50 cells per colony).</p></sec><sec id=\"S2.SS3\"><title>Cell Cycle Analyses</title><p>HCT116 and SW480 cells were treated with 9, 12, or 15 mg/ml FYY or PBS as a control for 24 h. The collected cells (1 × 10<sup>6</sup>) were fixed in cold ethanol and stored at 4°C overnight. The next day, the cells were washed twice with cold PBS; then 100 μl RNase A (25 μg/ml) and 400 μl propidium iodide (50 μg/ml, Sigma Aldrich; St. Louis, MO, United States) were added to each sample and incubated for 30 min in the dark. Measurements were taken using a flow cytometer (FACScan; BD Biosciences, Bedford, MA, United States), and the data were analyzed using FlowJo 7.6 software (Tree Star, Inc., Ashland, OR, United States).</p></sec><sec id=\"S2.SS4\"><title>Cell Apoptosis Analyses</title><p>Cell apoptosis was detected using an Apoptosis-Hoechst 33258 Staining Kit (Beyotime Biotechnology, Shanghai, China). Samples were fixed with 4% paraformaldehyde at room temperature for 10 min and stained with 10 mg/ml Hoechst 33258 at room temperature for 15 min. Then, fluorescence was detected under an Olympus IX50 microscope (Olympus Corp., Tokyo, Japan) at × 400 magnification. Apoptotic cells were identified using an Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit (Invitrogen<sup>TM</sup>/Molecular Probes, Eugene, OR, United States). After centrifugation at 300 <italic>g</italic> for 5 min, the cell density was counted and diluted in 1 × annexin-binding buffer to obtain 1 × 10<sup>6</sup> cells/ml (100 μl per assay). Cells were stained with 5 μl of annexin V-FITC and 1 μl propidium iodide at room temperature for 20 min in the dark, and then 400 μl of binding buffer was added. Measurements were taken using a flow cytometer, and the data were analyzed using FlowJo 7.6 software.</p></sec><sec id=\"S2.SS5\"><title>Network Pharmacology</title><p>Active FYY compounds were screened using the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP)<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup> (<xref rid=\"B24\" ref-type=\"bibr\">Ru et al., 2014</xref>). With the pharmacokinetic information retrieval filter based on the TCMSP platform, the oral bioavailability and drug-likeness were set to ≥30% and ≥0.18 to obtain qualified herbal compounds. The chemical structures of the compounds were drawn using ChemBioOffice 2010 (<xref rid=\"B14\" ref-type=\"bibr\">Kerwin, 2010</xref>). CRC targets were predicted and screened using the GeneCards database<sup><xref ref-type=\"fn\" rid=\"footnote2\">2</xref></sup> (<xref rid=\"B27\" ref-type=\"bibr\">Stelzer et al., 2016</xref>) and OMIM platform<sup><xref ref-type=\"fn\" rid=\"footnote3\">3</xref></sup> (<xref rid=\"B2\" ref-type=\"bibr\">Amberger and Hamosh, 2017</xref>). Venny 2.1.0<sup><xref ref-type=\"fn\" rid=\"footnote4\">4</xref></sup> (<xref rid=\"B31\" ref-type=\"bibr\">Venny, 2018</xref>) was used to screen for common targets between FYY and disease-related targets.</p><p>Drug compound–disease–target networks were built using Cytoscape (v 3.7.1) software (<xref rid=\"B25\" ref-type=\"bibr\">Shannon et al., 2003</xref>), and the merge function was used to analyze the core compounds. Protein interaction networks of the common FYY and CRC-related targets were built using the String database platform with medium confidence (0.7) and rejecting the target protein independent of the network (<xref rid=\"B29\" ref-type=\"bibr\">Szklarczyk et al., 2017</xref>).</p><p>Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using Metascape (<xref rid=\"B45\" ref-type=\"bibr\">Zhou et al., 2019</xref>). Enriched GO terms and relevant pathways with <italic>p</italic>-values < 0.05 were selected for better prediction and verification of the biological process and mechanism.</p></sec><sec id=\"S2.SS6\"><title>Western Blot Analysis</title><p>The following primary antibodies obtained from Cell Signaling Technology Inc. (Danvers, MA, United States) were used in the immunoblotting analysis: PI3K (p110α, #4255, 1:1,000), AKT (pan, #2920, 1:1,000), p-AKT (Ser473, #4060, 1:1,000), BCL-2 (#4223, 1:1,000), BCL-XL (#2762, 1:1,000), BAX (#5023, 1:1,000), P21 (#2947, 1:1,000), C-MYC (#18583, 1:1,000), and GAPDH (#5174, 1:1,000). Total proteins were extracted from cells and tissues using RIPA lysis buffer (CWBIO, Beijing, China). Equal amounts of protein from each sample were separated by 10% SDS-PAGE electrophoresis and then transferred onto 0.45-μm PVDF membranes (Bio-Rad Laboratories, Hercules, CA, United States). Subsequently, the membranes were blocked with 5% milk in PBS plus 0.1% Tween 20 (PBST) for 60 min, incubated with primary antibodies overnight at 4°C, and then incubated with goat anti-rabbit horseradish peroxidases (Abcam, Cambridge, MA, United States; 1:10,000) or goat anti-mouse horseradish peroxidases (Abcam, Cambridge, MA, United States; 1:10,000) for 2 h at room temperature. Finally, the band was detected using an enhanced chemiluminescence reagent and visualized with a Fusion FX7 System (Vilber Lourmat, France). ImageJ software was used to calculate the intensity (gray value) of each protein band, and GAPDH served as a control for normalization.</p></sec><sec id=\"S2.SS7\"><title>Tumor Xenografts in Nude Mice</title><p>Ten male BALB/c nude mice (4–5 weeks old, 20.2 ± 1.9 g) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The mice were housed at 24 ± 1°C under a 12-h light/dark cycle with free access to food and water. All animal experiments were completed at the specific-pathogen-free medical animal laboratory of The Affiliated Hospital of Qingdao University and approved by the Animal Ethics Committee of The Affiliated Hospital of Qingdao University (AHQU20180310A). HCT116 cells (1 × 10<sup>7</sup> cells per tumor) were subcutaneously injected into the right armpit of the nude mice. Seven days after tumor inoculation, the tumor size was measured using a Vernier caliper, and the mice were divided into two groups: the FYY treatment group and a control group (<italic>n</italic> = 5 mice per group). The FYY group was intragastrically administered 0.2 ml/10 g body weight daily in a primary concentration of 120 mg/ml. The control group was intragastrically administered an equivalent volume of PBS. Tumor sizes were measured every 3 days and calculated using the following formula: tumor volume (mm<sup>3</sup>) = 0.5 × length × width<sup>2</sup>. The nude mice were killed by cervical dislocation on day 36, and the tumors were excised, weighed, and photographed. Finally, tumor tissue and liver tissue were stored in 10% formalin or at −80°C for subsequent immunohistochemistry or western blot analyses, respectively.</p></sec><sec id=\"S2.SS8\"><title>Immunohistochemistry</title><p>Tumor and liver tissues of the nude mice were fixed with 10% paraformaldehyde for 12 h and then embedded in paraffin. Embedded paraffin sections were de-waxed in xylene and rehydrated in ethanol. Antigen retrieval was performed in 0.01 M citrate buffer (pH 6.0) using a pressure cooker followed by incubation for 3 min. Samples were then washed thrice with PBS and fixed in 95% ethanol for 30 min. Ki-67 antibody (Novus, Colorado, United States; 1:600) was stained with a streptavidin–peroxidase detection kit (ZSGB-BIO, Beijing, China) according to the kit instructions.</p></sec><sec id=\"S2.SS9\"><title>Statistical Analysis</title><p>Data analysis was performed using GraphPad Prism 6.0 software (San Diego, CA, United States). All experimental data were expressed as the mean ± SD. The statistical significance of the results was analyzed by one-way analysis of variance (ANOVA) for multiple group comparisons and Student’s <italic>t</italic>-test for two group comparisons. A value of <italic>p</italic> < 0.05 was considered statistically significant. All experiments were performed in triplicate.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>FYY Inhibited Proliferation and Promoted Apoptosis of CRC Cells <italic>in vitro</italic></title><p>Fufang Yiliu Yin significantly inhibited the growth of HCT116 and SW480 cells in a dose-dependent manner (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). The colony formation assay showed that the number of the colonies in the FYY group (12 and 15 mg/ml) was lower than that of the control group (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Fufang Yiliu Yin (FYY) inhibited colorectal cancer cell proliferation. <bold>(A)</bold> CCK-8 assay indicated that FYY inhibited the proliferation of HCT116 and SW480 cells in a dose- and time-dependent manner after 24 and 48 h of treatment. PBS was used for the control treatment (<italic>n</italic> = 6 per group). <bold>(B)</bold> Colony formation ability decreased after treatment with different concentrations of FYY for both HCT116 and SW480 (<italic>n</italic> = 3 per group). Values are shown as the mean ± SD, *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, and ***<italic>p</italic> < 0.001 vs. control group. The <italic>p</italic>-values were obtained using ANOVA.</p></caption><graphic xlink:href=\"fcell-08-00704-g001\"/></fig><p>Colony formation ability was significantly inhibited by >9 mg/ml (<italic>p</italic> = 0.035) and >12 mg/ml (<italic>p</italic> = 0.030) FYY for HCT116 and for SW480 cells, respectively. The cell cycle analysis showed no significant difference in the percentage of cells in S (<italic>p</italic> = 0.584 for 9 mg/ml) and G2/M phases (<italic>p</italic> = 0.864 for 9 mg/ml) in HCT116. However, a significant increase in G0/G1 phase was found after treatment with increasing concentrations of FYY (<italic>p</italic> = 0.013 for 9 mg/ml, <xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>) in HCT116. Similar results were obtained for SW480 cells. FYY blocked cell cycle at the G0/G1 phase in a concentration-dependent manner. FYY inhibited the expression of C-MYC (<italic>p</italic> < 0.001 for 9 mg/ml) and promoted the expression of P21 protein (<italic>p</italic> < 0.001 for 15 mg/ml, <xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>) in HCT116; similar results were observed in SW480 cells. This indicated an inhibitory effect on cell proliferation.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Fufang Yiliu Yin (FYY) significantly inhibited the colorectal cancer cell cycle. <bold>(A)</bold> FYY significantly inhibited the cell cycle progress of HCT116 and SW480, arresting them at the G2/M phase as shown by flow cytometry assay (<italic>n</italic> = 3 per group). <bold>(B)</bold> The expression of C-MYC decreased and P21 increased with FYY treatment (<italic>n</italic> = 3 per group). Values are shown as the mean ± SD, *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, and ***<italic>p</italic> < 0.001 vs. control group. The <italic>p</italic>-values were obtained using ANOVA.</p></caption><graphic xlink:href=\"fcell-08-00704-g002\"/></fig><p>Cell apoptosis, as shown by Hoechst staining, increased after FYY treatment (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>). Flow cytometry analysis showed that the early (<italic>p</italic> = 0.001 for 12 mg/ml) and late apoptosis (<italic>p</italic> = 0.019 for 9 mg/ml) of HCT116 cells were significantly promoted (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>) by FYY treatment. Similar results were obtained for SW480 cells (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Fufang Yiliu Yin (FYY) promoted colorectal cancer cell apoptosis. <bold>(A)</bold> Hoechst 33258 staining analysis indicated that FYY promoted apoptosis including chromatin condensation and nuclear fragmentation in HCT116 and SW480 cells (×400 magnification). <bold>(B)</bold> Flow cytometry indicated that FYY promoted the early and late apoptosis of HCT116 and SW480 cells (<italic>n</italic> = 3 per group). Values are shown as the mean ± SD, *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, and ***<italic>p</italic> < 0.001 vs. control group. The <italic>p</italic>-values were obtained using ANOVA.</p></caption><graphic xlink:href=\"fcell-08-00704-g003\"/></fig></sec><sec id=\"S3.SS2\"><title>Network Pharmacological Analysis of FYY Targeting CRC</title><p>A total of 218 compounds from FYY were retrieved (oral bioavailability ≥ 30% and drug likeness ≥ 0.18) from the TCMSP database (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 1</xref>). A total of 127 genes related to these compounds, and 1,005 genes related to CRC were screened out. Using Venny 2.1.0 (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>), 61 common targets were obtained (<xref ref-type=\"supplementary-material\" rid=\"TS2\">Supplementary Table 2</xref>).</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Network pharmacological analysis and biological functional enrichment analysis of Fufang Yiliu Yin (FYY). <bold>(A)</bold> Venn diagram showed 61 common targets of FYY in colorectal cancer (CRC); compound–disease–target networks of FYY against CRC. <bold>(B)</bold> Protein–protein interactions identified by STRING software. <bold>(C)</bold> The predicted key targets of FYY treatment of CRC. <bold>(D)</bold> GO and KEGG pathway enrichment analyses.</p></caption><graphic xlink:href=\"fcell-08-00704-g004\"/></fig><p>Data imported into Cytoscape 3.5.1 to construct compound–disease–target networks (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>) showed that 61 of the 218 FYY compounds may affect disease targets. The top 15 core compounds were screened based on the topological properties of degree as shown in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>. Quercetin, kaempferol, luteolin, beta-sitosterol, isorhamnetin, formononetin, calycosin, jaranol, acacetin, and naringenin were the top 10 active FYY ingredients against CRC. The other 46 active compounds are listed in <xref ref-type=\"supplementary-material\" rid=\"TS3\">Supplementary Table 3</xref>. Two networks were constructed for the top 15 core compounds and the remaining 46 active compounds (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>). The protein–protein interaction network built using STRING software (used to investigate the mechanisms of FYY) provided 61 common targets after setting the confidence level above 0.7 (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>). The prioritization of key targets was analyzed according to the degree of the node exported from the STRING database, and the top five targets were cyclin-D1, MAPK8, EGFR, MYC, and ESR1 (<xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>The top 15 bioactive compounds of Fufang Yiliu Yin are listed below according to the degree of similarity of the compound–disease–target networks.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><inline-graphic xlink:href=\"fcell-08-00704-i001.jpg\"/></td></tr></tbody></table><table-wrap-foot><attrib><italic>OB, oral bioavailability; DL, drug-likeness.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS3\"><title>Biological Function and Pathway Enrichment of FYY on CRC</title><p>The biological functions and signaling pathways from all core targets were enriched. The top 20 biological enrichment results are shown in <xref ref-type=\"fig\" rid=\"F4\">Figure 4D</xref>. FYY affected CRC through multiple GO biological processes, including apoptotic signaling pathway, response to steroid hormone, and response to inorganic substance. KEGG analysis results included cancer, prostate cancer, apoptosis, and PI3K/Akt signaling pathways.</p><p>We further investigated how the FYY mechanism promoted apoptosis using RT-PCR and western blot analysis of HCT116 and SW480 cells; FYY inhibited the relative expression of PI3K mRNA (<italic>p</italic> < 0.05, <xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>). FYY downregulated the expression of PI3K, p-AKT, BCL-2, and BCL-XL and upregulated the expression of BAX (<italic>p</italic> < 0.05, <xref ref-type=\"fig\" rid=\"F5\">Figures 5B,C</xref>). Taken together, these data support the idea that FYY induces CRC cell apoptosis by modulating the PI3K/Akt pathway and BCL-2 family proteins.</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Fufang Yiliu Yin (FYY) modulated the expression of the PI3K/Akt signaling pathway and BCL-2 family proteins. Relative PI3K mRNA expression was altered by FYY treatment in HCT116 and SW480 cells <bold>(A)</bold> (<italic>n</italic> = 3 per group). Expression levels of PI3K, AKT, p-AKT, BCL-2, BCL-XL, and BAX were altered by FYY treatment in HCT116 <bold>(B)</bold> and SW480 cells <bold>(C)</bold> (<italic>n</italic> = 3 per group). Values are shown as the mean ± SD, *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, and ***<italic>p</italic> < 0.001 vs. control group. The <italic>p</italic>-values were obtained using ANOVA.</p></caption><graphic xlink:href=\"fcell-08-00704-g005\"/></fig></sec><sec id=\"S3.SS4\"><title>FYY Inhibited Tumor Growth and Cell Proliferation <italic>in vivo</italic></title><p>The HCT116 cell xenograft model used to investigate the antitumor effect of FYY showed that FYY significantly inhibited tumor growth compared to the control (<xref ref-type=\"fig\" rid=\"F6\">Figure 6A</xref>). After 30 days of treatment, the average tumor volumes were 738.00 ± 442.38 mm<sup>3</sup> in the control group and 411.2.00 ± 54.87 mm<sup>3</sup> in FYY-treated group (<xref ref-type=\"fig\" rid=\"F6\">Figure 6B</xref>), while tumor weights were 678.00 ± 57.83 and 294.00 ± 73.66 mg, respectively. Ki-67 significantly decreased in the FYY-treated CRC tumor xenograft group (<xref ref-type=\"fig\" rid=\"F6\">Figure 6B</xref>). The expression of PI3K, p-AKT, BCL-2, and BCL-XL followed the same trend as the <italic>in vitro</italic> study results (<xref ref-type=\"fig\" rid=\"F6\">Figure 6C</xref>).</p><fig id=\"F6\" position=\"float\"><label>FIGURE 6</label><caption><p>Fufang Yiliu Yin (FYY) inhibited tumor growth <italic>in vivo.</italic>\n<bold>(A)</bold> Subcutaneous xenograft tumors after 36 days demonstrated that FYY inhibited xenograft tumor growth (<italic>n</italic> = 5 per group). <bold>(B)</bold> Tumor volume was significantly smaller after 14 days of FYY treatment (<italic>n</italic> = 5 per group). IHC analysis of Ki-67 expression in FYY-treated tumor and liver tissues (×400 magnification). The <italic>p</italic>-values were obtained using ANOVA. (C) Protein expression levels of PI3K, AKT, p-AKT, BCL-2, BCL-XL, and BAX in tumor tissues (<italic>n</italic> = 3 per group). Values are shown as the mean ± SD, *<italic>p</italic> < 0.05 and **<italic>p</italic> < 0.01 vs. control group. The <italic>p</italic>-values were obtained using Student’s <italic>t</italic>-test.</p></caption><graphic xlink:href=\"fcell-08-00704-g006\"/></fig></sec></sec><sec id=\"S4\"><title>Discussion</title><p>Both retrospective and prospective studies have proven the anticancer effects of TCM on CRC (<xref rid=\"B26\" ref-type=\"bibr\">Shi et al., 2017</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Xu et al., 2017</xref>). Here, we reported the anticancer effect of the FYY formula, which contains eight ingredients. FYY significantly inhibited cell proliferation and promoted CRC cell apoptosis <italic>in vitro</italic>. FYY also inhibited xenograft tumor growth <italic>in vivo</italic>. Using a network pharmacology analysis, we found that FYY may act on CRC through 61 active compounds targeting 61 CRC-related genes that regulate the apoptosis and PI3K/Akt signaling pathways.</p><p>To better understand the complementary effects of FYY formula ingredients, we retrieved a total of 218 compounds from the TCMSP database (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 1</xref>). Compound–disease–target networks showed that 61 of the 218 compounds may affect 61 CRC-related targets. By searching PubMed, we found that the top 10 compounds (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>) exhibit anti-CRC effects mainly by promoting apoptosis and inhibiting cell proliferation. For example, quercetin was mostly related to protective effects against CRC and is found in three of the eight remedies in FYY, Astragali Radix (Huang Qi), <italic>H. diffusa</italic> Willd (Bai Hua She She Cao), and <italic>G. glabra</italic> Linne (Gan Cao). Quercetin inhibits CRC progression by promoting cell apoptosis and autophagy, as well as inhibiting angiogenesis and inflammation (<xref rid=\"B8\" ref-type=\"bibr\">Darband et al., 2018</xref>). Quercetin induces apoptosis by inhibiting different signaling pathways including the MAPK/Erk, PI3K/Akt, and NF-κB signaling pathways (<xref rid=\"B41\" ref-type=\"bibr\">Zhang et al., 2015</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Xavier et al., 2019</xref>); it also inhibits the migration and invasion of CRC cells via regulating the toll-like receptor 4/NF-κB signaling pathway (<xref rid=\"B11\" ref-type=\"bibr\">Han et al., 2016</xref>). Further, kaempferol induces CRC cell apoptosis (<xref rid=\"B6\" ref-type=\"bibr\">Choi et al., 2018</xref>), while isorhamnetin, formononetin, and naringenin show anticancer effects by inhibiting cell proliferation (<xref rid=\"B17\" ref-type=\"bibr\">Li et al., 2014</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Abaza et al., 2015</xref>). The similarity of the effects provided by FYY compounds may provide a mutual enhancement effect, but this must be further tested using single or mixed compounds.</p><p>Fufang Yiliu Yin induced cell cycle arrest in CRC cells at the G0/G1 phase and promoted apoptosis in HCT116 and SW480 cells. To explain the mechanism by which FYY inhibits cell proliferation and promotes apoptosis, we performed protein–protein interaction network, KEGG, and GO pathway analyses. Protein–protein interaction network analysis indicated the top five targets were cyclin-D1, MAPK8, EGFR, C-MYC, and ESR1. Biological functional analysis indicated apoptosis and cancer-related pathways including the PI3K/Akt signaling pathway. Then, our experimental study confirmed the activation of the PI3K/Akt pathway and BCL-2 family proteins, as well as C-MYC expression.</p><p>Traditional Chinese medicine formulas reportedly inhibit cancer progression by different signaling pathways. A TCM formula, Jianpi Jiedu, inhibits CRC tumorigenesis and metastasis via the mTOR/HIF-1α/VEGF pathway (<xref rid=\"B22\" ref-type=\"bibr\">Peng et al., 2018</xref>). Another TCM formula, Huang Qin Ge Gen Tang, enhances the 5-fluorouracil anticancer effect by regulating the E2F1/TS pathway (<xref rid=\"B19\" ref-type=\"bibr\">Liu et al., 2018</xref>). The Zhi Zhen Fang formula reverses multidrug resistance mediated by the Hedgehog pathway in CRC (<xref rid=\"B28\" ref-type=\"bibr\">Sui et al., 2017</xref>). These formulas, as well as FYY, all contain Astragali Radix (Huang Qi), <italic>H. diffusa</italic> Willd (Bai Hua She She Cao), <italic>G. glabra</italic> Linne (Gan Cao), and Radix Panacis Quinquefolii (Xi Yang Shen). However, there have been no reports regarding the anticancer effect of TCM formulas acting through the apoptosis and PI3K/Akt pathways in CRC (<xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref>). In the current study, we found that FYY decreased the transcription and protein level of PI3K (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>) and further inhibited the phosphorylation of Akt in both the cells and tumor tissues (<xref ref-type=\"fig\" rid=\"F5\">Figures 5</xref>, <xref ref-type=\"fig\" rid=\"F6\">6</xref>). Accumulating evidence indicates that the PI3K/Akt pathway plays an important role in tumor development. PI3K can partially activate Akt at the Thr308 or Ser473 sites by inducing the translocation of Akt to the cell membrane via phosphoinositide-dependent kinase 1. Akt inhibition is usually indicated by a decrease in the p-Akt (Ser473) level and is mostly achieved by inhibiting PI3K using PI3K-specific inhibitors LY294002 or Wortmannin (<xref rid=\"B23\" ref-type=\"bibr\">Reener and Marti, 2013</xref>). The regulation of PI3K/Akt transcription and protein expression by a TCM treatment has been previously reported. TCM intervention decreased p-Akt levels following the concentration gradient of the TCM treatment, while the total overall Akt level was unchanged (<xref rid=\"B10\" ref-type=\"bibr\">Gu et al., 2017</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Zhao et al., 2018</xref>). Calycosin, a component of Astragali Radix, reportedly inhibits CRC proliferation through the ERβ-mediated regulation of the IGF-1R and PI3K/Akt signaling pathways (<xref rid=\"B43\" ref-type=\"bibr\">Zhao et al., 2016</xref>). Quercetin, kaempferol, and rutin in <italic>H. diffusa</italic> Willd also exhibit anticancer effects in CRC by regulating the PI3K/Akt signaling pathway (<xref rid=\"B3\" ref-type=\"bibr\">Cai et al., 2012</xref>).</p><fig id=\"F7\" position=\"float\"><label>FIGURE 7</label><caption><p>Schematic representation of the proposed PI3K/Akt signaling-induced cell cycle arrest and apoptosis triggered by Fufang Yiliu Yin (FYY). By combining the network pharmacological analysis and our results, we hypothesized that FYY activates the PI3K/Akt signaling pathway and modulates the expression of P21, C-MYC, and BCL-2 family proteins, thereby inducing cell cycle arrest and apoptosis.</p></caption><graphic xlink:href=\"fcell-08-00704-g007\"/></fig><p>We previously found that FYY inhibited cell proliferation, promoted cell apoptosis, and inhibited metastasis of hepatocellular carcinoma (<xref rid=\"B37\" ref-type=\"bibr\">Yang et al., 2018</xref>). FYY may have a similar effect on different types of cancer. Although we demonstrated both the anticancer effects of FYY and the action mechanism by which it operates, limitations of this study include the following: first, we did not investigate the antimetastatic effect of FYY on CRC. A migration and invasion assay and CRC liver metastasis model should be used to investigate this. Second, further studies should investigate whether mutual enhancement effects exist between the applications of FYY and regular chemotherapy and also examine its effect on drug resistance.</p><p>In conclusion, our study findings showed that FYY inhibited proliferation and promoted apoptosis in CRC cells by modulating the PI3K/Akt signaling pathway and BCL-2 family proteins. We believe that FYY could be a promising adjuvant therapy for CRC.</p></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>All data presented in this study are included in the article/<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Material</xref>.</p></sec><sec id=\"S6\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by Animal Ethics Committee of The Affiliated Hospital of Qingdao University (AHQU20180310A). Written informed consent was obtained from the owners for the participation of their animals in this study.</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>BD and CZ obtained funding, conducted the research, and prepared the manuscript. ZY and QJ performed the experiments. SZ prepared and provided the FYY formula. YW and HZ performed the network pharmacology analysis. CS designed the study and interpreted the data. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by the China Postdoctoral Science Foundation (grant numbers 2016M602098 and 2018M640615), the Taishan Scholars Program of Shandong Province (grant number 2019010668), the Shandong Higher Education Young Science and Technology Support Program (grant number 2020KJL005), and the Qingdao Postdoctoral Science Foundation (grant number 2016046), and the National Natural Science Foundation of China (grant number 81600490).</p></fn></fn-group><fn-group><fn id=\"footnote1\"><label>1</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://tcmspw.com/tcmsp.php\">http://tcmspw.com/tcmsp.php</ext-link></p></fn><fn id=\"footnote2\"><label>2</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.genecards.org/\">https://www.genecards.org/</ext-link></p></fn><fn id=\"footnote3\"><label>3</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.omim.org/\">https://www.omim.org/</ext-link></p></fn><fn id=\"footnote4\"><label>4</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://bioinfogp.cnb.csic.es/tools/venny/\">http://bioinfogp.cnb.csic.es/tools/venny/</ext-link></p></fn></fn-group><sec id=\"S9\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fcell.2020.00704/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fcell.2020.00704/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"TS1\"><media xlink:href=\"Table_1.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS2\"><media xlink:href=\"Table_2.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS3\"><media xlink:href=\"Table_3.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abaza</surname><given-names>M. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Mol Biosci</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Mol Biosci</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Mol. Biosci.</journal-id><journal-title-group><journal-title>Frontiers in Molecular Biosciences</journal-title></journal-title-group><issn pub-type=\"epub\">2296-889X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850972</article-id><article-id pub-id-type=\"pmc\">PMC7431656</article-id><article-id pub-id-type=\"doi\">10.3389/fmolb.2020.00185</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Molecular Biosciences</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Conjugative Coupling Proteins and the Role of Their Domains in Conjugation, Secondary Structure and <italic>in vivo</italic> Subcellular Location</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Álvarez-Rodríguez</surname><given-names>Itxaso</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/469651/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ugarte-Uribe</surname><given-names>Begoña</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1038526/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>de la Arada</surname><given-names>Igor</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Arrondo</surname><given-names>José Luis R.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1018109/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Garbisu</surname><given-names>Carlos</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/295149/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Alkorta</surname><given-names>Itziar</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/798076/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU)</institution>, <addr-line>Leioa</addr-line>, <country>Spain</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Instituto Biofisika (UPV/EHU, CSIC), University of the Basque Country (UPV/EHU), Spanish Research Council (CSIC)</institution>, <addr-line>Leioa</addr-line>, <country>Spain</country></aff><aff id=\"aff3\"><sup>3</sup><institution>NEIKER, Soil Microbial Ecology Group, Department of Conservation of Natural Resources</institution>, <addr-line>Derio</addr-line>, <country>Spain</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Tatiana Venkova, Fox Chase Cancer Center, United States</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Elisabeth Grohmann, Beuth Hochschule für Technik Berlin, Germany; Graciela Castro Escarpulli, National School of Biological Sciences, National Polytechnic Institute of Mexico (IPN), Mexico</p></fn><corresp id=\"c001\">*Correspondence: Itziar Alkorta, <email>itzi.alkorta@ehu.eus</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>Present address: Begoña Ugarte-Uribe, EMBL Heidelberg Cell Biology and Biophysics, Heidelberg, Germany</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Molecular Recognition, a section of the journal Frontiers in Molecular Biosciences</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>7</volume><elocation-id>185</elocation-id><history><date date-type=\"received\"><day>28</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>14</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Álvarez-Rodríguez, Ugarte-Uribe, de la Arada, Arrondo, Garbisu and Alkorta.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Álvarez-Rodríguez, Ugarte-Uribe, de la Arada, Arrondo, Garbisu and Alkorta</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Type IV Coupling Proteins (T4CPs) are essential elements in many type IV secretion systems (T4SSs). The members of this family display sequence, length, and domain architecture heterogeneity, being the conserved Nucleotide-Binding Domain the motif that defines them. In addition, most T4CPs contain a Transmembrane Domain (TMD) in the amino end and an All-Alpha Domain facing the cytoplasm. Additionally, a few T4CPs present a variable domain at the carboxyl end. The structural paradigm of this family is TrwB<sub>R388</sub>, the T4CP of conjugative plasmid R388. This protein has been widely studied, in particular the role of the TMD on the different characteristics of TrwB<sub>R388</sub>. To gain knowledge about T4CPs and their TMD, in this work a chimeric protein containing the TMD of TraJ<sub>pKM101</sub> and the cytosolic domain of TrwB<sub>R388</sub> has been constructed. Additionally, one of the few T4CPs of mobilizable plasmids, MobB<sub>CloDF13</sub> of mobilizable plasmid CloDF13, together with its TMD-less mutant MobBΔTMD have been studied. Mating studies showed that the chimeric protein is functional <italic>in vivo</italic> and that it exerted negative dominance against the native proteins TrwB<sub>R388</sub> and TraJ<sub>pKM101</sub>. Also, it was observed that the TMD of MobB<sub>CloDF13</sub> is essential for the mobilization of CloDF13 plasmid. Analysis of the secondary structure components showed that the presence of a heterologous TMD alters the structure of the cytosolic domain in the chimeric protein. On the contrary, the absence of the TMD in MobB<sub>CloDF13</sub> does not affect the secondary structure of its cytosolic domain. Subcellular localization studies showed that T4CPs have a unipolar or bipolar location, which is enhanced by the presence of the remaining proteins of the conjugative system. Unlike what has been described for TrwB<sub>R388</sub>, the TMD is not an essential element for the polar location of MobB<sub>CloDF13</sub>. The main conclusion is that the characteristics described for the paradigmatic TrwB<sub>R388</sub> T4CP should not be ascribed to the whole T4CP family. Specifically, it has been proven that the mobilizable plasmid-related MobB<sub>CloDF13</sub> presents different characteristics regarding the role of its TMD. This work will contribute to better understand the T4CP family, a key element in bacterial conjugation, the main mechanism responsible for antibiotic resistance spread.</p></abstract><kwd-group><kwd>coupling proteins</kwd><kwd>type IV secretion systems</kwd><kwd>bacterial conjugation</kwd><kwd>membrane proteins</kwd><kwd>antibiotic resistance spread</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Euskal Herriko Unibertsitatea<named-content content-type=\"fundref-id\">10.13039/501100003451</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"4\"/><table-count count=\"6\"/><equation-count count=\"0\"/><ref-count count=\"60\"/><page-count count=\"15\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Type IV coupling proteins (T4CPs) are essential elements in the conjugative type IV secretion systems (T4SSs) and are also key elements in many pathogenic T4SSs. The members of this family display a high sequence, length, and domain architecture heterogeneity being the Nucleotide-Binding Domain (NBD) the only conserved domain in all T4CPs. For this reason, they are classified according to the different domain architectures: (i) VirD4-type subfamily that are integral membrane proteins; (ii) TraG-J pairs, which are also integral membrane proteins but additionally present a physical and functional association with another membrane protein of the T4SS; (iii) T4CPs without Transmembrane Domain (TMD), which could or could not interact with other T4SS membrane proteins creating a VirD4-type complex, like the pair TraJ<sub>pIP501</sub> and TraI<sub>pIP501</sub>; (iv) FtsK-like T4CPs; and (v) Archaeal T4CPs (<xref rid=\"B35\" ref-type=\"bibr\">Llosa and Alkorta, 2017</xref>).</p><p>The structural paradigm of this family is the T4CP of conjugative plasmid R388, TrwB<sub>R388</sub>. It is a VirD4-type protein composed of a TMD at the N-terminus (consisting of two transmembrane α-helices connected through a small periplasmic loop) and a bulky globular cytosolic domain (CD). TrwB<sub>R388</sub> is the only full-length T4CP that has been successfully purified to date (<xref rid=\"B27\" ref-type=\"bibr\">Hormaeche et al., 2002</xref>; <xref rid=\"B44\" ref-type=\"bibr\">Redzej et al., 2017</xref>), while trials for purifying other membrane T4CPs have not rendered the sufficient amounts of high quality protein for performing <italic>in vitro</italic> assays (<xref rid=\"B14\" ref-type=\"bibr\">Chen et al., 2008</xref>). For this reason most of the <italic>in vitro</italic> studies of T4CPs have been achieved using deletion mutant proteins that lack the TMD (<xref rid=\"B45\" ref-type=\"bibr\">Schroder and Lanka, 2003</xref>; <xref rid=\"B50\" ref-type=\"bibr\">Tato et al., 2007</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Larrea et al., 2017</xref>). In this regard, the TMD deletion mutant protein of TrwB<sub>R388</sub>, TrwBΔN70, was resolved by X-ray crystallography, showing that the CD of TrwB<sub>R388</sub> contains an NBD with the Walker A and Walker B motifs and a small membrane-distal All-Alpha Domain (AAD) (<xref rid=\"B23\" ref-type=\"bibr\">Gomis-Rüth et al., 2001</xref>).</p><p>Comparative studies of the properties of TrwB<sub>R388</sub> and TrwBΔN70 showed significant differences regarding biological activity (such as <italic>in vivo</italic> function, <italic>in vitro</italic> nucleotide-binding properties, and <italic>in vitro</italic> ATPase activity), oligomerization pattern, subcellular location, and stability (<xref rid=\"B40\" ref-type=\"bibr\">Moncalián et al., 1999</xref>; <xref rid=\"B56\" ref-type=\"bibr\">Vecino et al., 2010</xref>, <xref rid=\"B54\" ref-type=\"bibr\">2011</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Hormaeche et al., 2002</xref>, <xref rid=\"B28\" ref-type=\"bibr\">2004</xref>, <xref rid=\"B29\" ref-type=\"bibr\">2006</xref>; <xref rid=\"B46\" ref-type=\"bibr\">Segura et al., 2013</xref>, <xref rid=\"B47\" ref-type=\"bibr\">2014</xref>). For this reason it has been concluded that the TMD of TrwB<sub>R388</sub> accomplishes a role beyond the anchorage of the protein to the membrane, influencing the location, stability, and activity of this protein.</p><p>To delve into the role of the TMD in T4CPs two different strategies have been followed. On the one hand, we have constructed a chimeric protein named TMD<sub>TraJ</sub>CD<sub>TrwB</sub> composed of the TMD of TraJ<sub>pKM101</sub>, the phylogenetically closest T4CP to TrwB<sub>R388</sub> from the conjugative plasmid pKM101 (<xref rid=\"B43\" ref-type=\"bibr\">Paterson et al., 1999</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Alvarez-Martinez and Christie, 2009</xref>) and the CD of TrwB<sub>R388</sub>. This strategy has already been used for the study of components of T4SSs, showing interesting results (<xref rid=\"B11\" ref-type=\"bibr\">Bourg et al., 2009</xref>). Specifically, through a chimeric protein approach the function of the AAD of VirD4<sub>At</sub> from the T-plasmid of <italic>Agrobacterium tumefaciens</italic> (<xref rid=\"B59\" ref-type=\"bibr\">Whitaker et al., 2016</xref>) and of the N-terminal HUH domain of TrwC<sub>R388</sub> (<xref rid=\"B2\" ref-type=\"bibr\">Agúndez et al., 2018</xref>) have been analyzed. On the other hand, the T4CP from the mobilizable plasmid CloDF13, MobB<sub>CloDF13</sub> and its deletion protein lacking the TMD, MobBΔTMD, have been constructed and studied. It is an interesting system to characterize since it is part of the rare MOB<sub>C1</sub> plasmid family, which are mobilizable plasmids that encode their T4CP (<xref rid=\"B49\" ref-type=\"bibr\">Smillie et al., 2010</xref>). Additionally, MobB<sub>CloDF13</sub> has been described as an atypical T4CP, due to its dual role in DNA transfer, since it acts as an accessory protein in CloDF13 relaxation process and also as a T4CP (<xref rid=\"B41\" ref-type=\"bibr\">Nuñez and de la Cruz, 2001</xref>).</p><p>We studied the functionality of these proteins in plasmid transfer, secondary structure, thermal stability, and subcellular localization. Our findings indicate that the TMD plays different roles in conjugative plasmid related and mobilizable plasmid related T4CPs. Specifically while the TMD could play a regulatory role in TrwB<sub>R388</sub> this cannot be inferred from the results about MobB<sub>CloDF13</sub>.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Materials</title><p>n-dodecyl β-D maltoside (DDM) was purchased from Anatrace (Santa Clara, CA, United States). Mouse anti-His (C-term) monoclonal antibody and Alexa Fluor goat anti-mouse antibody were purchased from Invitrogen (Carlsbad, CA, United States) and Molecular Probes (Eugene, OR, United States), respectively. All buffers employed in this work are shown in <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table S1</xref>.</p></sec><sec id=\"S2.SS2\"><title>Bacterial Strains and Bacterial Growth Conditions</title><p><italic>E. coli</italic> DH5α strain was used as host for plasmid constructions. This strain also served as the donor for mating experiments by hosting the plasmids of interest. <italic>E. coli</italic> UB1637 served as the recipient for mating experiments. <italic>E. coli</italic> Lemo21 (DE3), <italic>E. coli</italic> BL21 (DE3), and <italic>E. coli</italic> BL21C41 (DE3) strains were used for protein production, purification, and <italic>in vivo</italic> localization.</p><p><italic>E. coli</italic> strains were grown in LB medium and when necessary antibiotics were added at the following final concentrations: ampicillin (100 μg/mL), streptomycin (50 μg/mL), kanamycin (50 μg/mL), chloramphenicol (12.5–25 μg/mL), and thrimethoprim (10 μg/mL).</p></sec><sec id=\"S2.SS3\"><title>Plasmids</title><p>The plasmids and oligonucleotides used in this study are listed in <xref rid=\"T1\" ref-type=\"table\">Tables 1</xref>, <xref rid=\"T2\" ref-type=\"table\">2</xref>, respectively.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Plasmids employed in this work.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Plasmid</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Description</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phenotype</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">References</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pET24a (+)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Expression vector</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Kan<sup>R</sup>, C-terminal His tag</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Novagen</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R388</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Natural plasmid</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tmp<sup>R</sup>, TRA<sub><italic>W</italic></sub>, IncW</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B16\" ref-type=\"bibr\">Datta and Hedges, 1972</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pKM101</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Natural plasmid</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Amp<sup>R</sup>, TRA<sub><italic>N</italic></sub>, IncN</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B32\" ref-type=\"bibr\">Langer and Walker, 1981</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pKM101Δ<italic>traJ</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pKM101:Δ<italic>traJ</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Amp<sup>R</sup>, TRA<sub><italic>N</italic></sub>, IncN, TraJ<sup>–</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This work</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pKM101Spc<sup>r</sup>_Δ<italic>traJ</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pKM101:Δ<italic>traJ</italic> and Spt<sup>R</sup> resistance cassette</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Spt<sup>R</sup>, TRA<sub><italic>N</italic></sub>, IncN, TraJ<sup>–</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B59\" ref-type=\"bibr\">Whitaker et al., 2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Expression vector</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Amp<sup>R</sup>, C-terminal His tag</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B9\" ref-type=\"bibr\">Berrow et al., 2007</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE-3C-eGFP</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Expression vector</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Amp<sup>R</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OPPF-UK (Addgene)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE-3C-eGFP-<italic>mobB</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE-3C-eGFP:<italic>mobB</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE-3C-eGFP:MobB<sub>CloDF13</sub>GFP expression under T7 promoter</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This work</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE-3C-eGFP-<italic>MobBΔTMD</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE-3C-eGFP:<italic>MobBΔTMD</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE-3C-eGFP:MobBΔTMDGFP expression under T7 promoter</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This work</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE-<italic>mobB</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE:<italic>mobB</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE:MobB<sub>CloDF13</sub> expression under T7 promoter</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This work</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE-<italic>MobBΔTMD</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE:<italic>MobBΔTMD</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE:MobBΔTMD expression under T7 promoter</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This work</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pSU1456</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R388:Δ<italic>trwB</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Su<sup>R</sup>, Tmp<sup>R</sup>, TRA<sub><italic>W</italic></sub>, IncW, TrwB<sup>–</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B36\" ref-type=\"bibr\">Llosa et al., 1994</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pSU4814</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pSU19:<italic>mob</italic><sub>CloDF13</sub></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chl<sup>r</sup>, Rep (p15A), MOB (CloDF13)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B41\" ref-type=\"bibr\">Nuñez and de la Cruz, 2001</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pSU4833</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pSU4814:Δ<italic>mobB</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chl<sup>R</sup>, Rep (p15A), MobB<sup>–</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B41\" ref-type=\"bibr\">Nuñez and de la Cruz, 2001</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pUB9</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pWaldo-GFPe:<italic>trwB-GFP</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Kan<sup>R</sup>, TrwB-TEV-GFP-H<sub>8</sub>, expression under T7 promoter</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B47\" ref-type=\"bibr\">Segura et al., 2014</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pUBQ4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pET24a (+):<italic>traJTMD-trwBCD</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Kan<sup>R</sup>, TMD<sub>TraJ</sub>CD<sub>TrwB</sub>-H<sub>6</sub>, expression under T7 promoter</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This work</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pUBQ4 (K142T)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pET24a (+):<italic>traJTMD-trwBCD (K142T)</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Kan<sup>R</sup>, TMD<sub>TraJ</sub>CD<sub>TrwB</sub>-H<sub>6</sub> containing the K142T mutation in the CD<sub>TrwB</sub> domain, expression under T7 promoter</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This work</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pUBQ4GFP</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pWaldo-GFPe:<italic>traJTMD-trwBCD-GFP</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Kan<sup>R</sup>, TMD<sub>TraJ</sub>CD<sub>TrwB</sub>-TEV-GFP-H<sub>8</sub>, expression under T7 promoter</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">This work</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pWaldo-GFPe</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Expression vector</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Kan<sup>R</sup>, C-Terminal GFP and His tag</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B58\" ref-type=\"bibr\">Waldo et al., 1999</xref></td></tr></tbody></table></table-wrap><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Oligonucleotides used in this work.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Construct</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Template</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Protein</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Sequence (5′ → 3′)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cloning technology</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE-<italic>mobB</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">pSU4814</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MobB<sub>CloDF13</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">F:</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AGGAGATATACCATGTTTAATACGGATTCGCTTGCCTGGCAGTGG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">In-fusion<sup>a</sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R:</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GTGATGGTGATGTTTGTACAGCCCCGCAAAATCATCATCACCCG</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE-<italic>MobBΔTMD</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">pSU4814</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MobBΔTMD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">F:</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AGGAGATATACCATGGACGAGGCGGTGAACGCGAAAC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">In-fusion<sup>a</sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R:</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GTGATGGTGATGTTTGTACAGCCCCGCAAAATCATCATCAC</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE-3C-eGFP-<italic>mobB</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">pSU4814</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MobB<sub>CloDF13</sub>GFP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">F:</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AGGAGATATACCATGTTTAATACGGATTCGCTTGCCTGGCAGTGG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">In-fusion<sup>a</sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R:</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAGAACTTCCAGTTTGTACAGCCCCGCAAAATCATCATCACCCG</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pOPINE-3C-eGFP-<italic>MobBΔTMD</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">pSU4814</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MobBΔTMDGFP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">F:</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AGGAGATATACCATGGACGAGGCGGTGAACGCGAAAC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">In-fusion<sup>a</sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R:</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAGAACTTCCAGTTTGTACAGCCCCGCAAAATCATCATCAC</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pUBQ4 (K142T)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">pUBQ4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">TMD<sub>TraJ</sub>CD<sub>TrwB</sub> (K142T)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">F:</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AAGCAACACCGATGTACCCGTACCAGTGGCA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Site-Directed mutagenesis<sup><italic>b</italic></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R:</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GTGCCACTGGTACGGGTACATCGGTGTTGCTT</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pUBQ4GFP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">pUBQ4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">TMD<sub>TraJ</sub>CD<sub>TrwB</sub>GFP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">F:</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CCG<bold>CTCGAG</bold>ATGGACGATAGAGAAAGAGG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Restriction enzymes (<italic>Xho</italic>I and <italic>Bam</italic>HI)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R:</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CGC<bold>GGATCC</bold>GATAGTCCCCTCAAC</td><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic><sup>a</sup><xref rid=\"B9\" ref-type=\"bibr\">Berrow et al. (2007)</xref>. <sup>b</sup>QuikChange II Site-Directed Mutagenesis (Stratagene, San Diego, CA, United States).</italic></attrib></table-wrap-foot></table-wrap><p>pSU4814 and pSU4833 plasmids were kindly provided by Fernando de la Cruz. pOPINE (Addgene plasmid # 26043; <ext-link ext-link-type=\"uri\" xlink:href=\"https://scicrunch.org/resolver/RRID:Addgene_26043\">RRID: Addgene_26043</ext-link>)<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup> and pOPINE-3C-eGFP (Addgene plasmid # 41125; RRID: <ext-link ext-link-type=\"uri\" xlink:href=\"https://scicrunch.org/resolver/Addgene_41125\">Addgene_41125</ext-link>)<sup><xref ref-type=\"fn\" rid=\"footnote2\">2</xref></sup> plasmids were a gift from Ray Owens. pKM101Spc<sup>r</sup>_Δ<italic>traJ</italic> was kindly provided by Peter J. Christie. pKM101Amp<sup>r</sup>_Δ<italic>traJ</italic> plasmid was obtained by cleavage of the <italic>spc cassette of</italic> pKM101Spc<sup>r</sup>_Δ<italic>traJ</italic> plasmid using <italic>Eco</italic>RI restriction enzyme.</p></sec><sec id=\"S2.SS4\"><title>Cloning of T4CPs</title><p>To construct the chimeric protein TMD<sub>TraJ</sub>CD<sub>TrwB</sub> and the transmembrane deletion mutant protein of MobB<sub>CloDF13</sub>, MobBΔTMD, the sequences of TrwB<sub>R388</sub>, TraJ<sub>pKM101</sub>, and MobB<sub>CloDF13</sub>, were analyzed through bioinformatics tools. First, the different characteristics of the proteins, such as molecular weight and isoelectric point, were analyzed using <italic>ProtParam</italic>.<sup><xref ref-type=\"fn\" rid=\"footnote3\">3</xref></sup> Second, the topology of the membrane proteins was studied using <italic>Topcons</italic><sup><xref ref-type=\"fn\" rid=\"footnote4\">4</xref></sup> (<xref rid=\"B52\" ref-type=\"bibr\">Tsirigos et al., 2015</xref>).</p><p>Then different constructions were achieved as follows:</p><p>TMD<sub>TraJ</sub>CD<sub>TrwB</sub> chimeric protein consists of amino acids M<sub>1</sub>-D<sub>76</sub> from TraJ<sub>pKM101</sub> followed by amino acids L<sub>71</sub>-I<sub>507</sub> from TrwB<sub>R388</sub>. The <italic>tmd<sub>TraJ</sub>cd<sub>TrwB</sub></italic> sequence was synthesized <italic>de novo</italic> and inserted it into pET24a (+) plasmid vector using <italic>Nde</italic>I and <italic>Xho</italic>I restriction sites, rendering pUBQ4 plasmid to produce the chimeric protein. This construction was made by TOP Gene Technologies, Inc. (Saint-Lauren, QC-Canada). To study the role of the conserved lysine of the Walker A motif (K<sub>142</sub>), this residue was substituted with a threonine using the QuikChange II Site-Directed Mutagenesis Kit from Stratagene (San Diego, CA, United States) to obtain the TMD<sub>TraJ</sub>CD<sub>TrwB</sub> (K142T) protein. Additionally, to clone the <italic>tmd<sub>TraJ</sub>cd<sub>TrwB</sub>-eGFP</italic> gene <italic>tmd<sub>TraJ</sub>cd<sub>TrwB</sub></italic> sequence was inserted into the pWaldo-GFPe plasmid vector using <italic>Xho</italic>I and <italic>Bam</italic>HI restriction sites (<xref rid=\"B47\" ref-type=\"bibr\">Segura et al., 2014</xref>).</p><p>To clone MobB<sub>CloDF13</sub>-related proteins, the cloning of <italic>mobB, MobBΔTMD, mob-eGFP</italic>, and <italic>MobBΔTMD-eGFP</italic> genes was performed in the Oxford Protein Production Facility (OPPF-UK) using the High-throughput protocol described by <xref rid=\"B10\" ref-type=\"bibr\">Bird (2011)</xref>. Specifically, MobBΔTMD soluble mutant protein consists of amino acids D<sub>185</sub>-Y<sub>653</sub> of MobB<sub>CloDF13</sub> obtained after deletion of amino acids M<sub>1</sub>-A<sub>184</sub> from wild type MobB<sub>CloDF13</sub>.</p><p>All the oligonucleotides employed in the aforementioned cloning experiments are specified in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>.</p></sec><sec id=\"S2.SS5\"><title>Overexpression and Purification</title><p>The same purification protocol was followed for TMD<sub>TraJ</sub>CD<sub>TrwB</sub> and MobB<sub>CloDF13</sub> proteins. Briefly, <italic>E. coli</italic> BL21C41 (DE3) cells freshly transformed with plasmids pUBQ4 for TMD<sub>TraJ</sub>CD<sub>TrwB</sub> and pOPINE-<italic>mobB</italic> for MobB<sub>CloDF13</sub> were grown overnight in LB (8 flasks of 10 mL) supplemented with the corresponding antibiotics at 37°C with continuous shaking. Then, cells were diluted 1:50 (v/v) with fresh LB supplemented with antibiotics (8 flasks of 500 mL) and were grown at 37°C with continuous shaking until an OD<sub>600</sub> of 0.4–0.5 was achieved. Next, overexpression was induced by the addition of 1 mM isopropyl α-D-thiogalactopyranoside (IPTG) and cells were grown with continuous shaking at 25°C overnight. Cells were harvested by centrifugation at 8,000 <italic>g</italic> for 15 min at 4°C. The pellet was suspended in 80 mL of <italic>Cell buffer</italic>, frozen with liquid N<sub>2</sub> and stored at −80°C.</p><p>For purification, cells were thawed at 37°C and 0.02 mg/mL DNase I, 1 mM dithiothreitol (DTT), 0.07% (w/v) lysozyme, 1 mM MgCl<sub>2</sub>, 1 mM phenylmethanesulfonyl fluoride (PMSF) and two tablets of cOmplete<sup>TM</sup> EDTA-free Protease Inhibitor Cocktail from Sigma-Aldrich (San Luis, MO, United States) were added. From this point onward, the whole process was performed at 4°C to avoid aggregation of the proteins. After 45 min of incubation with agitation, cells were disrupted by sonication and centrifuged at 8,000 <italic>g</italic> for 15 min to remove non-lysed cell. The supernatant, containing the broken cells, was centrifuged at 138,000 <italic>g</italic> for 45 min to pellet the membrane fraction which was subsequently carefully resuspended in 30 mL of <italic>Cell buffer</italic>. Then DDM and NaCl were added to final concentrations of 19.6 mM and 600 mM, respectively, and the volume was adjusted to 40 mL. The mixture was incubated for 90 min with continuous stirring and then centrifuged at 138,000 <italic>g</italic> for 1 h.</p><p>The supernatant containing the protein to be purified was mixed 1:1 (v/v) with <italic>MP1 buffer</italic> to decrease the concentration of DDM and NaCl to 8.3 mM and 300 mM, respectively. Then, the sample was supplemented with 50 mM imidazole and loaded onto a 5 mL <italic>HisTrap<sup>TM</sup> FF</italic> (GE Life Sciences; Marlborough, MA, United States) column previously equilibrated with <italic>MP2 buffer</italic>. To increase the binding of the protein, the sample was left recirculating overnight. Next, it was connected to an <italic>ÄKTA-FPLC system</italic> and it was washed with 50 mL of <italic>MP2 buffer</italic> at a flow rate of 2 mL/min until the absorbance at 280 nm reached the baseline. Bound proteins were eluted with <italic>MP3 buffer</italic>, at a flow rate of 1.5 mL/min and fractions of 1 mL were collected. Obtained samples were analyzed by SDS-PAGE and the ones containing each target protein were pulled-down and concentrated using a centrifugal filter with a MWCO of 100 kDa. Then, 5 mL of the resulting samples were separately loaded onto a <italic>Superdex 200 HR 16/60</italic> column and the size-exclusion chromatography (SEC) was performed in <italic>MP purification buffer</italic> at 0.5 mL/min. The fractions corresponding to each target protein were pulled-down and concentrated as explained before. Glycerol was added to a final concentration of 20% (v/v) and protein concentration was determined by measuring absorption at 280 nm. Finally, aliquots were stored at −80°C.</p><p>When TMD<sub>TraJ</sub>CD<sub>TrwB</sub> was purified with the aim of performing infrared spectroscopy (IR) assays the DDM and NaCl concentrations of the MP purification buffer were changed to the ones described in the previously published purification protocols of TrwB<sub>R388</sub> and TrwBΔN50 (i.e., 0.2 mM DDM and 200 mM NaCl instead of 0.6 mM DDM and 300 mM NaCl) (<xref rid=\"B54\" ref-type=\"bibr\">Vecino et al., 2011</xref>, <xref rid=\"B55\" ref-type=\"bibr\">2012</xref>).</p><p>For MobBΔTMD purification, <italic>E. coli</italic> Lemo21 (DE3) cells freshly transformed with pOPINE-<italic>MobBΔTMD</italic> plasmid were grown in 4 L of LB supplemented with ampicillin at 37°C with continuous shaking until an OD<sub>600</sub> of 0.5–0.6 was reached. Expression was induced with 1 mM IPTG and performed for 20 h at 25°C. Cells were harvested and stored as explained previously.</p><p>For protein purification, the cell suspension was thawed and the lysis protocol previously described for TMD<sub>TraJ</sub>CD<sub>TrwB</sub> and MobB<sub>CloDF13</sub> was followed. Then, the sample was centrifuged at 138,000 <italic>g</italic> for 45 min to pellet the membrane fraction and the inclusion bodies. The supernatant with the soluble proteins was supplemented with 50 mM imidazole and loaded onto a 5 mL <italic>HisTrap<sup>TM</sup> FF</italic> (GE Life Sciences; Marlborough, MA, United States) column, previously equilibrated with <italic>MobBΔTMD1 buffer</italic>. Affinity chromatography was performed as previously described for MobB<sub>CloDF13</sub>, but using <italic>MobBΔTMD1 buffer</italic> for washing and <italic>MobBΔTMD2 buffer</italic> for elution. Fractions containing the target protein were pulled-down and concentrated using a <italic>Centricon YM-30</italic> to a final volume of 600 μL. The resulting sample was centrifuged to remove aggregates and loaded onto a <italic>Superdex 200 HR 10/30</italic> column. The SEC was performed in <italic>Cell buffer</italic> at 0.3 mL/min and 0.5 mL fractions were collected. Fractions that contained the protein of interest were pulled-down and the sample was centrifuged to discard the aggregates. Glycerol was added to a final concentration of 20% (v/v) and aliquots were made.</p></sec><sec id=\"S2.SS6\"><title>Mating Assays</title><p>Mating assays were performed as described by <xref rid=\"B37\" ref-type=\"bibr\">Llosa et al. (2003)</xref> with small modifications. Briefly, donors (<italic>E. coli</italic> DH5α co-transformed with the appropriate plasmids) and recipient cells (<italic>E. coli</italic> UB1637) were grown in LB supplemented with the corresponding antibiotics overnight at 37°C. For each mating assay 100 μL of the donor and the recipient cells were mixed, centrifuged, resuspended in 50 μL LB and placed onto a GS Millipore filter (0.22 μm pore size) settled on a pre-warmed LB-agar plate. After 1 h incubation at 37°C bacteria were washed from the filters in 2 mL LB by shaking at 450 rpm for 20 min and vortexing for 30 s. Then, 100 μL of the appropriate dilutions were plated on selective media for donors and transconjugants. The plates were incubated overnight at 37°C and the colonies were counted, normalizing the conjugation frequency as the number of transconjugants per donor cell.</p></sec><sec id=\"S2.SS7\"><title>Infrared Spectroscopy</title><p>To accomplish IR studies of different T4CPs and their variants (i.e., TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, MobB<sub>CloDF13</sub>, and MobBΔTMD) purification of each protein was carried out as previously described. The H-D exchange protocol was performed at 4°C and adapted for each protein. Briefly, TMD<sub>TraJ</sub>CD<sub>TrwB</sub> was diluted with the IR buffer, dialyzed against the same buffer using a <italic>D-Tube<sup>TM</sup> Dialyzer Midi</italic> (MWCO 3.5 kDa) (Merck; Darmstadt, Germany), diluted again in IR buffer and concentrated using an Amicon Ultra-0.5 mL centrifugal filter (MWCO: 100 kDa). A similar process was followed for MobB<sub>CloDF13</sub> except for the dialysis step. Regarding MobBΔTMD, sample was diluted, dialyzed and concentrated as described for TMD<sub>TraJ</sub>CD<sub>TrwB</sub> but using in MobBΔTMD IR buffer. Final protein samples were always above 1 mg/mL protein concentration.</p><p>Infrared spectra were recorded in a Thermo Nicolet Nexus 5700 (Thermo Fisher Scientific; Waltham, MA, United States) spectrometer equipped with a liquid nitrogen-refrigerated mercury-cadmium-telluride detector using a Peltier-based temperature controller (TempComp<sup>TM</sup>, BioTools; Wauconda, IL, United States), and a 25 μm optical path. Typically 370 scans for each, background and sample, were collected at 2 cm<sup>–1</sup> resolution and averaged after each minute. Spectra were collected with OMNIC software (Nicolet) and data processing was performed with OMNIC and SpectraCalc, following previously resolved methods (<xref rid=\"B6\" ref-type=\"bibr\">Arrondo et al., 1993</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Arrondo and Goñi, 1999</xref>).</p><p>The information about the secondary structure and about the thermal denaturation of T4CPs and their derivatives was obtained by IR spectroscopy through analysis of the infrared amide I band that corresponds mainly to the C = O stretching vibrations of the peptide bonds and which is located between 1700 and 1600 cm<sup>–1</sup> region of the IR spectrum. Amide I band is conformationally sensitive and can be used to monitor the protein secondary structure composition and changes induced by thermal denaturation. Secondary structure studies were made at 20°C and band decomposition of the amide I was performed as previously reported (<xref rid=\"B54\" ref-type=\"bibr\">Vecino et al., 2011</xref>, <xref rid=\"B55\" ref-type=\"bibr\">2012</xref>). For thermal stability studies samples were heated from 20 to 80°C at a rate of 1°C/min.</p></sec><sec id=\"S2.SS8\"><title>Subcellular Location of T4CPs</title><p>Subcellular location of different T4CPs and their variants (i.e., TrwB<sub>R388</sub>, TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, MobB<sub>CloDF13</sub>, and MobBΔTMD) was achieved by confocal fluorescence microscopy. To do so, two different approaches were used: (i) eGFP-labeling (<xref rid=\"B15\" ref-type=\"bibr\">Cormack et al., 1996</xref>) and (ii) immunofluorescence. Since the eGFP moiety only emits fluorescence when properly folded (<xref rid=\"B19\" ref-type=\"bibr\">Drew et al., 2005</xref>), the eGFP based approach allowed visualizing only properly folded proteins.</p><p>Prior to localization assays, the <italic>in vivo</italic> activity of the T4CP-eGFP fusion-proteins was proved by mating assays as previously described (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table S2</xref>). Afterward, T4CP-eGFP fusion-proteins were expressed in <italic>E. coli</italic> BL21C41 (DE3) strain, except for MobBΔTMD that was expressed in BL21 (DE3) strain. To do so cells were transformed with pUBQ4, by induction with 1 mM IPTG at OD<sub>600</sub> 0.4–0.5 for the membrane proteins and OD<sub>600</sub> 0.5–0.6 for MobBΔTMD. Protein expression was performed for 4 and 20 h at 25°C. Additionally, the subcellular location of TrwB<sub>R388</sub>-related proteins was determined in the presence of pSU1456 plasmid, which codes for all the R388 conjugative proteins except TrwB<sub>R388</sub>. Similarly, MobB<sub>CloDF13</sub> was also expressed in the presence of plasmid pSU1456 and to mimic the <italic>in vivo</italic> transfer of CloDF13, its location was additionally studied in the presence of plasmids pSU1456 (R388 plasmid that lacks TrwB<sub>R388</sub> protein) and pSU4833 (CloDF13 plasmid that contains its mobilization region except for MobB<sub>CloDF13</sub> protein). Sample handling was performed as described by <xref rid=\"B47\" ref-type=\"bibr\">Segura et al. (2014)</xref>. The images were acquired in a Leica TCS SP5 confocal fluorescence microscope, with a 60× oil immersion objective. Sample excitation was performed with 488 nm wavelength, while fluorescence emission was measured between 505 and 525 nm. The images were analyzed using Huygens and ImageJ softwares. To ease the counting process and better distinguish the different locations the images were treated with the preset ICE filter of ImageJ software; in this manner five different locations for the T4CP-eGFP fusion-proteins were described (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Figure S1</xref>).</p><p>For immunofluorescence assays protein expression was performed as with the eGFP fusion-proteins. Sample collection and handling was performed as described by <xref rid=\"B47\" ref-type=\"bibr\">Segura et al. (2014)</xref>. Cells were immunostained with mouse anti-His (C-term) monoclonal antibody as primary antibody, and Alexa Fluor goat anti-mouse as secondary antibody. Image acquisition was performed in an Olympus FluoviewTM 500 confocal fluorescence microscope at the “Analytical and high-resolution microscopy in biomedicine” facility (SGIker, UPV/EHU).</p></sec></sec><sec id=\"S3\"><title>Results</title><p>Bacterial conjugation is one of the main processes responsible for the horizontal dissemination of antibiotic resistance genes among bacteria. One of the essential proteins in this process is the T4CP, which is ubiquitous in all conjugative systems. Despite its importance, the only widely studied T4CP is TrwB<sub>R388</sub>. Given its central role in bacterial conjugation, detailed knowledge of the T4CP family could contribute to the development of new strategies against the spread of antibiotic resistance among bacteria.</p><p>Previously published papers have highlighted the role of the TMD on different characteristics of TrwB<sub>R388</sub>, such as plasmid conjugation (<xref rid=\"B40\" ref-type=\"bibr\">Moncalián et al., 1999</xref>), subcellular localization (<xref rid=\"B47\" ref-type=\"bibr\">Segura et al., 2014</xref>), nucleotide-binding (<xref rid=\"B29\" ref-type=\"bibr\">Hormaeche et al., 2006</xref>), hexamerization (<xref rid=\"B27\" ref-type=\"bibr\">Hormaeche et al., 2002</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Matilla et al., 2010</xref>), protein stability (<xref rid=\"B28\" ref-type=\"bibr\">Hormaeche et al., 2004</xref>), interaction with other proteins of the T4SS of R388 (<xref rid=\"B46\" ref-type=\"bibr\">Segura et al., 2013</xref>), and ATP hydrolase activity (<xref rid=\"B51\" ref-type=\"bibr\">Tato et al., 2005</xref>, <xref rid=\"B50\" ref-type=\"bibr\">2007</xref>). From all these studies it was inferred that the TMD of TrwB<sub>R388</sub> has a role beyond the mere anchorage in the membrane.</p><p>To gain more knowledge about different T4CPs, and in particular about the role of their TMD in T4CP features, in this work a TrwB<sub>R388</sub> chimeric protein that combines its CD with the TMD of its phylogenetically closest T4CP, TraJ<sub>pKM101</sub>, has been studied. Also one of the few T4CPs of mobilizable plasmids, MobB<sub>CloDF13</sub>, and its TMD deletion mutant protein, MobBΔTMD, have been studied. Plasmid transfer, secondary structure, thermal stability, and subcellular location studies have been carried out to shed light on the functioning of this protein family and in the role of their TMD.</p><sec id=\"S3.SS1\"><title>Cloning of Soluble Mutant and Chimeric Proteins</title><p>The membrane protein topologies obtained after the bioinformatic analysis performed with Topcons software of TrwB<sub>R388</sub>, TraJ<sub>pKM101</sub>, and MobB<sub>CloDF13</sub> are shown in <xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>. TrwB<sub>R388</sub> and TraJ<sub>pKM101</sub> have similar size and organization of their TMDs that consist of about 70 residues and contain two α-helixes connected by a small periplasmic loop. In contrast, the TMD of MobB<sub>CloDF13</sub> is larger (about 150 amino acids) and is organized into three α-helixes. This information was used to design the chimeric and mutant proteins studied in this work (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). The chimeric protein TMD<sub>TraJ</sub>CD<sub>TrwB</sub> was made by combination of the TMD of the T4CP TraJ<sub>pKM101</sub> and the CD of TrwB<sub>R388</sub>. In addition, in this work the T4CP of the mobilizable plasmid CloDF13, MobB<sub>CloDF13</sub>, and its TMD-deletion protein MobBΔTMD, were constructed (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). The theoretical molecular weights of these proteins, necessary for their purification process, were calculated using <italic>ProtParam</italic><sup><xref ref-type=\"fn\" rid=\"footnote3\">3</xref></sup> bioinformatic tool. The estimated molecular weights were 58.28, 73.95, and 53.13 kDa for TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, MobB<sub>CloDF13</sub>, and MobBΔTMD, respectively. Finally, the eGFP fusion-proteins (i.e., TMD<sub>TraJ</sub>CD<sub>TrwB</sub>-eGFP, MobB<sub>CloDF13</sub>-eGFP, and MobBΔTMD-eGFP) were constructed and since they emitted a fluorescent signal, it was deduced that they were correctly folded (<xref rid=\"B18\" ref-type=\"bibr\">Drew et al., 2006</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p><bold>(A)</bold> Predicted membrane topology of TrwB<sub>R388</sub>, TraJ<sub>pKM101</sub>, and MobB<sub>CloDF13</sub> proteins. Membrane topology of the different T4CPs was predicted using Topcons software. The black lines represent the inner bacterial membrane. M1, amino-terminus; COOH, carboxy-terminus. The first and last residues of each transmembrane helix are shown indicating their position in the sequence. Proteins from R388, pKM101, and CloDF13 plasmids are shown in green, blue, and purple, respectively. <bold>(B)</bold> Schematic representation of the different T4CPs and their variants used in the present study. Proteins from R388, pKM101, and CloDF13 plasmids are shown in green, blue and purple, respectively. The transmenbrane α-helices (H) and the small periplasmic loops connecting α-helices are indicated in dark boxes and stripped boxes, respectively.</p></caption><graphic xlink:href=\"fmolb-07-00185-g001\"/></fig></sec><sec id=\"S3.SS2\"><title>Functionality and Dominance Experiments</title><p>Through mating assays two different properties of TMD<sub>TraJ</sub>CD<sub>TrwB</sub> were analyzed: (i) its capacity to complement the conjugative process in the absence of another T4CP (<italic>functionality studies</italic>) and (ii) its effect on each native conjugative system (R388 or pKM101 plasmids), being the corresponding T4CP present (TrwB<sub>R388</sub> or TraJ<sub>pKM101</sub>, respectively) (<italic>dominance studies</italic>). Results obtained in mating assays are summarized in <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>.</p><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>Conjugation and dominance experiments with TMD<sub>TraJ</sub>CD<sub>TrwB</sub>. Transfer frequencies of plasmids pSU1456 and pKM101Δt<italic>raJ</italic> complemented with TMD<sub>TraJ</sub>CD<sub>TrwB</sub> protein have been studied.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Plasmids in donors</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">T4CP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Transfer frequency</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R388 (+)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Wild type TrwB<sub>R388</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><sup><italic>a</italic></sup>2.1 10<sup>–1</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R388Δ<italic>trwB</italic> (pSU1456) (−)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Ø</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 10<sup>–8</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pKM101 (+)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Wild type TraJ<sub>pKM101</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><sup>b</sup>2.2 10<sup>–1</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pKM101Δ<italic>traJ</italic> (−)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Ø</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 10<sup>–8</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R388Δ<italic>trwB</italic> (pSU1456) pUBQ4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">TMD<sub>TraJ</sub>CD<sub>TrwB</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.82 10<sup>–4</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pKM101Δ<italic>traJ</italic> pUBQ4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">TMD<sub>TraJ</sub>CD<sub>TrwB</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 10<sup>–8</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R388Δ<italic>trwB</italic> (pSU1456) pUBQ4 (K142T)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">TMD<sub>TraJ</sub>CD<sub>TrwB</sub> (K142T)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 10<sup>–8</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R388 pUBQ4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Wild type TrwB<sub>R388</sub> TMD<sub>TraJ</sub>CD<sub>TrwB</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.13 10<sup>–2</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pKM101 pUBQ4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Wild type TraJ<sub>pKM101</sub> TMD<sub>TraJ</sub>CD<sub>TrwB</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.10 10<sup>–2</sup></td></tr></tbody></table><table-wrap-foot><attrib><italic>Additionally, the effect of the chimeric protein in the conjugative process of R388 and pKM101 has been analyzed (dominance assays, shaded in gray). E. coli DH5α and UB1637 strains were used as donor and recipient cells, respectively. Transfer frequencies were normalized to the number of transconjugants per donor and are the mean value of at least five independent experiments. (+) Positive control; (–) negative control; Ø: no T4CP. <sup>a</sup>Data from <xref rid=\"B46\" ref-type=\"bibr\">Segura et al. (2013)</xref>. <sup>b</sup>Data from <xref rid=\"B59\" ref-type=\"bibr\">Whitaker et al. (2016)</xref>.</italic></attrib></table-wrap-foot></table-wrap><p>Our results showed that TMD<sub>TraJ</sub>CD<sub>TrwB</sub> efficiently complemented the Δ<italic>trwB</italic> mutation in R388 transfer but to a lower rate than native R388 (0.21 vs. 1.82 10<sup>–4</sup> transconjugants per donor, respectively). On the contrary, TMD<sub>TraJ</sub>CD<sub>TrwB</sub> was unable to complement the Δ<italic>traJ</italic> mutation in pKM101 transfer (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). These results are in agreement with the necessary specific interactions between the CD of the T4CP and its cognate relaxase for transfer to happen as reported previously (<xref rid=\"B12\" ref-type=\"bibr\">Cabezón et al., 1997</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Hamilton et al., 2000</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Llosa et al., 2003</xref>).</p><p>It has been reported that mutation of the conserved lysine in the Walker A motif rendered a transfer deficient mutant protein TrwBK136T (<xref rid=\"B29\" ref-type=\"bibr\">Hormaeche et al., 2006</xref>). Similarly, an equivalent mutant of the soluble protein TrwBΔN70, TrwBΔN70 (K136T), lacked ATPase activity (<xref rid=\"B40\" ref-type=\"bibr\">Moncalián et al., 1999</xref>), underlying the essential role of this amino acid in the activity of TrwB<sub>R388</sub>. Here we studied the effect of the equivalent point mutation in the Walker A domain, TMD<sub>TraJ</sub>CD<sub>TrwB</sub> (K142T), on the transfer capacity of chimeric protein. As expected, TMD<sub>TraJ</sub>CD<sub>TrwB</sub> (K142T) was unable to complement the Δ<italic>trwB</italic> mutation in R388 plasmid transfer (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). This is in agreement with the crucial role of the K residue as it has been reported with homologous mutants in other T4CPs (<xref rid=\"B40\" ref-type=\"bibr\">Moncalián et al., 1999</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Kumar and Das, 2002</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Gunton et al., 2005</xref>).</p><p>Next, to accomplish dominance assays, the transfer frequencies of plasmids R388 or pKM101 in the presence of the cognate T4CP and the chimeric protein were measured. It was observed that TMD<sub>TraJ</sub>CD<sub>TrwB</sub> reduced the transfer frequency of R388 or pKM101 by an order of magnitude (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>).</p></sec><sec id=\"S3.SS3\"><title>Mobilization Experiments</title><p>CloDF13 mobilization experiments were achieved to know whether the TMD of MobB<sub>CloDF13</sub> was essential for the mobilization of the plasmid as happens with TrwB<sub>R388</sub> or not as it has been described for TcpA<sub><italic>pcW</italic>3</sub>, whose TMD-less mutant can perform conjugation although at a lower frequency (<xref rid=\"B42\" ref-type=\"bibr\">Parsons et al., 2007</xref>). First of all, the transfer frequency of the mobilizable region of CloDF13 (plasmid pSU4814) mediated by the T4SS of R388 was analyzed. Afterwards, the complementation experiments were performed in the presence of both pSU4833 (the mobilizable region of CloDF13 without functional MobB<sub>CloDF13</sub>) and pSU1456 plasmid (encoding for R388 conjugative system except for TrwB<sub>R388</sub>). Results obtained in mobilization assays are summarized in <xref rid=\"T4\" ref-type=\"table\">Table 4</xref>. It was observed that cloned MobB<sub>CloDF13</sub> was functional when the T4SS of R388 was used, in agreement with what has been previously published for TrwB<sub>R388</sub> (<xref rid=\"B29\" ref-type=\"bibr\">Hormaeche et al., 2006</xref>). Similarly, the deletion of the TMD, MobBΔTMD mutant, rendered a non-functional phenotype as it occurs with other T4CP mutants that lack the TMD, such as TrwBΔN70 and PcfCΔN103 (<xref rid=\"B40\" ref-type=\"bibr\">Moncalián et al., 1999</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Chen et al., 2008</xref>).</p><table-wrap id=\"T4\" position=\"float\"><label>TABLE 4</label><caption><p>Mobilization experiments.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Plasmids in donors</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">T4CP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Transfer frequency</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R388Δ<italic>trwB</italic> (pSU1456) pSU4814 (+)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Wild type MobB<sub>CloDF13</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.42 10<sup>–2</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R388Δ<italic>trwB</italic> (pSU1456) MOB<sub>CloDF13</sub>Δ<italic>mobB</italic> (pSU4833) (−)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Ø</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><10<sup>–8</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R388Δ<italic>trwB</italic> (pSU1456) MOB<sub>CloDF13</sub>Δ<italic>mobB</italic> (pSU4833) pOPINE-<italic>mobB</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MobB<sub>CloDF13</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.75 × 10<sup>–2</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R388Δ<italic>trwB</italic> (pSU1456) pSU4833</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Ø</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><10<sup>–8</sup></td></tr></tbody></table><table-wrap-foot><attrib><italic>Transfer frequency of pSU4833 plasmid complemented with MobB<sub>CloDF13</sub> or MobBΔTMD mediated by the secretion channel of R388 conjugative system has been studied. E. coli DH5α and UB1637 strains were used as donor and recipient cells, respectively. Transfer frequencies were normalized to the number of transconjugants per donor and are the mean value of at least five independent experiments. (+) Positive control; (−) Negative control; Ø: no T4CP.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS4\"><title>Secondary Structure of T4CPs and Their Variants</title><p>Analysis of the secondary structure components of TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, MobB<sub>CloDF13</sub>, and MobBΔTMD were performed by IR spectroscopy through analysis of the IR amide I band.</p><p>The secondary structure of TMD<sub>TraJ</sub>CD<sub>TrwB</sub> was compared to those of the native TrwB<sub>R388</sub> and its mutants TrwBΔN50 and TrwBΔN70 (<xref rid=\"B55\" ref-type=\"bibr\">Vecino et al., 2012</xref>). <xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref> shows the original spectra and the curve-fitting decomposition corresponding to TMD<sub>TraJ</sub>CD<sub>TrwB</sub> purified in the presence of detergent. Band position, percentage area, and structure assignation corresponding to the deconvolved spectrum of the amide I region are summarized in <xref rid=\"T5\" ref-type=\"table\">Table 5</xref> together with those previously reported of TrwB<sub>R388</sub>, TrwBΔN50, and TrwBΔN70 (<xref rid=\"B55\" ref-type=\"bibr\">Vecino et al., 2012</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p><bold>(A)</bold> Amide I region of the infrared spectra of TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, MobB<sub>CloDF13</sub>, and MobBΔTMD. Proteins were purified, dialyzed against the corresponding buffer in D<sub>2</sub>O and analyzed by IR spectroscopy as explained in “Materials and Methods” section. Obtained spectra were curve-fitted to show the different secondary structure components as detailed in <xref rid=\"T5\" ref-type=\"table\">Table 5</xref>. <bold>(B)</bold> Thermal denaturation of TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, MobB<sub>CloDF13</sub>, and MobBΔTMD as seen by IR spectroscopy. The widths at half-height (WHH) of the amide I bands are plotted as a function of temperature (°C). Thermal denaturation is marked by an abrupt increase in bandwidth. Mid-point denaturation temperature (Tm) values corresponding to each protein are detailed in <xref rid=\"T5\" ref-type=\"table\">Table 5</xref>.</p></caption><graphic xlink:href=\"fmolb-07-00185-g002\"/></fig><table-wrap id=\"T5\" position=\"float\"><label>TABLE 5</label><caption><p>Secondary structure components and mid-point denaturation temperatures (Tm) of TrwB<sub>R388</sub>, TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, TrwBΔN50, TrwBΔN70, MobB<sub>CloDF13</sub>, and MobBΔTMD.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">TrwB<sub>R388</sub><sup>a</sup><hr/></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">TMD<sub>TraJ</sub>CD<sub>TrwB</sub><hr/></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">TrwBΔN50<sup>a</sup><hr/></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">TrwBΔN70<sup>a</sup><hr/></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">MobB<sub>CloDF13</sub><hr/></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">MobBΔTMD<hr/></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Assignment</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Position (cm<sup>–1</sup>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Area (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Position (cm<sup>–1</sup>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Area (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Position (cm<sup>–1</sup>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Area (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Position (cm<sup>–1</sup>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Area (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Position (cm<sup>–1</sup>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Area (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Position (cm<sup>–1</sup>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Area (%)</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">β-T + β-S</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1676</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1671</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1676</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1675</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1677</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1682</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">β-T</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1661</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">/</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">/</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1665</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1665</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1666</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1665</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">α-H</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1647</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1654</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1654</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1653</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1653</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1652</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Unordered + β-S</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">/</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">/</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1640</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1641</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1640</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1639</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1639</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">β-S</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1631</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1626</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1628</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1627</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">/</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">/</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">/</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">/</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tm (°C)</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">48</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">51</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">48</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">63<sup>b</sup></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">56</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">62</td></tr></tbody></table><table-wrap-foot><attrib><italic>Secondary structure components were obtained from band decomposition of the amide I band of IR spectra in D<sub>2</sub>O medium. The spectra are shown in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>. Only areas above 5% are shown. For details on assignments, see the text. <sup>a</sup>Data from <xref rid=\"B54\" ref-type=\"bibr\">Vecino et al. (2011</xref>, <xref rid=\"B55\" ref-type=\"bibr\">2012)</xref>. <sup>b</sup>Unpublished data from our group. β-T, β-turns; β-S, β-sheet; α-H, α-helix; Tm, mid-point denaturation temperature.</italic></attrib></table-wrap-foot></table-wrap><p>The spectrum of TMD<sub>TraJ</sub>CD<sub>TrwB</sub> purified in the presence of detergent exhibited four bands related to protein structure at 1671, 1654, 1640, and 1626 cm<sup>–1</sup> (<xref rid=\"T5\" ref-type=\"table\">Table 5</xref>). Interpretation of the results must be done taking into account that band assignation is not always a straightforward process since its position can be altered by the environment (<xref rid=\"B5\" ref-type=\"bibr\">Arrondo and Goñi, 1999</xref>). The component at 1654 cm<sup>–1</sup> was assigned to α-helix, the bands at 1626 and 1671 cm<sup>–1</sup> were associated with the low and high-frequency vibrations of β-sheet, respectively, although it should be noted that the later is also assigned to β-turns. And the band at 1640 cm<sup>–1</sup> was assigned to flexible, non-periodic elements.</p><p>When these results were compared to previously reported ones (i.e., TrwB<sub>R388</sub> TrwBΔN50, and TrwBΔN70) (<xref rid=\"B55\" ref-type=\"bibr\">Vecino et al., 2012</xref>) it can be observed that the proportion of the α-helix (35%) is lower than the one observed in the native protein TrwB<sub>R388</sub> purified in detergent (41%) and higher than the one shown in the deletion mutant proteins (26%). This result can be directly associated to the presence of a TMD both in TrwB<sub>R388</sub> and TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, even if in the later belongs to another T4CP such as TraJ<sub><italic>pMK</italic>101</sub>. Regarding bands associated to β-sheet and β-turns, it is remarkable the absence of a band around 1661–1665 cm<sup>–1</sup> in TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, as it was observed in TrwB<sub>R388</sub>, TrwBΔN50, and TrwBΔN70. Nevertheless the total proportion of the different bands associated to β-sheet and β-turns of TMD<sub>TraJ</sub>CD<sub>TrwB</sub> (31%) is similar to the proportion seen in the soluble mutant proteins and significantly lower than that of TrwB<sub>R388</sub> (59%). Finally, a sizeable proportion (35%) of the structure of TMD<sub>TraJ</sub>CD<sub>TrwB</sub> gave off a signal centered at 1640 cm<sup>–1</sup> (assigned to flexible, non-periodic elements) as seen in the deletion mutant proteins TrwBΔN50 and TrwBΔN70 but not in TrwB<sub>R388</sub> (<xref rid=\"B55\" ref-type=\"bibr\">Vecino et al., 2012</xref>). Previous studies about TrwBΔN70 and TrwBΔN50 showed that this band at 1640 cm<sup>–1</sup> also had a β-sheet component (<xref rid=\"B55\" ref-type=\"bibr\">Vecino et al., 2012</xref>). And it was published that at higher temperatures the band at 1640 cm<sup>–1</sup> split showing a β-sheet related band, which would not happen if the band was purely composed of unordered structures (<xref rid=\"B4\" ref-type=\"bibr\">Andraka et al., 2017</xref>). To elucidate if this also happened in TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, IRS experiments at different temperatures were performed (20, 40, 60, and 80°C). With the increase of temperature, the 1640 cm<sup>–1</sup> band shifts to 1645 cm<sup>–1</sup> and that at 1626 cm<sup>–1</sup> increases both its contribution and width, suffering also a shift to higher wavenumbers. This behavior indicates that although those two not resolved bands included in that at 1640 cm<sup>–1</sup> do not directly split into two bands, there is a transfer of the β-sheet contribution to the 1626 cm<sup>–1</sup> only β-sheet band, confirming that the 1640 cm<sup>–1</sup> band in the chimeric protein was composed of both unordered and β-sheet elements.</p><p>With the aim of further studying if the deletion of the TMD has the same effect in all T4CPs, the same study was carried out with MobB<sub>CloDF13</sub> and its deletion mutant MobBΔTMD to be compared with TrwB<sub>R388</sub> and its variants. <xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref> shows the original spectra and their curve-fitting decomposition corresponding to MobB<sub>CloDF13</sub> purified in the presence of detergent and its deletion mutant MobBΔTMD. In this case, both proteins exhibited four main bands. Band position, percentage area, and structure assignation corresponding to the deconvolved spectrum of the amide I region are summarized in <xref rid=\"T5\" ref-type=\"table\">Table 5</xref>. It can be observed that the component at 1653 and 1652 cm<sup>–1</sup> was assigned to α-helix in MobB<sub>CloDF13</sub> and MobBΔTMD, respectively. But it should be pointed out that the proportion of the α-helix in MobBΔTMD (29%) was lower than in native protein (35%), as expected taking into account the three transmembrane α-helices postulated for the native protein (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). The bands at 1666 or 1665 cm<sup>–1</sup> were associated with β-turns which were also observed in the proteins studied in <xref rid=\"B55\" ref-type=\"bibr\">Vecino et al. (2012)</xref>, but not in TMD<sub>TraJ</sub>CD<sub>TrwB</sub>. Nevertheless, proportions were slightly different (13 and 22% for MobB<sub>CloDF13</sub> and MobBΔTMD, respectively). On the contrary, the signal centered at 1677 or 1682 cm<sup>–1</sup> assigned to low-frequency vibrations of β-sheet and β-turns showed similar proportions in MobB<sub>CloDF13</sub>, MobBΔTMD (7 and 3%, respectively) and in the proteins studied in <xref rid=\"B55\" ref-type=\"bibr\">Vecino et al. (2012)</xref> but not in TMD<sub>TraJ</sub>CD<sub>TrwB</sub> where the proportion of this component was 14%. Finally, as observed in TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, TrwBΔN50 and TrwBΔN70, both MobB<sub>CloDF13</sub> and MobBΔTMD had a band at 1639 cm<sup>–1</sup> assigned to unordered structures that represented 30% of the structure, a band that is missing in TrwB<sub>R388</sub>.</p></sec><sec id=\"S3.SS5\"><title>Thermal Stability of T4CPs and Their Variants</title><p>The information regarding the denaturation of TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, MobB<sub>CloDF13</sub>, and MobBΔTMD was obtained through analysis of the amide I band (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). Specifically, two bands appear at 1615–1620 cm<sup>–1</sup> and 1680 cm<sup>–1</sup> when the protein aggregates. The appearance of these bands allows monitoring the denaturation process of the protein and the calculation of the mid-point denaturation temperature (Tm) (<xref rid=\"T5\" ref-type=\"table\">Table 5</xref>). Data treatment and band decomposition of the original amide I have been described previously (<xref rid=\"B54\" ref-type=\"bibr\">Vecino et al., 2011</xref>, <xref rid=\"B55\" ref-type=\"bibr\">2012</xref>).</p><p>The thermal stability of TMD<sub>TraJ</sub>CD<sub>TrwB</sub> purified in detergent was compared to TrwB<sub>R388</sub>, TrwBΔN50, and TrwBΔN70 (<xref rid=\"B55\" ref-type=\"bibr\">Vecino et al., 2012</xref>). As observed in <xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>, the thermal denaturation of TMD<sub>TraJ</sub>CD<sub>TrwB</sub> started at 35°C showing a mid-point denaturation temperature of 51.1°C at the tested conditions. This result is similar to TrwB<sub>R388</sub> and to the TrwBΔN50 (<xref rid=\"B55\" ref-type=\"bibr\">Vecino et al., 2012</xref>; <xref rid=\"T5\" ref-type=\"table\">Table 5</xref>).</p><p>Thermal stability of MobB<sub>CloDF13</sub> and MobBΔTMD was also studied by IR spectroscopy. As depicted in <xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>, the denaturation of the native protein starts at 47°C, achieving its Tm at 56°C, while the denaturation of MobBΔTMD starts at 55°C, achieving its Tm at 62°C (<xref rid=\"T5\" ref-type=\"table\">Table 5</xref>).</p></sec><sec id=\"S3.SS6\"><title>Subcellular Location</title><p>Studies about subcellular location of different T4CPs reported up to now have rendered ambiguous results (<xref rid=\"B31\" ref-type=\"bibr\">Kumar and Das, 2002</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Gunton et al., 2005</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Segura et al., 2014</xref>). To gain a deeper knowledge of this matter, specifically regarding the role of the TMD, we have studied the subcellular location of TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, MobB<sub>CloDF13</sub>, and MobBΔTMD under different experimental conditions. Subcellular location was analyzed by confocal fluorescence microscopy using eGFP-labeling and immunofluorescence techniques. Since eGFP proteins are only fluorescent when they are properly folded, their visualization ensures the analysis of functional proteins, excluding those that are denatured or included in inclusion bodies (<xref rid=\"B18\" ref-type=\"bibr\">Drew et al., 2006</xref>). In both studies similar results were obtained but eGFP-labeling rendered better quality images (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Figure S2</xref>).</p><p>To study the effect of expression times in the absence of the rest of T4SS proteins, the subcellular location of each protein was visualized after induction with 1 mM IPTG for 4 or 20 h. Different patterns were observed in the location of each protein at different expression times (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref> and <xref rid=\"T6\" ref-type=\"table\">Table 6</xref>). After 4 h of expression, TrwB<sub>R388</sub> and TMD<sub>TraJ</sub>CD<sub>TrwB</sub> were mainly located along the whole cell membrane and switched to a single-pole after 20 h, being this change less pronounced in the case of the chimeric protein. MobB<sub>CloDF13</sub> was predominantly located at both poles both at 4 and 20 h, showing a little increase in one pole location after 20 h (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref> and <xref rid=\"T6\" ref-type=\"table\">Table 6</xref>). On the contrary, most of the cells (95%) showed MobBΔTMD located on a single pole in the cytosol at both tested times.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Subcellular location of TrwB<sub>R388</sub>GFP and TMD<sub>TraJ</sub>CD<sub>TrwB</sub>GFP fusion-proteins by confocal fluorescence microscopy. Proteins were expressed in <italic>E. coli</italic> BL21C41 (DE3) strain by induction with 1 mM IPTG for 4 (left panels) and 20 h (right panels) at 25°C. Subcellular location of TrwB<sub>R388</sub>GFP and TMD<sub>TraJ</sub>CD<sub>TrwB</sub>GFP was determined in <italic>E. coli</italic> strains containing plasmid pSU1456 that expresses all R388 conjugative proteins except TrwB<sub>R388</sub>, or without plasmid pSU1456. The images were acquired in a <italic>Leica TCS SP5</italic> confocal fluorescence microscope, with a 60× oil immersion objective. Sample excitation was performed with 488 nm wavelength, while fluorescence emission was measured between 505 and 525 nm. The images were analyzed using Huygens and ImageJ softwares. Arrrowheads indicate the eGFP fluorescence through the periphery (1st column) and at a single cell pole (2nd column). Scale bar: 5 μm.</p></caption><graphic xlink:href=\"fmolb-07-00185-g003\"/></fig><table-wrap id=\"T6\" position=\"float\"><label>TABLE 6</label><caption><p>Subcellular location of different T4CP-eGFP fusion-proteins at different expression times in the absence or presence of T4SS<sub>R388</sub>.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Expression time (h)</td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Without T4SS (%)<hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">T4SS<sub>R388</sub> (%)<hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">T4SS<sub>R388</sub> and MOB<sub>CloDF13</sub> (%)<hr/></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2P</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TrwB<sub>R388</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>97 (77)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11 (9)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18 (14)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>34 (84)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3 (8)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3 (8)</td><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\">–</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31 (15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>155 (75)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29 (10)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10 (6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>138 (90)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6 (4)</td><td valign=\"top\" colspan=\"3\" rowspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TMD<sub>TraJ</sub>CD<sub>TrwB</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>85 (83)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11 (11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6 (6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>55 (76)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13 (18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (6)</td><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\">–</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">49 (40)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>62 (51)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10 (8)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18 (14)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>85 (67)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23 (18)</td><td valign=\"top\" colspan=\"3\" rowspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MobB<sub>CloDF13</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18 (18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8 (8)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>73 (74)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22 (19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16 (14)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>80 (68)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7 (5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19 (13)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>122 (82)</bold></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20 (18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24 (21)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>69 (61)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16 (15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16 (15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>74 (70)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7 (4)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">43 (22)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>143 (74)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MobBΔTMD<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>40 (95)</bold><sup>b</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (5)<sup>b</sup></td><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\">N.d.</td><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\">N.d.</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>35 (95)</bold><sup>b</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (5)<sup>b</sup></td><td valign=\"top\" colspan=\"3\" rowspan=\"1\"/><td valign=\"top\" colspan=\"3\" rowspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic>The eGFP variants of the proteins in the first column were expressed in E. coli BL21C41 (DE3) strain by induction with 1 mM IPTG for 4 and 20 h. The numbers represent the amount of cells showing each location. Total number of cells counted for each sample was between 40 and 200. Numbers between parentheses represent the percentages calculated up to the total amount of cells counted for each sample. The main location for each condition is marked in bold. M, location through the perimeter of the cell membrane; 1P, location at a single-pole; 2P, location at both poles. –, without relevance for this study. N.d., not determined. <sup>a</sup>Protein expressed in E. coli BL21 (DE3). <sup>b</sup>Polar location in the cytosol.</italic></attrib></table-wrap-foot></table-wrap><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Subcellular location of MobB<sub>CloDF13</sub>GFP and MobBΔTMDGFP fusion-proteins by confocal fluorescence microscopy. MobB<sub>CloDF13</sub>GFP and MobBΔTMDGFP proteins were expressed in <italic>E. coli</italic> BL21C41 (DE3) and <italic>E. coli</italic> BL21 (DE3) strains, respectively. Subcellular location of these proteins was determined by induction with 1 mM IPTG after 4 (left panels) and 20 h (right panels) at 25°C. Additionally, subcelular location was analyzed in the presence of pSU1456 plasmid, which expresses all R388 conjugative proteins except TrwB<sub>R388</sub>, or without plasmid pSU1456. MobB<sub>CloDF13</sub> was also expressed in the presence of both pSU1456 and pSU4833 that codes for the mobilization proteins of plasmid CloDF13 (MOB<sub>CloDF13</sub>), except for MobB<sub>CloDF13</sub>. The images were acquired in a <italic>Leica TCS SP5</italic> confocal fluorescence microscope, with a 60× oil immersion objective. Sample excitation was performed with 488 nm wavelength, while fluorescence emission was measured between 505 and 525 nm. The images were analyzed using <italic>Huygens</italic> and <italic>ImageJ</italic> software. Arrrowheads indicate the eGFP fluorescence foci at both cell poles (MobB<sub>CloDF13</sub>) and at a single cell pole (MobBΔTMD). Scale bar: 5 μm.</p></caption><graphic xlink:href=\"fmolb-07-00185-g004\"/></fig><p>Since the interaction with other conjugative proteins of the T4SS could modify the subcellular location pattern (<xref rid=\"B47\" ref-type=\"bibr\">Segura et al., 2014</xref>), the proteins that were active <italic>in vivo</italic> (i.e., TrwB<sub>R388</sub>, TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, and MobB<sub>CloDF13</sub>) were also observed in the presence of a T4SS lacking a functional T4CP that could interfere with the studied eGFP variant. On the one hand, TrwB<sub>R388</sub> and TMD<sub>TraJ</sub>CD<sub>TrwB</sub> were analyzed in the presence of the remaining conjugative proteins of R388 (i.e., in the presence of plasmid pSU1456 that lacks functional TrwB<sub>R388</sub> but contains the remaining conjugative proteins). On the other hand, since CloDF13 needs the T4SS of a co-resident conjugative plasmid to be mobilized, the subcellular location of MobB<sub>CloDF13</sub> was studied in the presence of R388 lacking functional TrwB<sub>R388</sub> (plasmid pSU1456) and also in the presence of both, R388 lacking functional TrwB<sub>R388</sub> (plasmid pSU1456) and the mobility region of CloDF13 lacking functional MobB<sub>CloDF13</sub> (plasmid pSU4833) (<xref rid=\"T6\" ref-type=\"table\">Table 6</xref>). In these experiments (<xref ref-type=\"fig\" rid=\"F3\">Figures 3</xref>, <xref ref-type=\"fig\" rid=\"F4\">4</xref>) the location pattern of each protein was the same in the absence or presence of a conjugative system (<xref rid=\"T6\" ref-type=\"table\">Table 6</xref>). However, although MobB<sub>CloDF13</sub> kept two poles as its main location in all the tested conditions, in the presence of R388 lacking functional TrwB<sub>R388</sub> it did not partially switch to a single-pole after 20 h, as in the absence of it.</p><p>The general observed pattern for all studied T4CPs was that the presence of T4SS enhanced the percentage of cells with the T4CP at the predominant location shown in the absence of the T4SS for each protein after 20 h (<xref ref-type=\"fig\" rid=\"F3\">Figures 3</xref>, <xref ref-type=\"fig\" rid=\"F4\">4</xref>). Specifically, the percentage of cells showing TrwB<sub>R388</sub> and TMD<sub>TraJ</sub>CD<sub>TrwB</sub> at a single pole increased and so did the percentage of cells showing MobB<sub>CloDF13</sub> at both poles. Moreover, regarding MobB<sub>CloDF13</sub>, the additional presence of its cognate mobilization region enhanced the effect produced by T4SS<sub>R388</sub>.</p></sec></sec><sec id=\"S4\"><title>Discussion</title><p>The increase of multidrug-resistant bacteria has become one of the major health concerns in our society (<xref rid=\"B60\" ref-type=\"bibr\">World Health Organization [WHO], 2019</xref>), being bacterial conjugation one of the key mechanisms responsible for the spread of antibiotic resistance genes among bacteria (<xref rid=\"B8\" ref-type=\"bibr\">Bello-López et al., 2019</xref>). This process is performed through a T4SS, a multiprotein complex that transfers the nucleoprotein substrate from a donor into a recipient bacterium (<xref rid=\"B57\" ref-type=\"bibr\">Waksman, 2019</xref>). T4CPs are essential proteins during conjugation, as they connect the substrates to be transferred in the cytosol with the secretion machinery in the membrane (<xref rid=\"B21\" ref-type=\"bibr\">Gomis-Ruth et al., 2005</xref>). Despite their importance, as membrane proteins are challenging to be studied, their characterization has been mostly accomplished using mutants that lack their TMD (<xref rid=\"B45\" ref-type=\"bibr\">Schroder and Lanka, 2003</xref>; <xref rid=\"B50\" ref-type=\"bibr\">Tato et al., 2007</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Larrea et al., 2017</xref>). However, several studies performed with TrwB<sub>R388</sub>, the full-length T4CP of the conjugative plasmid R388, have proven that the TMD is more than a mere anchor to the membrane and that it has a role in protein activity, stability, oligomerization and subcellular localization (<xref rid=\"B40\" ref-type=\"bibr\">Moncalián et al., 1999</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Hormaeche et al., 2002</xref>, <xref rid=\"B28\" ref-type=\"bibr\">2004</xref>, <xref rid=\"B29\" ref-type=\"bibr\">2006</xref>; <xref rid=\"B56\" ref-type=\"bibr\">Vecino et al., 2010</xref>, <xref rid=\"B54\" ref-type=\"bibr\">2011</xref>; <xref rid=\"B46\" ref-type=\"bibr\">Segura et al., 2013</xref>, <xref rid=\"B47\" ref-type=\"bibr\">2014</xref>).</p><p>The aim of this work has been to provide new data about different T4CPs that will contribute to infer general conclusions on their functioning to develop strategies to inhibit them and control the spread of antibiotic resistance genes. To do so, we studied the <italic>in vivo</italic> functionality, secondary structure, thermal stability, and subcellular location of TrwB<sub>R388</sub>, its chimeric protein TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, and the T4CP of the mobilizable plasmid CloDF13, MobB<sub>CloDF13</sub>, and its TMD-less mutant, MobBΔTMD.</p><sec id=\"S4.SS1\"><title><italic>In vivo</italic> Functionality</title><p>TMD<sub>TraJ</sub>CD<sub>TrwB</sub> was able to complement the Δ<italic>trwB</italic> mutation for R388 transfer, although with a lower transfer frequency than TrwB<sub>R388</sub>. This decrease in conjugation frequency may be due to a combination of effects such as conformational changes in the CD of TrwB<sub>R388</sub> due to its chimeric nature that render a less active protein and the heterologous interaction between TMD<sub>TraJ</sub> and the T4SS of R388 (<xref rid=\"B37\" ref-type=\"bibr\">Llosa et al., 2003</xref>). Thus, TMD<sub>TraJ</sub>CD<sub>TrwB</sub>-mediated transfer of R388 would occur through a specific interaction between CD<sub>TrwB</sub> with its cognate relaxase, TrwC<sub>R388</sub>, and an unspecific interaction of TMD<sub>TraJ</sub> with the heterologous T4SS from R388. This could explain why TMD<sub>TraJ</sub>CD<sub>TrwB</sub> did not complement the Δ<italic>traJ</italic> mutation (pKM101Δt<italic>raJ</italic>) since CD<sub>TrwB</sub> could not recognize the heterologous TraI<sub>pKM101</sub> relaxase, even if TMD<sub>TraJ</sub> interacted with its cognate T4SS<sub>pKM101</sub>.</p><p>Moreover, TMD<sub>TraJ</sub>CD<sub>TrwB</sub> showed negative dominance in the presence of the native T4CPs TrwB<sub>R388</sub> or TraJ<sub>pKM101</sub>. Since it has been reported that TMD<sub>TraJ</sub> can interact with both T4SS<sub>R388</sub> and T4SS<sub>pKM101</sub> (<xref rid=\"B37\" ref-type=\"bibr\">Llosa et al., 2003</xref>; <xref rid=\"B17\" ref-type=\"bibr\">De Paz et al., 2010</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Celaya et al., 2017</xref>), a possible explanation for the observed negative dominance could be that competition for the T4SS occurred. According to this hypothesis, TMD<sub>TraJ</sub>CD<sub>TrwB</sub> would have interacted with the secretion channel, sequestering it from interacting with the native T4CPs and reducing the conjugative rate of the wild type system. This hypothesis comes into agreement with the fact that the transfer of pKM101 using TraJ<sub>pKM101</sub> and T4SS<sub>R388</sub> is one order of magnitude lower than using T4SS<sub>pKM101</sub> (<xref rid=\"B37\" ref-type=\"bibr\">Llosa et al., 2003</xref>). Another explanation compatible with dominance experiments with both systems would be that non-functional heteroligomers were made between the native and the chimera proteins, competing for the conjugative machinery and therefore lowering the transfer frequency. Any of these alternatives or a combination of them would have caused a decrease in the plasmid transfer rate, as observed in the dominance mating assays. It must be underlined that a point mutation in the Walker A domain, TMD<sub>TraJ</sub>CD<sub>TrwB</sub> (K142T), resulted in a non-functional phenotype. Hence, the K mutation totally arrested transfer capacity as previously reported for other T4CPs (<xref rid=\"B40\" ref-type=\"bibr\">Moncalián et al., 1999</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Kumar and Das, 2002</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Gunton et al., 2005</xref>), suggesting that concerning its NBD TMD<sub>TraJ</sub>CD<sub>TrwB</sub> is functionally similar to TrwB<sub>R388</sub> despite the results obtained in the complementation and dominance studies.</p><p>Regarding CloDF13 system, it was observed that the deletion of the TMD rendered a non-functional MobBΔTMD as it occurs with other TMD-less mutants such as TrwBΔN70 and PcfCΔN103 (<xref rid=\"B14\" ref-type=\"bibr\">Chen et al., 2008</xref>). However, a TcpA mutant lacking the TMD, but not the N-term cytosolic residues, TcpA<sub>Δ46–104</sub>, was able to perform conjugation, although at a frequency lower than the wild type plasmid. In this regard, unpublished experiments with TrwBΔN8, which lacks the N-term cytosolic eight residues, showed a decrease in the transfer rate of more than three orders of magnitude (<xref rid=\"B53\" ref-type=\"bibr\">Vecino, 2009</xref>). Taken together these results, it seems that not only the TMD but also the N-term cytosolic residues have an important role in the transfer capacity of T4CPs. In this context, the importance of this small region in specific interactions with the relaxosome has already been described (<xref rid=\"B37\" ref-type=\"bibr\">Llosa et al., 2003</xref>; <xref rid=\"B45\" ref-type=\"bibr\">Schroder and Lanka, 2003</xref>). In the case of MobB<sub>CloDF13</sub> its N-term is located in the periplasm (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>), which could be an important feature for recognition and interaction with conjugative secretion channels.</p></sec><sec id=\"S4.SS2\"><title>Secondary Structure and Thermal Stability</title><p>As it has been described that the TMD influences the secondary structure of the CD and the thermal stability of TrwB<sub>R388</sub> (<xref rid=\"B28\" ref-type=\"bibr\">Hormaeche et al., 2004</xref>; <xref rid=\"B54\" ref-type=\"bibr\">Vecino et al., 2011</xref>, <xref rid=\"B55\" ref-type=\"bibr\">2012</xref>), in this work we have studied whether this behavior can be observed in TMD<sub>TraJ</sub>CD<sub>TrwB</sub> and in MobB<sub>CloDF13</sub>.</p><p>Regarding TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, one of the most important differences in comparison with TrwB<sub>R388</sub> was the appearance of a band at 1640 cm<sup>–1</sup>, mainly assigned to flexible structures (non-periodic elements) related to a less compact overall structure (<xref rid=\"B20\" ref-type=\"bibr\">Echabe et al., 1998</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Agopian et al., 2016</xref>). As the crystal structure of TrwBΔN70 shows flexible loops (<xref rid=\"B22\" ref-type=\"bibr\">Gomis-Rüth et al., 2002</xref>), the presence of the 1640 cm<sup>–1</sup> band present at similar percentages (30–37%) in TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, TrwBΔN50, and TrwBΔN70 mutant proteins but missing in TrwB<sub>R388</sub>, could be explained as the loss of the compact structure of TrwB<sub>R388</sub> due to the deletion of its cognate TMD (<xref rid=\"B28\" ref-type=\"bibr\">Hormaeche et al., 2004</xref>).</p><p>Taking into account the values related to all the bands associated to β-sheet elements (1671, 1640, and 1626 cm<sup>–1</sup>) it could be concluded that the total percentage of β-sheets in TMD<sub>TraJ</sub>CD<sub>TrwB</sub> is just slightly smaller to that of TrwB<sub>R388</sub>. Concerning β-turns, the band assigned to them in the TrwB<sub>R388</sub> related proteins, 1661–1665 cm<sup>–1</sup>, was not observed in TMD<sub>TraJ</sub>CD<sub>TrwB</sub>. However, it can be postulated that the increase seen in TMD<sub>TraJ</sub>CD<sub>TrwB</sub> of the band at 1671 cm<sup>–1</sup>, was partially related to the β-turns component seen in the mutants at 1665 cm<sup>–1</sup>. These would imply that the β-turns component of the chimeric protein is lower than the one of the native protein and similar to the TMD deletion mutants. Finally, TMD<sub>TraJ</sub>CD<sub>TrwB</sub> shows a decrease in α-helix percentage (35%) in comparison with TrwB<sub>R388</sub> (41%), but an increase in comparison to the mutants (26%).</p><p>Therefore it seems that TMD<sub>TraJ</sub>CD<sub>TrwB</sub> presents qualitative and quantitative features in between the native protein and the deletion mutants. These results suggest that the presence of a heterologous full-length TMD does provide a more compact and ordered structure to the T4CP in comparison to the TMD-less mutants, even if it does not reach the level of the native protein. This result comes in agreement with the transfer capacity reduction of the chimeric protein that could be explained partially by the observed structural changes reported here.</p><p>To test if the results described for TrwB<sub>R388</sub> could be extrapolated to other T4CPs, the secondary structures of MobB<sub>CloDF13</sub> and MobBΔTMD were studied (<xref rid=\"T5\" ref-type=\"table\">Table 5</xref>). Surprisingly, MobBΔTMD presented an IR spectrum quite similar to the one obtained for MobB<sub>CloDF13</sub>. It presented smaller helical structure percentages and higher β-turns percentages but both showed similar unordered and β-sheet percentages. This suggests that in MobB<sub>CloDF13</sub> the presence of the TMD does not have an effect on the structure of the CD as it does in TrwB<sub>R388</sub>. As MobB<sub>CloDF13</sub> has to interact with heterologous T4SSs, it could be that its TMD has to interact with heterologous T4SSs without altering the structure of its CD where the specific interaction with its cognate relaxosome occurs.</p><p>Concerning the thermal denaturation of TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, its Tm was similar to the ones described for TrwB<sub>R388</sub> and the mutant lacking the first transmembrane helix, TrwBΔN50 (<xref rid=\"T5\" ref-type=\"table\">Table 5</xref>). This result suggests that although the secondary structures of the mutants differ from that of the native protein, their overall thermal stability is similar. Additionally, when comparing the results between both studied systems, as expected due to the high instability of purified membrane proteins (<xref rid=\"B24\" ref-type=\"bibr\">González Flecha, 2017</xref>) the soluble proteins showed higher mid-point denaturation temperatures than the full-length proteins. Specifically, MobBΔTMD and TrwBΔN70 showed similar Tm values (Tm 62 and 63°C, respectively); on the contrary, MobB<sub>CloDF13</sub> was more stable than TrwB<sub>R388</sub> against thermal denaturation (Tm 56 and 48°C, respectively). This could be related to different buffer compositions that had to be used when MobB<sub>CloDF13</sub> and TrwB<sub>R388</sub> were analyzed.</p></sec><sec id=\"S4.SS3\"><title>Subcellular Location</title><p>The polar location of proteins in bacteria underlines their sophisticated internal organization, being important in many processes like chemotaxis and cellular division (<xref rid=\"B30\" ref-type=\"bibr\">Howard, 2004</xref>). Similarly, subcellular location has been considered important in bacterial conjugation (<xref rid=\"B14\" ref-type=\"bibr\">Chen et al., 2008</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Leonetti et al., 2015</xref>). Sequence analysis, cell fractionation, and protein purification experiments proved that TrwB<sub>R388</sub>, TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, and MobB<sub>CloDF13</sub> are located in the bacterial membrane, while MobBΔTMD is located in the cytosol (data not shown). As studies in the literature do not show a consensus pattern either in the location of the T4CPS nor in the role of each domain in this property (<xref rid=\"B31\" ref-type=\"bibr\">Kumar and Das, 2002</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Bauer et al., 2011</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Segura et al., 2014</xref>), the subcellular location of TrwB<sub>R388</sub>, TMD<sub>TraJ</sub>CD<sub>TrwB</sub>, MobB<sub>CloDF13</sub>, and MobBΔTMD was investigated.</p><p>In this work we observed TrwB<sub>R388</sub> located along the membrane after 4 h of induction and only after 20 h it focused at single pole. These results differ from our previous results where the polar location of TrwB<sub>R388</sub> was observed after 4 h (<xref rid=\"B47\" ref-type=\"bibr\">Segura et al., 2014</xref>). However, the induction OD<sub>600</sub> values were different (0.4 vs. 0.7, this work and previous work, respectively), probably rendering populations at different growth phase. On the contrary MobB<sub>CloDF13</sub> was located at both poles in the membrane at 4 and 20 h after induction.</p><p>Previous studies have reported that in the absence of other conjugative proteins, T4CPs that lacked the whole TMD or even the periplasmic loop located in the cytosol or at the membrane periphery, respectively (<xref rid=\"B31\" ref-type=\"bibr\">Kumar and Das, 2002</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Segura et al., 2014</xref>). Moreover, the TMD alone of TrwB<sub>R388</sub> located at the membrane poles without the need for the CD (<xref rid=\"B47\" ref-type=\"bibr\">Segura et al., 2014</xref>), suggesting a leading role for the TMD in the subcellular location of the T4CPs. Surprisingly, MobBΔTMD located at a single cell pole in the cytosol even in the absence of other conjugative proteins. These results suggest that mobilizable plasmid-related T4CPs could use different mechanisms than VirD4-type T4CPs for subcellular location. It has been speculated that the polar location of T4CPs could be related to interactions with the cardiolipin enriched membrane poles (<xref rid=\"B39\" ref-type=\"bibr\">Mileykovskaya and Dowhan, 2009</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Segura et al., 2014</xref>), but at the same time mediated by complex and dynamic changes in transduction, cytoskeleton proteins, etc. (<xref rid=\"B48\" ref-type=\"bibr\">Shapiro et al., 2002</xref>). Since interactions between mobilizable plasmid-related T4CPs and T4SSs are not specific, these T4CPs could have evolved to develop different mechanisms to interact with the membrane and ensure their polar location.</p><p>Previous studies (<xref rid=\"B31\" ref-type=\"bibr\">Kumar and Das, 2002</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Gunton et al., 2005</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Segura et al., 2014</xref>) have reported that the location of native T4CPs is independent of the presence of the rest of the conjugative proteins. Our results suggest that the presence of a complete conjugative system (i.e., mobilizable region and secretion channel) seems to enhance the polar location of wild type T4CPs. Moreover, although the location of MobB<sub>CloDF13</sub> at both poles was enhanced in the presence of T4SS<sub>R388</sub>, it was further enhanced when MOB<sub>CloDF13</sub> was also present.</p><p>Taking all together, it seems that that no universal location patterns can be attributed to T4CPs. Nevertheless, three conclusions can be undertaken regarding subcellular location: (i) T4CPs localize either at a single pole or both poles, depending on the system; (ii) the presence of a TMD is not essential for the polar location of a mobilizable plasmid associated T4CP, and (iii) the presence of a conjugative system enhances the polar location of full-length T4CPs.</p><p>To sum up, the comparative study between the conjugative system related TrwB<sub>R388</sub> and the mobilizable plasmid-related MobB<sub>CloDF13</sub> and their variants has underlined that the characteristics described for the paradigmatic conjugative plasmid related VirD4-type T4CPs and their TMDs should not be ascribed to the whole T4CP family.</p></sec></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.</p></sec><sec id=\"S6\"><title>Author Contributions</title><p>IÁ-R, CG, and IAl contributed to the design of the work (text and figures) and the acquisition of the data, writing and revision of the content, approval of the last version, and ensuring accuracy and integrity of the work. IAr and JA contributed to acquisition of the data, revision of the content, and approval of the last version of the work. BU-U contributed to writing, revision of the content, and approval of the last version. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was in part supported by grants from the University of the Basque Country (GIU18/229 and COLAB19/08) and the Industry Department of the Basque Government (ELKARTEK 2020 KK-2020/00007). IÁ-R was a pre-doctoral student supported by the Basque Government.</p></fn></fn-group><ack><p>We would like to sincerely thank Dr. Lide Arana for her invaluable help and contribution to this work, to Unai Lorenzo for his help with the confocal microscope and image treatment, and to Dr. Louise Bird for her help with the high-throughput cloning at OPPF-UK.</p></ack><fn-group><fn id=\"footnote1\"><label>1</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://n2t.net/addgene:26043\">http://n2t.net/addgene:26043</ext-link></p></fn><fn id=\"footnote2\"><label>2</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://n2t.net/addgene:41125\">http://n2t.net/addgene:41125</ext-link></p></fn><fn id=\"footnote3\"><label>3</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"https://web.expasy.org/protparam/\">https://web.expasy.org/protparam/</ext-link></p></fn><fn id=\"footnote4\"><label>4</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://topcons.cbr.su.se/\">http://topcons.cbr.su.se/</ext-link></p></fn></fn-group><sec id=\"S9\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fmolb.2020.00185/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fmolb.2020.00185/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"TS1\"><media xlink:href=\"Table_1.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Agopian</surname><given-names>A.</given-names></name><name><surname>Quetin</surname><given-names>M.</given-names></name><name><surname>Castano</surname><given-names>S.</given-names></name></person-group> (<year>2016</year>). <article-title>Structure and interaction with lipid membrane models of Semliki Forest virus fusion peptide.</article-title>\n<source><italic>Biochim. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Bioeng Biotechnol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Bioeng Biotechnol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Bioeng. Biotechnol.</journal-id><journal-title-group><journal-title>Frontiers in Bioengineering and Biotechnology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-4185</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850770</article-id><article-id pub-id-type=\"pmc\">PMC7431657</article-id><article-id pub-id-type=\"doi\">10.3389/fbioe.2020.00961</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Bioengineering and Biotechnology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Formulation of Bio-Based Washing Agent and Its Application for Removal of Petroleum Hydrocarbons From Drill Cuttings Before Bioremediation</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Arpornpong</surname><given-names>Noulkamol</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1022349/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Padungpol</surname><given-names>Rattiya</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1021316/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Khondee</surname><given-names>Nichakorn</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/994966/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Tongcumpou</surname><given-names>Chantra</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Soonglerdsongpha</surname><given-names>Suwat</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Suttiponparnit</surname><given-names>Komkrit</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Luepromchai</surname><given-names>Ekawan</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/864779/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Natural Resources and Environment, Faculty of Agriculture, Natural Resources and Environment, Naresuan University</institution>, <addr-line>Phitsanulok</addr-line>, <country>Thailand</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Microbial Technology for Marine Pollution Treatment Research Unit, Department of Microbiology, Faculty of Science, Chulalongkorn University</institution>, <addr-line>Bangkok</addr-line>, <country>Thailand</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Environmental Research Institute, Chulalongkorn University</institution>, <addr-line>Bangkok</addr-line>, <country>Thailand</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Research Program on Remediation Technologies for Petroleum Contamination, Center of Excellence on Hazardous Substance Management, Chulalongkorn University</institution>, <addr-line>Bangkok</addr-line>, <country>Thailand</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Environmental Technology Research Department, Innovation Institute, PTT Public Company Limited</institution>, <addr-line>Bangkok</addr-line>, <country>Thailand</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Rudolf Hausmann, University of Hohenheim, Germany</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Carla Silva, University of Minho, Portugal; Harumi Veny, Universiti Teknologi MARA, Malaysia</p></fn><corresp id=\"c001\">*Correspondence: Ekawan Luepromchai, <email>ekawan.l@chula.ac.th</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Industrial Biotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>961</elocation-id><history><date date-type=\"received\"><day>24</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>24</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Arpornpong, Padungpol, Khondee, Tongcumpou, Soonglerdsongpha, Suttiponparnit and Luepromchai.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Arpornpong, Padungpol, Khondee, Tongcumpou, Soonglerdsongpha, Suttiponparnit and Luepromchai</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Drill cuttings from petroleum exploration and production sites can cause diverse environmental problems. Total petroleum hydrocarbons (TPHs) are a major pollutant from the use of polyolefin-based mud. As an alternative to incineration, this study investigated the application of surfactant-enhanced washing technology prior to bioremediation. The washing step was necessary because the initial TPH concentrations were quite high at approximately 15% (w/w). Washing agents were formulated by varying the concentration of lipopeptide biosurfactant (in foamate or cell-free broth), Dehydol LS7TH (fatty alcohol ethoxylate 7EO, oleochemical surfactant) and butanol (as a lipophilic linker) at different salinities. The most efficient formula produced a Winsor Type I microemulsion (oil-in-water microemulsion) with polyolefin and contained only 20% (v/v) foamate and 2% (v/v) Dehydol LS7TH in water. Due to the synergistic behavior between the anionic lipopeptides and non-ionic Dehydol LS7TH, the formula efficiently removed 92% of the TPHs from the drill cuttings when applied in a jar test. To reduce the cost, the concentrations of each surfactant should be reduced; thus, the formula was optimized by the simplex lattice mixture design. In addition, cell-free broth, at a pH of 10, containing 3.0 g/L lipopeptides was applied instead of foamate because it was easy to prepare. The optimized formula removed 81.2% of the TPHs and contained 72.0% cell-free broth and 1.4% Dehydol LS7TH in water. A 20-kg soil washing system was later tested where the petroleum removal efficiency decreased to 70.7% due to polyolefin redeposition during separation of the washing solution. The remaining TPHs (4.5%) in the washed drilled cuttings were further degraded by a mixture of <italic>Marinobacter salsuginis</italic> RK5, <italic>Microbacterium saccharophilum</italic> RK15 and <italic>Gordonia amicalis</italic> JC11. To promote TPH biodegradation, biochar and fertilizer were applied along with bacterial consortia in a microcosm experiment. After 49-day incubation, the TPHs were reduced to 0.9% by both physical and biological mechanisms, while the TPHs in the unamended samples remained unaffected. With the use of the formulated bio-based washing agent and bioremediation approach, the on-site treatment of drill cuttings could be conducted with an acceptable cost and low environmental impacts.</p></abstract><kwd-group><kwd>drilling waste</kwd><kwd>soil washing</kwd><kwd>biosurfactants</kwd><kwd>polyolefin biodegradation</kwd><kwd>sequential treatment</kwd></kwd-group><counts><fig-count count=\"7\"/><table-count count=\"4\"/><equation-count count=\"4\"/><ref-count count=\"57\"/><page-count count=\"16\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Drilling waste from petroleum exploration and production sites can cause environmental problems due to the presence of petroleum hydrocarbons, heavy metals and inorganic salts in the drill cuttings (<xref rid=\"B45\" ref-type=\"bibr\">Sharif et al., 2017</xref>). The management options for drilling waste include incineration, thermal desorption, disposal in landfills, reuse in construction without prior treatment, stabilization/solidification, surfactant-enhanced washing, bioremediation and phytoremediation (<xref rid=\"B30\" ref-type=\"bibr\">Leonard and Stegemann, 2010</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Ball et al., 2011</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Fan et al., 2014</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Kogbara et al., 2017</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Liu et al., 2019</xref>). In Thailand, onshore petroleum production sites usually adopt synthetic based mud during operation and later transport the drill cuttings to waste management companies for incineration. The disadvantages of the current treatment options are their high cost, high energy use, high time consumption and notable environmental unsustainability (<xref rid=\"B30\" ref-type=\"bibr\">Leonard and Stegemann, 2010</xref>). Consequently, this study aimed to develop an alternative on-site treatment by formulating a bio-based washing agent and then applying it to reduce the concentration of petroleum hydrocarbons in drilling waste prior to bioremediation.</p><p>Petroleum hydrocarbons are the major contaminants in drilling waste with concentrations ranging from 1.5–15% (w/w). For example, 15,300 and 17,125 mg/kg of TPHs were present in drilling wastes from an active drilling operation in Sichuan, China and at a disposal facility in the Niger Delta region, Nigeria, respectively (<xref rid=\"B14\" ref-type=\"bibr\">Fan et al., 2014</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Kogbara et al., 2017</xref>). <xref rid=\"B30\" ref-type=\"bibr\">Leonard and Stegemann (2010)</xref> reported a total petroleum hydrocarbon (TPH) concentration of 66,700 mg/kg in an unidentified onshore drilling operation. In addition, <xref rid=\"B15\" ref-type=\"bibr\">Fernandez et al. (2008)</xref> investigated a drilling waste sample that had been stored for 20–30 years in open cesspits in Tabasco, Mexico and measured a high TPH concentration of 135,400 mg/kg. This information suggests that the indigenous microorganisms in drill cuttings have a low TPH-degrading activity and that natural attenuation is not an appropriate option for drilling waste treatment. Bioremediation of oil-based cuttings, such as by composting, biopiling, slurry bioreactors and phytoremediation, has been performed as a cost-effective treatment; however, TPH biodegradation occurs slowly and may require up to 12 months for treatment of waste with high initial petroleum concentrations (<xref rid=\"B2\" ref-type=\"bibr\">Alavi et al., 2014</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Fan et al., 2014</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Chaîneau et al., 1995</xref>).</p><p>Surfactant-enhanced washing technology using either synthetic surfactants or biosurfactants has been considered an effective and rapid technique for the removal of petroleum hydrocarbons from soils and contaminated sites (<xref rid=\"B49\" ref-type=\"bibr\">Trellu et al., 2016</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Befkadu and Chen, 2018</xref>). A single type of surfactant is usually applied as the washing agent; for example, <xref rid=\"B15\" ref-type=\"bibr\">Fernandez et al. (2008)</xref> found that 4% SDS (an anionic surfactant) and 1% ENP (a non-ionic surfactant) could remove 55.7% and 52.2%, respectively of the TPHs (at 13.5%) from drill cuttings stored for 20 to 30 years. <xref rid=\"B55\" ref-type=\"bibr\">Yan et al. (2011)</xref> used 0.03% rhamnolipid to remove 83% of 8.5% (w/w) petroleum in washed drill cuttings from the Liaohe oilfield in China. Recently, mixtures of anionic and non-ionic surfactants have been applied as their synergistic behavior can potentially increase pollutant washing efficiency. <xref rid=\"B47\" ref-type=\"bibr\">Shi et al. (2015)</xref> reported that a mixture of Triton X-100 and SDS increased the solubilization of polycyclic aromatic hydrocarbons (PAHs) and effectively removed PAHs from highly contaminated soil collected from an abandoned coke oven plant in China.</p><p>Another potential washing agent is a surfactant-based microemulsion, which can be formulated by mixing different surfactants in water or saline water and is based on the experimental phase behavior of a surfactant-oil-water system (Winsor Type region). The removal of petroleum usually occurs via two mechanisms: solubilization via a Winsor Type I microemulsion and mobilization via a Winsor Type III microemulsion (<xref rid=\"B22\" ref-type=\"bibr\">Javanbakht and Goual, 2016</xref>). Microemulsion-based washing agents can be achieved with a low surfactant amount; thus, the application of microemulsions could reduce the potential risk of new contaminants being released into the environment and ensure the economic practicality of the washing process (<xref rid=\"B3\" ref-type=\"bibr\">Arpornpong et al., 2018</xref>). In addition, the enhancement of petroleum solubility by microemulsions is linearly proportional to the concentration of microemulsion, while conventional surfactants promote solubilization of petroleum only in the vicinity of its critical micelle concentration (CMC) (<xref rid=\"B29\" ref-type=\"bibr\">Lau et al., 2014</xref>).</p><p>In this study, bio-based washing agents were formulated by investigating the microemulsion phase behavior of polyolefin, the major component in synthetic-based mud, and solutions of mixed bio-based surfactants at different salinities. Lipopeptides from <italic>Bacillus subtilis</italic> GY19 and Dehydol LS7TH, a fatty alcohol ethoxylate oleochemical surfactant, were selected because they are bio-based surfactants and have been applied to solubilize hydrophobic compounds such as crude petroleum and vegetable oil (<xref rid=\"B43\" ref-type=\"bibr\">Rongsayamanont et al., 2017</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Arpornpong et al., 2018</xref>). The addition of butanol as a lipophilic linker was also investigated. <xref rid=\"B37\" ref-type=\"bibr\">Nguyen and Sabatini (2009)</xref> reported that increasing the surfactant/linker concentration in biosurfactant-based microemulsions enhances the lipophilicity of surfactant aggregates and leads to a lower salinity requirement in the microemulsion system. The TPH removal efficiency of bio-based washing agents was determined with drill cuttings in jar tests as well as a scale-up washing system. The washed drilled cuttings were further remediated by adding a mixture of <italic>Marinobacter salsuginis</italic> RK5, <italic>Microbacterium saccharophilum</italic> RK15, and <italic>Gordonia amicalis</italic> JC11, which are locally isolated petroleum-degrading bacteria. Compared to a single bioremediation or washing treatment, the two-stage remedial system can reduce treatment time and increase treatment efficiency (<xref rid=\"B55\" ref-type=\"bibr\">Yan et al., 2011</xref>). To promote TPH biodegradation, biochar and fertilizer were applied along with mixed bacteria to the washed drilled cuttings. Biochar can improve soil fertility and hydraulic properties as well as enhance contaminant immobilization and transformation (<xref rid=\"B31\" ref-type=\"bibr\">Lim et al., 2016</xref>; <xref rid=\"B57\" ref-type=\"bibr\">Zhu et al., 2017</xref>), while fertilizer can enhance the degradation potential of bacterial populations (<xref rid=\"B50\" ref-type=\"bibr\">Tyagi et al., 2011</xref>). With the use of the newly formulated bio-based washing agent and bioremediation approach, the on-site treatment of drill cuttings can become feasible.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Drill Cuttings, Surfactants, Bacteria and Chemicals</title><p>The surfactants studied in this work are classified into two main groups: biosurfactants and synthetic surfactants. <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> lists the properties of all the surfactants used in this study. The lipopeptide biosurfactant (anionic) was produced by chitosan-immobilized <italic>Bacillus</italic> sp. GY19 using waste glycerol and palm oil as substrates according to <xref rid=\"B24\" ref-type=\"bibr\">Khondee et al. (2015)</xref>. The production medium was passed through a stainless steel screener (<0.5 mm), and bacterial cells were removed from the culture medium by centrifugation at 8,000 rpm for 20 min. Cell-free broth was sterilized by autoclaving at 121°C for 15 min. Lipopeptides as foamate were separated and concentrated following the foam fractionation method of <xref rid=\"B24\" ref-type=\"bibr\">Khondee et al. (2015)</xref> and <xref rid=\"B43\" ref-type=\"bibr\">Rongsayamanont et al. (2017)</xref>. The properties of the foamate and cell-free broth are summarized in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Properties of surfactants.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Parameter</bold></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><bold>Biosurfactant</bold><hr/></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Dehydol LS7TH</bold></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Foamate (pH 7)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Cell-free broth (pH 7)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Cell-free broth (pH 10)</bold></td><td rowspan=\"1\" colspan=\"1\"/></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Head group</td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Anionic</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Non-ionic</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Classification</td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Lipopeptides</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Fatty alcohol C<sub>12</sub><sub>–</sub><sub>14</sub> with 7 moles ethoxylate</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Concentration</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.9 g/L</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.4 g/L</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.4 g/L</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.7<sup>%a</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CMC at 25°C</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.3 g/L<sup>b</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.4 g/L<sup>b</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.2 g/L</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.02 g/L</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surface tension at CMC (mN/m)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.4<sup>b</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.9<sup>b</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CMD at 25<sup>o</sup>C (dilution times)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.5<sup>c</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ND</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surface tension at CMD (mN/m)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.1<sup>c</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ND</td></tr></tbody></table><table-wrap-foot><attrib><italic><sup><italic>a</italic></sup>Data was provided from manufacturer. <sup><italic>b</italic></sup>Data was obtained from <xref rid=\"B43\" ref-type=\"bibr\">Rongsayamanont et al. (2017)</xref>. <sup><italic>c</italic></sup>Data was obtained from <xref rid=\"B24\" ref-type=\"bibr\">Khondee et al. (2015)</xref>. ND = no data, CMC = Critical micelle concentration; CMD = Critical micelle dilution.</italic></attrib></table-wrap-foot></table-wrap><p>Dehydol LS7TH, a fatty alcohol C<sub>12</sub><sub>–</sub><sub>14</sub> with 7 ethoxylate groups (99.7% purity), was purchased from the Thai Ethoxylate Co., Ltd., (Thailand). It is a non-ionic synthetic surfactant derived from palm oil. 1-butanol (99% purity, Fisher Scientific, United Kingdom Limited) was used as a hydrophilic linker. Chloroform (99%, +), 1-hexane (99%, +), methanol (99%, +), and dichloromethane (99%, +) were purchased from Sigma–Aldrich (Saint Louis, MO) and used as solvents. A saline solution [4% (w/v)] was prepared by dissolving 4 g sodium chloride (NaCl, 99%, +) in 100 mL deionized water. All other chemicals were of analytical grade.</p><p>Synthetic-based mud (SBM), drill cutting, and linear C<sub>9</sub> to C<sub>21</sub> α-olefins (LAOs, polyolefin) were obtained from an onshore oilfield in Thailand. Polyolefin is the main component in SBM. It was used as a surrogate for residual SBM present in the drill cutting. The drill cuttings were excavated from the onshore drilling operation with a well depth of 3,000–3,500 m from the surface. Particle diameter of the drill cuttings as determined by sieve analysis (ASTM D422) was smaller than 75 μm. The initial TPH concentration in the drill cuttings was 151,572 mg/kg cuttings (>15% w/w) (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). Total hydrocarbons present in the drill cuttings were mainly asphaltenes (50.5% w/w) and saturated hydrocarbons (49.5% w/w).</p><p><italic>Marinobacter salsuginis</italic> RK5 and <italic>Microbacterium saccharophilum</italic> RK15 were isolated from seawater as crude oil-degrading bacteria. They were maintained in Zobell Marine Broth 2216 (HiMedia Laboratories) and deposited at the MSCU culture collection, Thailand Bioresource Research Center (TBRC), as MSCU 1054 and 1055, respectively. <italic>Marinobacter salsuginis</italic> RK5 had a low cell hydrophobicity (8%) but a high EPS producing activity (95%), while both cell hydrophobicity and EPS producing activity of <italic>Microbacterium saccharophilum</italic> RK15 were high at 78% and 97%, respectively. To increase their polyolefin-degrading activity, they were mixed with a fuel oil-degrading bacterium, <italic>Gordonia amicalis</italic> JC11 (previously <italic>Gordonia</italic> sp. JC11), which had been studied for oil spill remediation in the presence of biosurfactants (<xref rid=\"B28\" ref-type=\"bibr\">Laorrattanasak et al., 2016</xref>). Marine bacteria were initially selected to avoid the salt stress due to the high conductivity of drill cuttings (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). Biochar derived from wood and NPK fertilizer (30:5:5) were purchased from local agriculture companies.</p></sec><sec id=\"S2.SS2\"><title>Development of the Bio-Based Washing Agent Using Microemulsion Phase Behavior Experiments</title><p>The flowchart of experimental procedure and outcomes is presented in <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>. The development of bio-based washing agent was carried out using microemulsion phase behavior experiments to identify the appropriate compositions for an efficient formulation. Previously, researchers in our laboratory have applied the hydrophilic-lipophilic deviation (HLD) concept of binary anionic surfactant mixtures to formulate crude oil spill dispersants by mixing lipopeptide biosurfactants with sodium dihexyl sulfosuccinate (<xref rid=\"B43\" ref-type=\"bibr\">Rongsayamanont et al., 2017</xref>). However, no HLD concept exists for a mixture of anionic and non-ionic surfactants. Therefore, in this study a bio-based washing agent based on microemulsion phase behavior experiments was formulated. The microemulsion phase experiments were performed in 1 mL flat-bottom glass vials with PTFE caps. This method was adapted from <xref rid=\"B48\" ref-type=\"bibr\">Tongcumpou et al. (2003)</xref>. In this study, 0.2 mL of polyolefin and 0.8 mL of the surfactant solution with different saline concentrations were added to the vials. The surfactant solution contained varying concentrations of foamate, Dehydol LS7TH and butanol. All samples were mixed with a vortex mixer for 1 min and then left to reach equilibrium at 25°C for 24 h. After the systems reached equilibrium, the relative phase volumes were quantified for each sample to determine the microemulsion type. The phase structure and characteristics of each microemulsion have been reported in literature (<xref rid=\"B44\" ref-type=\"bibr\">Salager et al., 2013</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Arpornpong et al., 2018</xref>). The dynamic interfacial tension (IFT) of the SBM and bio-based washing formulations were measured by a spinning drop tensiometer (SVT 20, Dataphysic Instruments).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Flowchart of experimental procedure and outcomes.</p></caption><graphic xlink:href=\"fbioe-08-00961-g001\"/></fig></sec><sec id=\"S2.SS3\"><title>Soil Washing Experiment in a Small System and Jar Tests</title><p>In a small system test, ten grams of dried drill cutting was suspended in a vial containing 20 mL of the bio-based washing agent. The suspension was then mixed for 30 min by placing on an orbital shaker at 200 rpm. The sample was centrifuged for 30 min at 3,000 rpm to separate the TPHs from the drill cutting and the washing agent. The drill cutting was rinsed twice with distilled water to remove the remaining TPHs and washing solution. The TPHs remaining in the washed cutting were determined to calculate the TPH removal efficiency over the initial TPHs in drill cuttings.</p><p>A jar test experiment was conducted to investigate the impacting factors on TPH removal of the selected formulations under varying washing conditions. The washing conditions were varied by altering the washing time and the cuttings-to-washing agent ratio. Initially, 100 grams of dried SBM drill cutting was loaded into a 1 L glass beaker containing different volumes of washing agent (ranging from 200–400 mL) to achieve cuttings (g) to washing agent (mL) ratios of 1:2, 1:3, and 1:4. In the jar test, the suspension was then mixed at a speed of 150 rpm for 5–30 min. Further washing steps were performed using the same washing procedure as in the small system test. All washing experiments were performed in triplicates.</p></sec><sec id=\"S2.SS4\"><title>Optimization of the Bio-Based Washing Agent for the Scale-up System</title><p>Statistica 8.0 software was used to obtain the optimum formulation of simplex lattice mixture comprising lipopeptides, Dehydol LS7TH, and water, for the semi-pilot scale experiment. The concentration ranges for lipopeptide and Dehydol LS7TH solutions were selected based on the microemulsion phase behavior experiments. A 14-run design matrix was generated (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>) and a triangular surface with three factors, three polynomial degrees and augmented with interior points was constructed. The TPH removal efficiency from batch washing experiments was the response variable in the experimental design. Each experiment was performed in duplicates. The most suitable mathematical fitting model was selected by comparing various statistical parameters provided by the one-way analysis of variance (ANOVA), such as <italic>p</italic>-value, lack of fit and <italic>R</italic>-squared value (<xref rid=\"B51\" ref-type=\"bibr\">Visetvichaporn et al., 2020</xref>). The selected model was then applied to predict the suitable formulation of the bio-based washing agent.</p><p>Since the production of foamate by a large-scale foam fractional process is difficult and rather costly, cell-free broth obtained from the production process was applied instead of foamate. The pH of the cell-free broth was adjusted to 10 with NaOH. Batch washing experiments were conducted in vials. The cuttings were vigorously mixed with the washing solution in a vortex mixer for 10 min. The cuttings-to-liquid ratio was fixed to 1:4 based on the results of the jar test experiment. The washing solution was separated from the cuttings via centrifugation at 5,000 rpm for 10 min. The washed cuttings were rinsed twice with deionized water. The TPHs remaining in the washed cutting was determined to calculate the TPH removal efficiency over the initial TPHs in the cuttings.</p></sec><sec id=\"S2.SS5\"><title>Drill Cutting Treatment Process in the Scale-up System</title><p>The scale-up washing process for the removal of TPHs from cuttings consisted of a rotary screener, hydrocyclone, agitation mixing tank and rotary vacuum-drum filter. The formulation of the bio-based washing agent and the cuttings-to-liquid ratio were obtained from the mixture design and jar test experiments, respectively. The maximum capacity of this process was 40 kg/day. The drill cuttings were transferred into the rotary screener to remove any coarse bits of gravel (>10 mm) at a flow rate of 40 L/min. The fined cuttings were mixed with the washing solution in the hydrocyclone and then transported to the agitation mixing tank, which operated at a contact time of 30 min and agitation rate of 250 rpm. The washing solution was separated from the cuttings using the rotary vacuum-drum filter at a rotation rate of 0.6 rpm with water spraying, which was repeated twice. The TPHs remaining in the washed cuttings were determined to calculate the TPH removal efficiency over the initial TPH in the cuttings.</p></sec><sec id=\"S2.SS6\"><title>Polyolefin Biodegradation Experiment</title><p>After the washing process, residual TPHs and bio-based washing components might remain in the drill cuttings. This study therefore investigated the polyolefin degradation efficiency of mixed <italic>Marinobacter salsuginis</italic> RK5, <italic>Microbacterium saccharophilum</italic> RK15, and <italic>Gordonia amicalis</italic> JC11 in the presence of the bio-based washing solution. The polyolefin biodegradation test was conducted in a 125 mL flask containing 45 mL MSM medium, which consisted of 2.5 g/L NH<sub>4</sub>Cl, 5.46 g/L KH<sub>2</sub>PO<sub>4</sub>, 4.76 g/L Na<sub>2</sub>HPO<sub>4</sub>, 0.20 g/L MgSO<sub>4</sub>, and 5.0 g/L NaCl (<xref rid=\"B5\" ref-type=\"bibr\">Arulazhagan and Vasudevan, 2011</xref>). The concentration of NaCl was lower than that of seawater because the drill cuttings attained a decreased conductivity after washing (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). The concentration of polyolefin in the medium was varied at 0.1, 0.25, and 0.5% (v/v), while the bio-based washing formulation was added based on a dispersant-to-oil ratio (DOR) of 1:10. The DOR was adopted to represent the optimal amount of washing formulation when applied to disperse the polyolefin on the water surface and promote its solubilization. To prepare the bacterial inoculum, each strain was cultivated in Zobell Marine Broth and incubated at room temperature with shaking at 200 rpm. The 24 h culture of each bacterial inoculum was harvested, adjusted to a concentration of 10<sup>9</sup> CFU/mL and mixed at an equal volume. The mixed inoculum was added to the medium at 5 mL/flask, while the control experiment consisted of an uninoculated sample for determining the polyolefin loss by abiotic processes. All experiments were conducted in triplicates and incubated with shaking at 200 rpm. The concentration of residual TPHs and the bacterial number were analyzed from the flasks sacrificed on days 0, 7, and 14.</p></sec><sec id=\"S2.SS7\"><title>Bioremediation of Washed Drill Cutting Experiment</title><p>Bioremediation of the washed drill cutting was performed in a soil microcosm under aerobic condition. Prior to bioremediation, the washed drill cutting samples were dried and ground to 2 mm particles. Their soil texture, conductivity, organic matter content, bacterial number and available nutrients were characterized using standard methods. The washed drill cuttings had a silty clay texture with a low nutrient level and small bacterial number (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). Consequently, mixed bacterial inoculum, fertilizer and biochar were applied for bioremediation to promote TPH biodegradation and improve soil properties. There were five treatments: (1) unamended, (2) biochar, (3) biochar and mixed bacteria, (4) biochar and fertilizer, and (5) biochar, fertilizer and mixed bacteria. In each treatment, the washed drill cuttings (soil) were mixed with the amendments to obtain a total weight of 200 g, and then placed in a 9 × 14 × 5 cm<sup>3</sup> plastic box with a solid lid. The proportion of soil and biochar was 20% (w/w) or 50% (v/v), while the fertilizer was applied to achieve a C:N:P ratio of 100:10:1. The moisture content of the inoculated and uninoculated microcosms was adjusted to 25% WHC by adding bacterial inoculum and distilled water, respectively. The mixed bacterial inoculum was prepared as in the polyolefin biodegradation experiment. The soil microcosm experiments were conducted in triplicates and incubated at room temperature. The boxes were opened every week, and the soil was manually mixed to provide aeration, while the moisture content was adjusted by adding distilled water. On days 0, 7, 21, 35, and 49, soil samples (6 g each) were collected from two locations in the box and mixed well before determining the TPH concentration and bacterial number.</p></sec><sec id=\"S2.SS8\"><title>Analytical Methods</title><p>The amount of TPHs in the drill cuttings was analyzed by thin layer chromatography-flame ionization detector (TLC-FLD, with an Iatroscan<sup>TM</sup> MK-6/6S, Mitsubishi Kagaku Iatron, Inc., Japan) and gas chromatography-flame ionization detector (GC-FID, with a Hewlett-Packard 6890, Agilent Technologies, United States) in the washing and bioremediation experiments, respectively. The TPHs were extracted from the cuttings by mixing twice with chloroform at a ratio of 2:1 and the chloroform was separated from the cutting via centrifugation at 5,000 rpm for 20 min. The concentration of TPHs in the chloroform was analyzed by TLC-FID and GC-FID according to <xref rid=\"B25\" ref-type=\"bibr\">Khondee et al. (2012)</xref>. The concentrations of TPHs were calculated based on a standard polyolefin curve.</p><p>The surface tension, critical micelle concentration (CMC) and critical micelle dilution (CMD) were determined with a digital tensiometer (Kruss, K10ST, Germany) at 25°C via the plate method. The CMC was obtained from the cross-section of the plot between the surface tension and concentration of crude lipopeptides, while the CMD was obtained from the cross-section of the plot between the surface tension and serial dilution of the cell-free supernatant.</p><p>The bacterial number in the polyolefin biodegradation experiments was determined on Zobell Marine agar because the bacteria were previously cultivated in MSM medium containing NaCl. During bioremediation of the washed drilled cuttings, the total bacteria were counted on tryptone soya agar (TSA) to include all the soil heterotrophic bacteria, while the oil-degrading bacteria were counted on NSW agar overlaid with 50 μL polyolefin. The bacteria were dislodged from the soil by sonicating the soil suspension for 2 min followed by 1 min of vortex mixing. The process was repeated twice, and serial dilutions were then prepared before plate counting.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Development of the Bio-Based Washing Formulations</title><p>Microemulsion phase behavior experiments can reveal different microemulsion types depending on the composition of the aqueous and oil phases. From literature, surfactant solutions can form different types of microemulsions (Type I, III, and II) by varying the concentrations of cosurfactant (<xref rid=\"B19\" ref-type=\"bibr\">Hayes et al., 2013</xref>), lipophilic linker (<xref rid=\"B9\" ref-type=\"bibr\">Bourrel et al., 1980</xref>; <xref rid=\"B44\" ref-type=\"bibr\">Salager et al., 2013</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Phaodee et al., 2018</xref>), salinity (<xref rid=\"B1\" ref-type=\"bibr\">Acosta and Bhakta, 2009</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Arpornpong et al., 2018</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Phaodee et al., 2018</xref>) or temperature (<xref rid=\"B44\" ref-type=\"bibr\">Salager et al., 2013</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Arpornpong et al., 2018</xref>) in the system. The phase behavior of polyolefin with either lipopeptide biosurfactant (as foamate) or Dehydol LS7TH indicated the occurrence of microemulsion Type I (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table 1</xref>). However, the mixtures of foamate and Dehydol LS7TH generated Type I to III microemulsions with polyolefin when the concentrations of NaCl or butanol increased (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Figures 1</xref>, <xref ref-type=\"supplementary-material\" rid=\"DS1\">2</xref>). The phase transition was due to the increase in hydrophobicity of the system. The minimum amount of Dehydol LS7TH required in the surfactant mixture to form Type III microemulsion was 2% (v/v) (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Figure 3</xref>).</p><p>To formulate the bio-based washing agents, the volume of foamate was varied to achieve lipopeptide concentrations below and above the CMC. <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref> shows the phase behavior and TPH removal efficiency of the bio-based washing agents at various foamate concentrations containing 2% Dehydol LS7TH and 4% butanol in saline water. Phase transition was observed from Type III to I with increasing foamate concentration. This occurs because the increased foamate concentration leads to a decrease in hydrophobicity of the system. A similar trend has been observed for anionic surfactants with a strong hydrophilicity (<xref rid=\"B53\" ref-type=\"bibr\">Wade et al., 1978</xref>). According to <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>, the TPH removal efficiency sharply increased as the foamate concentration increased in the system containing 2% Dehydol LS7TH and 4% butanol in saline water. The greater TPH removal was observed as the foamate concentration increased beyond its CMD value (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). The efficiency reached its highest value of 92.9 ± 1.0% at a foamate concentration of 20% or approximately 5x CMD. The concentration of foamate at 20% was therefore selected to formulate bio-based washing agents (F1 to F5 formulations).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Microemulsion phase behavior of polyolefin and surfactant solution containing 2% Dehydol LS7TH, 4% butanol and varying volumes of foamate in saline water. The petroleum hydrocarbon removal efficiency of each formulation was conducted with drill cutting in a batch experiment. The ratio of cutting (g) to washing agent (mL) was 1:2 and the washing time was 30 min.</p></caption><graphic xlink:href=\"fbioe-08-00961-g002\"/></fig><p>Due to complex mixtures of drill cuttings (e.g., cutting, drilling mud, inorganic and organic matters), they originally contained some amount of dissolved salts (e.g., Ca<sup>2+</sup>, Mg<sup>2+</sup>, and Na<sup>+</sup>). NaCl or CaCl<sub>2</sub> was added to the drilling mud as an alkalinity controller and brine enhancer during the drilling process. Calcite (CaCO<sub>3</sub>), hydrophilite (CaCl<sub>2</sub>) and halite (NaCl) are the main components found in the drill cuttings as reported in literature (<xref rid=\"B16\" ref-type=\"bibr\">Filippov et al., 2009</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Mansour et al., 2016</xref>; <xref rid=\"B40\" ref-type=\"bibr\">Poyai et al., 2020</xref>). The initial salinity of drill cutting was indirectly measured using an electrical conductivity meter. The electrical conductivity (EC) of the drill cuttings was 4,290 μs/cm (∼0.5% (w/v) salinity). It was thus assumed that the salinity of the drill cutting would not significantly affect the total concentration of NaCl in the bio-based washing agents. The role of DI and saline water (4% w/v NaCl) in bio-based washing agents were compared in the washing experiment.</p><p>The TPH removal efficiency increased from 50.3% (only foamate) and 61.9% (only Dehydol LS7TH) to 99.2% (20% foamate and 2% Dehydol LS7TH in formulation F1) (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). The efficiency of F1 to F5 formulations containing 20% foamate and 2% Dehydol LS7TH as the major components were compared and evaluated based on the ability to form microemulsion and decrease the residual TPHs in the drill cuttings. Many investigations reported that the formations of microemulsion Type I (near the Type I to III transition) and Type III can be applied for enhanced petroleum recovery (<xref rid=\"B12\" ref-type=\"bibr\">Childs et al., 2005</xref>), site remediation (<xref rid=\"B54\" ref-type=\"bibr\">Wu et al., 2000</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Quraishi et al., 2015</xref>), and detergency (<xref rid=\"B39\" ref-type=\"bibr\">Phaodee et al., 2018</xref>). The most efficient formulation, F1, produced a Type I microemulsion with polyolefin and contained only 20% (v/v) foamate and 2% (v/v) Dehydol LS7TH in water. The F3 formulation contained similar surfactant mixtures and produced a Type I microemulsion as with the F1 formulation. However, it had a significantly lower TPH removal efficiency than that of the F1 formulation, which was probably due to the use of saline water. On the other hand, the effect of saline water was different in the formulations containing butanol. The F2 formulation (forming Type III microemulsion in saline water) had higher TPH removal efficiency than the F4 formulation (forming Type I microemulsion in water). When the amount of butanol in the formulations increased, i.e., from 4% butanol in formulation F2 to 8% butanol in formulation F5, the TPH removal efficiency decreased. However, these formulations produced a microemulsion Type III with polyolefin and attained a low dynamic interfacial tension (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Figure 4</xref>).Thus, microemulsion formation is not the only parameter governing oil removal efficiency, but other parameters also play a role in oil washing.</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Effectiveness of bio-based washing agents on removal of petroleum hydrocarbons from drill cuttings in a batch experiment.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Formulation<sup>a</sup></bold></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Composition</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Microemulsion type<sup>b</sup></bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>TPH Removal efficiency<sup>c</sup> (%)</bold></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Deionized water (DI)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Not occur</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.9 ± 0.6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Foamate (20% v/v)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Type I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50.3 ± 1.0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dehydol LS7TH (2% v/v)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Type I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">61.9 ± 1.3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">F1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">20% Foamate + 2% Dehydol LS7TH + 78% DI</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Type I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.2 ± 0.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">F2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">20% Foamate + 2% Dehydol LS7TH + 4% Butanol + 74% saline water</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Type III</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92.9 ± 1.0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">F3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">20% Foamate + 2% Dehydol LS7TH + 78% saline water</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Type I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">74.8 ± 2.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">F4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">20% Foamate + 2% Dehydol LS7TH + 4% Butanol + 74% DI</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Type I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">81.2 ± 6.0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">F5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">20% Foamate + 2% Dehydol LS7TH + 8% Butanol + 70% saline water</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Type III</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">78.6 ± 0.7</td></tr></tbody></table><table-wrap-foot><attrib><italic><sup><italic>a</italic></sup>Compositions of each formulation were reported as volume of each component. The saline water contains 4% (w/v) NaCl. <sup><italic>b</italic></sup>Microemulsion type was tested with polyolefin. <sup><italic>c</italic></sup>Ratio of cutting (g) to washing agent (mL) was 1:2 and washing time was 30 min.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS2\"><title>Application of the Bio-Based Washing Formulations in Jar Tests</title><p>For a scale-up experiment, the effects of the drill cuttings-to-washing agent ratio and washing time on the performance of the two selected bio-based washing agents in removing TPHs from the drill cuttings were investigated via jar tests. The F1 and F2 formulations were chosen based on their high oil removal efficiency and they were representatives of the formulations that generated microemulsion Type I and III, respectively. <xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref> shows the TPH removal efficiency as a function of the cuttings-to-washing agent ratio. The results show that when the washing agent loading of the system was increased, the TPH removal efficiency tended to increase. This occurs because the system contains sufficient surfactant to penetrate the cuttings with less coalescence between the surfactant and oil (<xref rid=\"B36\" ref-type=\"bibr\">Naksuk et al., 2009</xref>). The cuttings-to-washing agent ratio of 1:4 had the maximum TPH removal efficiency (up to 91.6%). <xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref> reveals that a washing time of 30 min for the cuttings was considered the optimum time for the TPHs to detach from the cuttings and solubilize into the micelles. The results exhibit a similar trend for both formulations. As expected, the F1 formulation provided a higher TPH removal efficiency than the F2 formulation. These results were consistent with those of the previous experiments in this study.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Effect of drill cutting-to-washing agent ratio <bold>(A)</bold> and washing time <bold>(B)</bold> on the petroleum hydrocarbon removal efficiency of the bio-based washing formulations, F1 (20% Foamate + 2% Dehydol LS7TH + 78% DI) and F2 (20% Foamate + 2% Dehydol LS7TH + 4% Butanol + 74% saline water) in a jar test experiments.</p></caption><graphic xlink:href=\"fbioe-08-00961-g003\"/></fig></sec><sec id=\"S3.SS3\"><title>Optimization of the Bio-Based Washing Formulation for the Scale-up System</title><p>According to the small-scale study, the F1 formulation containing only lipopeptide and Dehydol LS7TH was appropriate for bio-based washing agent. The formulation is simple but the cost might be high due to the high concentration of each composition. Consequently, the F1 formulation was further optimized via the simplex lattice mixture design to obtain an efficient formulation with lower amounts of surfactants. In addition, cell-free broth containing lipopeptides was used instead of foamate to avoid difficulties associated with biosurfactant recovery via foam fractionation process. The initial concentration of lipopeptides in the cell-free broth was 3.0 g/L, and its surface activity was enhanced by adjusting the pH to 10 (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). The Dehydol LS7TH solution was prepared at 5% (v/v).</p><p>A range of cell-free broth and Dehydol LS7TH concentrations were prepared based on the results from the microemulsion phase behavior experiments; therefore, 16.70–100.00% cell-free broth (pH = 10) and 0.84–5.00% Dehydol LS7TH were chosen. <xref rid=\"T3\" ref-type=\"table\">Table 3</xref> lists the experimental plan from the simplex lattice mixture design and the obtained responses for each run. The empirical coefficient values and ANOVA outcomes, as well as the statistical criteria for the responses, are presented in the <xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Data</xref>. The maximum <italic>R</italic>-squared value of the full cubic regression model demonstrated the best fit between the biobased washing agent compositions (independent variables) and TPH removal (dependent variable) compared to other models (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table 2</xref>). The lack of fit <italic>p</italic>-value of higher than 0.05 confirms the applicability of this model. The fitted full cubic equation is as follows:</p><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>Simplex lattice mixture design of three components and results of response values for optimizing of the F1 formulation.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Mixtures</bold></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><bold>Independent variables (%)</bold><hr/></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Dependent variables (TPH removal efficiency, %)</bold></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Cell-free-broth, pH 10 (100%)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Dehydol LS7TH (5%)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Water (100%)</bold></td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">44.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21.0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">66.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">78.4</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">66.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">44.1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">66.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">46.8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">66.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53.8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">66.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">79.9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">66.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">54.1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">81.0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">66.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">74.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">66.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">55.6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">66.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53.1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">75.8</td></tr></tbody></table></table-wrap><disp-formula id=\"S3.Ex1\"><mml:math id=\"M1\"><mml:mrow><mml:mpadded width=\"+2.8pt\"><mml:mi>TPH</mml:mi></mml:mpadded><mml:mpadded width=\"+2.8pt\"><mml:mi>removal</mml:mi></mml:mpadded><mml:mrow><mml:mo>(</mml:mo><mml:mo>%</mml:mo><mml:mo rspace=\"5.3pt\">)</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:mn>43.619</mml:mn><mml:mpadded width=\"+2.8pt\"><mml:mi>X</mml:mi></mml:mpadded><mml:mo>+</mml:mo><mml:mn> 21.185</mml:mn><mml:mi>Y</mml:mi></mml:mrow></mml:math></disp-formula><disp-formula id=\"S3.Ex2\"><mml:math id=\"M2\"><mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:mn> 29.441</mml:mn><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>Z</mml:mi></mml:mpadded></mml:mrow></mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:mn> 145.122</mml:mn><mml:mo>⁢</mml:mo><mml:mi>X</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>Y</mml:mi></mml:mpadded></mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:mn> 120.076</mml:mn><mml:mo>⁢</mml:mo><mml:mi>X</mml:mi><mml:mo>⁢</mml:mo><mml:mi>Z</mml:mi></mml:mrow></mml:mrow></mml:math></disp-formula><disp-formula id=\"S3.Ex3\"><mml:math id=\"M3\"><mml:mrow><mml:mrow><mml:mi/><mml:mo>+</mml:mo><mml:mrow><mml:mn> 108.118</mml:mn><mml:mo>⁢</mml:mo><mml:mi>Y</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>Z</mml:mi></mml:mpadded></mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:mn> 84.404</mml:mn><mml:mo>⁢</mml:mo><mml:mi>X</mml:mi><mml:mo>⁢</mml:mo><mml:mi>Y</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>Z</mml:mi></mml:mpadded></mml:mrow></mml:mrow><mml:mo>-</mml:mo><mml:mrow><mml:mn> 153.632</mml:mn><mml:mo>⁢</mml:mo><mml:mi>X</mml:mi><mml:mo>⁢</mml:mo><mml:mi>Y</mml:mi><mml:mo>⁢</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mi>X</mml:mi><mml:mo>-</mml:mo><mml:mi>Y</mml:mi></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:mrow></mml:math></disp-formula><disp-formula id=\"S3.E1\"><label>(1)</label><mml:math id=\"M4\"><mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:mn> 195.865</mml:mn><mml:mo>⁢</mml:mo><mml:mi>X</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>Z</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mi>X</mml:mi><mml:mo>-</mml:mo><mml:mi>Z</mml:mi></mml:mrow><mml:mo rspace=\"5.3pt\" stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:mrow><mml:mo>-</mml:mo><mml:mrow><mml:mn>3.866</mml:mn><mml:mo>⁢</mml:mo><mml:mi>Y</mml:mi><mml:mo>⁢</mml:mo><mml:mi>Z</mml:mi><mml:mo>⁢</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mi>Y</mml:mi><mml:mo>-</mml:mo><mml:mi>Z</mml:mi></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:mrow></mml:math></disp-formula><p>where X = cell-free broth (lipopeptide 0.3%, pH 10), Y = water and Z = Dehydol LS7TH (5%).</p><p>A sequential <italic>p</italic>-value below 0.05 indicates that the model terms are statistically significant. Both the cell-free broth and Dehydol LS7TH have a significant effect on the TPH removal efficiency (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table 3</xref>). The coefficients X to Z are positive, indicating the synergistic effect of the lipopeptide and Dehydol LS7TH mixture. The optimal formulation of the bio-based washing agent consisted of 72.0% cell-free broth and 1.4% Dehydol LS7TH in water (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). The predicted response from this optimal formulation was a TPH removal efficiency of 81.2%. The developed bio-based washing agent was applied to a scale-up washing experiment for TPH removal from cuttings. The obtained TPH removal efficiency was 70.7% at an initial TPH concentration of 151,572 mg/kg (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). The loss of washing capability likely occurred during the scale-up process. For example, polyolefins were redeposited with the cutting particles during the separation of washing solution from the washed cuttings using the rotary vacuum-drum filter.</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Contour response surface plot for optimizing the components of F1 formulation. Lipopeptide solution was used as cell-free-broth (pH = 10). The initial concentration of Dehydol LS7TH was 5% (v/v). The ratio of cutting (g) to washing agent (mL) was 1:4.</p></caption><graphic xlink:href=\"fbioe-08-00961-g004\"/></fig><table-wrap id=\"T4\" position=\"float\"><label>TABLE 4</label><caption><p>Characteristics of drill cuttings after washing in a scale-up system and after mixing with biochar.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><inline-graphic xlink:href=\"fbioe-08-00961-i001.jpg\"/></td></tr></tbody></table><table-wrap-foot><attrib><italic><sup><italic>a</italic></sup>ND = not determined.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS4\"><title>Biodegradation of Polyolefin by the Mixed Bacteria</title><p><italic>Marinobacter salsuginis</italic> RK5, <italic>Microbacterium saccharophilum</italic> RK15, and <italic>Gordonia amicalis</italic> JC11 slightly degraded polyolefin when applied as a single strain (data not shown). The mixture of these bacteria was expected to impose a synergistic effect on the polyolefin biodegradation efficiency because each strain should have different characteristics and catabolic pathways. The LAOs in all inoculated samples decreased over time. After 14 days, the mixed bacterial inoculum showed the highest removal efficiency with 0.1% LAOs (97%), followed by 0.25% LAOs (84%) and 0.5% LAOs (70%), while the control treatment only attained a removal efficiency of 9–18% (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>). The bacterial number in the 0.1% LAO system increased by approximately 1 magnitude to 9.07 log CFU/mL at the end of the study, corresponding to a decrease in the LAOs (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>). On the other hand, the bacterial number in the 0.25% LAO system remained constant at 8.38 log CFU/mL, while it was slightly reduced to 7.73 log CFU/mL in the 0.5% LAO system. The low cell numbers might be due to the formation of aggregated cells, as indicated by the orange cell clumps in the 0.25% LAO system and the attached film in the 0.5% LAO system (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Figure 5</xref>), which interfered with the dilution of bacterial cells during the plate count technique. The difference in cell aggregation could be due to the presence of the bio-based washing formulation, which was applied according to the amount of oil in each flask. The different concentrations of biosurfactant molecules might promote different forms of aggregation of polyolefin, bacterial cells and bacterial metabolites. The presence of viable cells suggested that longer incubation time could increase the biodegradation efficiency of the mixed bacterial inoculum. Since the mixed bacterial inoculum was able to efficiently degrade low concentrations of polyolefin, they were applied for bioremediation of the washed drilled cuttings.</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Degradation of polyolefin (LAOs) by mixed bacterial inoculum in MSM medium containing 0.5% NaCl <bold>(A)</bold> and the changes in bacterial number during the experiment <bold>(B)</bold>.</p></caption><graphic xlink:href=\"fbioe-08-00961-g005\"/></fig></sec><sec id=\"S3.SS5\"><title>Bioremediation of the Washed Drill Cuttings</title><p>In this study, biochar was initially mixed with the washed drilled cuttings to improve soil characteristics such as water holding capacity and available nutrients (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). The biochar<italic>-</italic>amended washed drill cuttings had a notable decrease in conductivity from 1,721 to 4.31 μS/cm, while the available potassium and phosphorus increased from 155 and 96 ppm to 6,155 and 688 ppm, respectively. The concentration of the TPHs in biochar<italic>-</italic>amended washed drill cuttings decreased by approximately half (from 44,468 mg/kg to 26,359 mg/kg) due to the dilution effect (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). Other characteristics, such as bacterial number and organic matter and total nitrogen contents, in the biochar<italic>-</italic>amended washed drill cuttings were similar to those of the initial sample. The unamended sample or the natural attenuation treatment attained only 1% TPH removal efficiency, and TPHs remained at a concentration of 43,989 mg/kg after 49 days of incubation (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>). On the other hand, treatment combining biochar, fertilizer and mixed bacteria achieved the highest TPH removal efficiency of 71%. This TPH removal efficiency was calculated from the TPH concentrations on day 0 and day 49, which were 26,359 and 7,515 mg/kg, respectively (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>). The second most efficient treatment was the biochar- and mixed bacteria-amended microcosms, which removed 66% of the TPHs with 8,968 mg/kg of TPHs remaining at the end of experiment. Without bacterial addition, the removal of TPHs was inefficient, of which the biochar-fertilizer treatment and biochar treatment removed 39 and 28% of TPHs, respectively. The results indicated that the addition of mixed bacteria played an important role in TPH removal from the biochar-amended washed drill cutting. The changes in alkane composition as revealed by the GC-FID chromatograms on day 49 also confirmed that the TPHs were significantly reduced by both physical and biological mechanisms in the treatments containing mixed bacteria (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Figure 6</xref>).</p><fig id=\"F6\" position=\"float\"><label>FIGURE 6</label><caption><p>Efficiency of bioremediation approaches comprising of mixed bacterial inoculum, biochar and fertilizer on the removal of petroleum hydrocarbons (TPHs) from drill cuttings after washing in a scale-up system.</p></caption><graphic xlink:href=\"fbioe-08-00961-g006\"/></fig><p>In all microcosms, the growth curves of both total and olefin-degrading bacteria showed a similar trend, but the numbers of olefin-degrading bacteria were slightly lower at each time point (<xref ref-type=\"fig\" rid=\"F7\">Figures 7A,B</xref>). The initial washed drilled cuttings had a low bacterial number; thus, the addition of mixed bacteria was necessary. The rapid decrease in TPHs in the biochar and mixed bacteria treatment (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>) corresponded with the numbers of total and olefin-degrading bacteria after incubation, which were maintained throughout the incubation period at 8.51 and 8.47 log CFU/g soil, respectively (<xref ref-type=\"fig\" rid=\"F7\">Figures 7A,B</xref>). The addition of fertilizer also promoted the growth of indigenous bacteria in the washed drill cuttings from day 0 to 21; however, they later competed with the added bacterial inoculum (<xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref>) and led to delayed TPH degradation (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>). The average bacterial number in the treatment with biochar, fertilizer and mixed bacteria decreased to 7.33 log CFU/g soil on day 49, which was lower than that in the treatment with only biochar and mixed bacteria. Competition between different indigenous populations might also occur since the bacterial numbers in biochar and fertilizer treatment were lower than that in the treatment with biochar only. However, the number of indigenous olefin-degrading bacteria in the treatments with no bacterial addition increased from an average of 5.25 log CFU/g soil to 7.92 log CFU/g soil at the end of the study (<xref ref-type=\"fig\" rid=\"F7\">Figure 7B</xref>). The TPH removal efficiencies in the uninoculated microcosms, i.e., soil only, biochar and biochar-fertilizer treatments were low to moderate (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>). The results indicated that the TPH-degrading activity of the indigenous olefin-degrading bacteria was lower than that of added mixed bacterial inoculum. Consequently, bioaugmentation with mixed bacterial inoculum was appropriate for the treatment of washed drill cuttings.</p><fig id=\"F7\" position=\"float\"><label>FIGURE 7</label><caption><p>Changes in number of total bacteria <bold>(A)</bold> and polyolefin-degrading bacteria <bold>(B)</bold> during the bioremediation of drill cuttings after washing in a scale-up system.</p></caption><graphic xlink:href=\"fbioe-08-00961-g007\"/></fig></sec></sec><sec id=\"S4\"><title>Discussion</title><p>Microemulsion phase behavior studies were conducted to develop suitable bio-based washing formulations. The formation of microemulsions between foamate (highly hydrophilic character) and polyolefin (highly hydrophobic character) implies the attainment of a low interfacial tension and the possibility of substantial oil solubilization. This occurs because of the balance between the hydrophilic water and hydrophobic oil phases. The best washing formulation F1, comprised of 20% foamate and 2% Dehydol LS7TH in water, could form Type I microemulsion with polyolefin (near the transition region between Type I and Type III). The high TPH removal efficiency by Type I microemulsion might be due to the super-solubilization effect, where the swollen micelles incorporate a large amount of oil into their hydrophobic core (<xref rid=\"B48\" ref-type=\"bibr\">Tongcumpou et al., 2003</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Javanbakht and Goual, 2016</xref>). The same trend was found in the study by <xref rid=\"B54\" ref-type=\"bibr\">Wu et al. (2000)</xref>, who reported that the microemulsion Type I (near the Type I to III boundary) and Type III had similar oil solubilization capacities. Since there was no butanol or salt in formulation F1, it was considered a bio-based and environment-friendly washing agent.</p><p>The present study found that butanol reduced the TPH removal efficiency of formulations (Formulation F2, F4, and F5). One possibility is that butanol reduces the micellar cavity where the SBM and polyolefin prefer to solubilize in micelles. The results obtained in this study correspond to those reported by <xref rid=\"B9\" ref-type=\"bibr\">Bourrel et al. (1980)</xref> and <xref rid=\"B48\" ref-type=\"bibr\">Tongcumpou et al. (2003)</xref>, whereby an increased lipophilic linker of the system can decrease oil solubilization. Moreover, saline water was not required in the bio-based washing formulation. The presence of salt and cation (Na<sup>+</sup>) probably influenced anionic surfactant adsorption on drill cuttings and precipitation between Na<sup>+</sup> and foamate anionic surfactant. Increase in surfactant adsorbed onto cutting, lead to an increased surfactant loss during the washing process. The effect of salinity on anionic surfactant adsorption at the solid–liquid interface has been discussed by many researchers (<xref rid=\"B12\" ref-type=\"bibr\">Childs et al., 2005</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Arpornpong et al., 2010</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Belhaj et al., 2020</xref>). In addition, <xref rid=\"B3\" ref-type=\"bibr\">Arpornpong et al. (2018)</xref> showed scanning electron microscopy images of spent bleaching earth (SBE) surface before and after surfactant-based washing procedure and found that a high salinity washing solution (15% w/v NaCl) increased the adsorption of ethoxylate non-ionic surfactant on SBE surface.</p><p>The TPH removal efficiency of surfactant mixtures was higher than either lipopeptides or Dehydol LS7TH alone. These results confirmed that the higher efficiency was due to the synergistic effect. <xref rid=\"B47\" ref-type=\"bibr\">Shi et al. (2015)</xref> also reported that the mixed anionic–non-ionic surfactants considerably enhanced the oil solubilization capacity and interfacial tension reduction, which resulted in a decrease in the total amount of surfactant used in a particular application, which in turn reduced both the cost and the environmental impact. A single-biosurfactant-based washing agent usually attains less than 90% TPH removal from drill cutting. For example, <xref rid=\"B55\" ref-type=\"bibr\">Yan et al. (2011)</xref> reported that the maximum TPH removal efficiency of oil-based mud from drill cuttings was 83% at a rhamnolipid concentration up to 360 mg/L, a solid:liquid ratio of 1:3 and a 20 min washing time, while <xref rid=\"B38\" ref-type=\"bibr\">Olasanmi and Thring (2020)</xref> attained an 85.4% TPH reduction from drill cuttings with a rhamnolipid solution (500 mg/L) at an optimal agitation rate of 100 rpm for 30 min and a ratio of cutting-to-washing agent of 1:1.</p><p>When formulation F1 was applied in the jar tests, the results indicated an optimal washing condition consisting of a 30 min washing time at a drill cutting to bio-based washing agent ratio of 1:4. However, the efficiency of formulation F1 was reduced to 91.6% due to the large amount of drill cutting in a jar test, which caused a reduction in the mixing speed and redeposition of TPHs during solid-liquid phase separation. The same trend was found in other studies (<xref rid=\"B23\" ref-type=\"bibr\">Kantar and Honeyman, 2006</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Elgh-Dalgren et al., 2009</xref>). The jar test suggested that a large volume of formulation F1 was required for washing drill cuttings on site. To reduce the cost of formulation F1, the concentration of each component was optimized via the simplex lattice mixture design, and cell-free broth was used instead of foamate. The drawback of a lower lipopeptide concentration in cell-free broth than that in foamate was resolved by increasing the pH of cell-free broth solution from 7 to 10. Alkaline condition can reduce the adsorption of anionic surfactants on drill cuttings by increasing electrical negative charge on the surface of rock solids (<xref rid=\"B27\" ref-type=\"bibr\">Kurnia et al., 2020</xref>). Thus, the surfactant activity increased, resulting in a decrease in the amount of surfactant required in the bio-based washing agents. Moreover, the alkali might react with the acid components in SBM present in the cuttings to produce soap (<xref rid=\"B46\" ref-type=\"bibr\">Sheng, 2014</xref>). The presence of soap helps in the reduction of IFT, providing a greater oil mobilization from the cuttings during the washing process. Although the washing efficiency obtained with cell-free broth (81.2%) was lower than that obtained with foamate (91.6%), the TPH removal efficiency was comparable to that of other studies (<xref rid=\"B55\" ref-type=\"bibr\">Yan et al., 2011</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Olasanmi and Thring, 2020</xref>). The application of non-purified lipopeptides in foamate and cell-free broth indicates an economic advantage of the developed bio-based washing agents for drill cutting treatment.</p><p>The bio-based washing agents can be applied to other cleaning applications, for example, washing of petroleum-contaminated soil. The formulation can be further optimized by increasing the salt or hydrophobic surfactant concentration for heavier crude oil-contaminated soil. For saline soil with different salinity and hardness levels, the washing agent can be formulated by adjusting the salt concentration or adding appropriate amounts of builder or lime-soap dispersing agent. <xref rid=\"B12\" ref-type=\"bibr\">Childs et al. (2005)</xref> evaluated the performance of surfactant-based washing agents for the treatment of oil-based drill cuttings. They found that the addition of C<sub>8</sub>-sulfobetaine, a lime soap dispersing agent (LSDA), and sodium metasilicate (Na<sub>2</sub>SiO<sub>3</sub>), a builder, with a small amount of added salt in the surfactant-based washing agent, could enhance the oil removal efficiency from drill cuttings. For a long storage period, potassium sorbate preservative maybe added to the bio-based washing agents, as reported by <xref rid=\"B17\" ref-type=\"bibr\">Freitas et al. (2016)</xref>, to maintain their activities.</p><p>To date, there have been few studies on the scale-up of drill cutting treatment by the surfactant washing technique. <xref rid=\"B13\" ref-type=\"bibr\">Elgh-Dalgren et al. (2009)</xref> attained a lower PAH removal by soil washing at the pilot scale than that attained at the laboratory scale due to the variation in environmental conditions such as the ambient temperature. This research also found a decrease in TPH removal from 81.2% at the laboratory scale to 70.7% at the semi-pilot scale. Therefore, the combination of biosurfactant washing and subsequent treatment, such as bioremediation was required to enhance the TPH removal from the drill cuttings obtained from the oilfield. Considering the cost of wastewater management due to the surfactant washing approach, the recovery and reusability of bio-based washing solutions should be studied further. Nevertheless, the biological treatment of wastewater containing bio-based washing solutions should be easier than that of wastewater containing chemical-based washing solutions due to its low toxicity, high biodegradability, and high biocompatibility of lipopeptide biosurfactants.</p><p>The residual TPHs in the bio-based washing solutions as well as in the washed drilled cuttings should be biodegraded before disposal. Polyolefin, the main component in SBM, is a mixture of unsaturated hydrocarbons with medium chain lengths. It is relatively persistent under natural conditions. The degradation of olefins in a marine anaerobic biodegradation test lasted longer than 9 months of incubation and required sediment containing a large number of sulfate reducing bacteria, general anaerobes and methanogens as a source of inoculum (<xref rid=\"B20\" ref-type=\"bibr\">Herman and Roberts, 2005</xref>). Solid-phase tests revealed that synthetic drilling fluids containing olefins are slowly degraded in mud and coarse sand by indigenous microorganisms, where their half-life increases as the nominal concentration increases (<xref rid=\"B35\" ref-type=\"bibr\">Munro et al., 1998</xref>). Non-etheless, bioremediation can increase the olefin biodegradation rate. Approximately 96% of the 14,720 mg/kg linear alpha-olefin in a subsoil sample was reduced by providing a nitrogen source under aerobic condition, and the treated soil after 93 days of incubation showed low toxicity to plants, earthworms and microorganisms (<xref rid=\"B52\" ref-type=\"bibr\">Visser et al., 2002</xref>). It is thus possible to further enhance polyolefin biodegradation by adding efficient bacterial inoculum under aerobic condition. This study found that the mixture of <italic>Marinobacter salsuginis</italic> RK5, <italic>Microbacterium saccharophilum</italic> RK15, and <italic>Gordonia amicalis</italic> JC11 was able to degrade the polyolefin in the liquid medium containing a biobased washing agent. The uses of mixed bacteria probably promote the success of bioaugmentation because they have complementary catabolic pathways as well as the ability to enhance the oil dispersion and hydrocarbon bioavailability (<xref rid=\"B34\" ref-type=\"bibr\">McGenity et al., 2012</xref>). The extent of polyolefin removal in the bioaugmented sample was much higher than that of the abiotic process. However, the degrading activity of mixed bacteria decreased at high polyolefin concentrations (>2.5%); thus, the bacterial inoculum was applied for bioremediation of drill cuttings only after the washing process.</p><p>Bioremediation of washed drill cuttings was examined in a microcosm study with varying amendments for 49 days. The results indicated that only mixed polyolefin-degrading bacteria and biochar were required for an enhanced TPH removal from the washed drill cuttings. Although the above treatment resulted in more remaining TPHs (0.89%) than that with additional fertilizer (0.75%), it attained a higher biodegradation rate, and its cost was relatively lower. Biochar enhanced the activity of the mixed bacterial inoculum probably by providing a habitat for microorganisms and improving the bulk density, pH and movement of air, water and nutrients within the soil matrix (<xref rid=\"B18\" ref-type=\"bibr\">Gul et al., 2015</xref>). In addition, biochar can sorb petroleum hydrocarbons and their degrading intermediates, which leads to a considerable reduction in soil toxicity and allows continuous petroleum biodegradation (<xref rid=\"B41\" ref-type=\"bibr\">Qin et al., 2013</xref>). The presence of biosurfactant molecules in the washed drill cuttings might also increase the bioavailability of TPHs. <xref rid=\"B10\" ref-type=\"bibr\">Brown et al. (2017)</xref> reported that biochar and rhamnolipid biosurfactant imposed a synergistic action on enhancing oil biodegradation in soil during landfarming. During bioremediation, the number of added bacteria decreased over time, probably due to their inability to compete with indigenous bacteria. <xref rid=\"B56\" ref-type=\"bibr\">Zhang et al. (2016)</xref> reported that biochar-immobilized <italic>Corynebacterium variabile</italic> HRJ4 achieved high TPH biodegradation, which made the bioremediation process more robust against environmental factors and other competitors. Thus, the bacterial inoculum should be immobilized on biochar before application to washed drill cuttings.</p><p>The developed sequential bio-based washing and bioremediation system removed TPHs in the drill cuttings. The TPH concentration in the drill cuttings was reduced from 151,572 mg/kg to 8,968 mg/kg, i.e., TPH removal efficiency of 94.1%. The efficiency of this system was comparable to that of <xref rid=\"B55\" ref-type=\"bibr\">Yan et al. (2011)</xref>, where oil-based drill cuttings with an initial TPH concentration of 85,000 mg/kg were washed with a rhamnolipid solution and then subjected to bioremediation by adding sawdust, as a bulking agent, and mixed bacterial culture. They found that the concentration of TPHs decreased to 5,470 mg/kg, i.e., 93.5% TPH removal efficiency (<xref rid=\"B55\" ref-type=\"bibr\">Yan et al., 2011</xref>). Consequently, the sequential remedial system could be applied in onshore drilling operations. It is expected that the sequential bio-based washing and bioremediation system will have lower cost and environmental impacts than the current incineration technique. The treated drill cuttings contained less than 1% TPHs and could be further treated on site by rhizoremediation. The technology uses synergistic interactions of plant roots and rhizospheric microorganisms to degrade hydrocarbons and is considered one of the most suitable treatment methods for petroleum-polluted soils over a large area (<xref rid=\"B21\" ref-type=\"bibr\">Hussain et al., 2018</xref>).</p></sec><sec id=\"S5\"><title>Conclusion</title><p>Drill cuttings with high concentrations of TPHs (>15%) were sequentially treated with a bio-based washing agent and bioremediation. The formulation of the bio-based washing agents was guided by the experimental phase behavior of the mixed bio-based surfactants and polyolefin and further optimized via experimental design. The efficient formulation contained only lipopeptides and Dehydol LS7TH in water, which is considered environmentally friendly and cost effective. When the bio-based washing agents were applied in a large-scale system, the TPH washing efficiency decreased. It is thus necessary to treat the remaining TPHs by bioremediation. From a microcosm study, only mixed polyolefin-degrading bacteria and biochar were required for an enhanced TPHs removal from washed drill cuttings. The sequential remedial system can be applied on site without high energy use in contrast to incineration. It is also possible to use the bio-based washing agents for cleaning petroleum-contaminated soil and other polluted environments.</p></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>All datasets presented in this study are included in the article/<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Material</xref>.</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>NA designed and formulated the bio-based washing agent. RP investigated biodegradation and bioremediation experiments. NK produced biosurfactant and optimized the bio-based washing agent. CT, SS, and KS supervised and conceptualized the bio-based washing formulations and washing systems. NA, NK, and EL analyzed the data and wrote the manuscript. EL designed the overall project, acquired funding and performed manuscript editing. All authors contributed to the preparation of manuscript.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This research was funded by the Innovation Institute, PTT Public Company Limited and the 90<sup>th</sup> Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund).</p></fn></fn-group><ack><p>The authors are grateful to Parisarin Nawavimarn and Witchaya Rongsayamanont for their kind assistance on surfactant characterization.</p></ack><sec id=\"S10\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fbioe.2020.00961/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fbioe.2020.00961/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"DS1\"><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Acosta</surname><given-names>E. 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Biotechnol.</journal-id><journal-title-group><journal-title>Frontiers in Bioengineering and Biotechnology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-4185</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850768</article-id><article-id pub-id-type=\"pmc\">PMC7431658</article-id><article-id pub-id-type=\"doi\">10.3389/fbioe.2020.00955</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Bioengineering and Biotechnology</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Cells, Materials, and Fabrication Processes for Cardiac Tissue Engineering</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Montero</surname><given-names>Pilar</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Flandes-Iparraguirre</surname><given-names>María</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Musquiz</surname><given-names>Saioa</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/951439/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Pérez Araluce</surname><given-names>María</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Plano</surname><given-names>Daniel</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/941369/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Sanmartín</surname><given-names>Carmen</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/998873/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Orive</surname><given-names>Gorka</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/153257/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Gavira</surname><given-names>Juan José</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"aff\" rid=\"aff7\"><sup>7</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Prosper</surname><given-names>Felipe</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"aff\" rid=\"aff8\"><sup>8</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/739114/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Mazo</surname><given-names>Manuel M.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"aff\" rid=\"aff8\"><sup>8</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/385466/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Regenerative Medicine Program, Cima Universidad de Navarra, Foundation for Applied Medical Research</institution>, <addr-line>Pamplona</addr-line>, <country>Spain</country></aff><aff id=\"aff2\"><sup>2</sup><institution>NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country – UPV/EHU</institution>, <addr-line>Vitoria-Gasteiz</addr-line>, <country>Spain</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Pharmaceutical Technology and Chemistry, University of Navarra</institution>, <addr-line>Pamplona</addr-line>, <country>Spain</country></aff><aff id=\"aff4\"><sup>4</sup><institution>IdiSNA, Navarra Institute for Health Research</institution>, <addr-line>Pamplona</addr-line>, <country>Spain</country></aff><aff id=\"aff5\"><sup>5</sup><institution>University Institute for Regenerative Medicine and Oral Implantology – UIRMI (UPV/EHU – Fundación Eduardo Anitua)</institution>, <addr-line>Vitoria-Gasteiz</addr-line>, <country>Spain</country></aff><aff id=\"aff6\"><sup>6</sup><institution>Singapore Eye Research Institute</institution>, <addr-line>Singapore</addr-line>, <country>Singapore</country></aff><aff id=\"aff7\"><sup>7</sup><institution>Cardiology Department, Clínica Universidad de Navarra</institution>, <addr-line>Pamplona</addr-line>, <country>Spain</country></aff><aff id=\"aff8\"><sup>8</sup><institution>Hematology and Cell Therapy Area, Clínica Universidad de Navarra</institution>, <addr-line>Pamplona</addr-line>, <country>Spain</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Susanna Sartori, Politecnico di Torino, Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Karina Nakayama, Oregon Health and Science University, United States; Diana Massai, Politecnico di Torino, Italy</p></fn><corresp id=\"c001\">*Correspondence: Manuel M. Mazo, <email>mmazoveg@unav.es</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>These authors have contributed equally to this work</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Tissue Engineering and Regenerative Medicine, a section of the journal Frontiers in Bioengineering and Biotechnology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>955</elocation-id><history><date date-type=\"received\"><day>10</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>23</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Montero, Flandes-Iparraguirre, Musquiz, Pérez Araluce, Plano, Sanmartín, Orive, Gavira, Prosper and Mazo.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Montero, Flandes-Iparraguirre, Musquiz, Pérez Araluce, Plano, Sanmartín, Orive, Gavira, Prosper and Mazo</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Cardiovascular disease is the number one killer worldwide, with myocardial infarction (MI) responsible for approximately 1 in 6 deaths. The lack of endogenous regenerative capacity, added to the deleterious remodelling programme set into motion by myocardial necrosis, turns MI into a progressively debilitating disease, which current pharmacological therapy cannot halt. The advent of Regenerative Therapies over 2 decades ago kick-started a whole new scientific field whose aim was to prevent or even reverse the pathological processes of MI. As a highly dynamic organ, the heart displays a tight association between 3D structure and function, with the non-cellular components, mainly the cardiac extracellular matrix (ECM), playing both fundamental active and passive roles. Tissue engineering aims to reproduce this tissue architecture and function in order to fabricate replicas able to mimic or even substitute damaged organs. Recent advances in cell reprogramming and refinement of methods for additive manufacturing have played a critical role in the development of clinically relevant engineered cardiovascular tissues. This review focuses on the generation of human cardiac tissues for therapy, paying special attention to human pluripotent stem cells and their derivatives. We provide a perspective on progress in regenerative medicine from the early stages of cell therapy to the present day, as well as an overview of cellular processes, materials and fabrication strategies currently under investigation. Finally, we summarise current clinical applications and reflect on the most urgent needs and gaps to be filled for efficient translation to the clinical arena.</p></abstract><kwd-group><kwd>cardiac tissue engineering</kwd><kwd>human pluripotent stem cells</kwd><kwd>material properties</kwd><kwd>cell differentiation</kwd><kwd>fabrication strategies</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Instituto de Salud Carlos III<named-content content-type=\"fundref-id\">10.13039/501100004587</named-content></funding-source><award-id rid=\"cn001\">Red TERCEL RETIC RD16/0011/0005</award-id><award-id rid=\"cn001\">PI 19/01350</award-id><award-id rid=\"cn001\">Nanoreheart</award-id></award-group><award-group><funding-source id=\"cn002\">Ministerio de Economía, Industria y Competitividad, Gobierno de España<named-content content-type=\"fundref-id\">10.13039/501100010198</named-content></funding-source><award-id rid=\"cn002\">Program RETOS Cardiomesh RTC-2016-4911-1</award-id></award-group><award-group><funding-source id=\"cn003\">Departamento de Educación, Gobierno de Navarra<named-content content-type=\"fundref-id\">10.13039/501100003425</named-content></funding-source><award-id rid=\"cn003\">0011-1383-2019-000006</award-id><award-id rid=\"cn003\">0011-1383-2018-000011</award-id></award-group><award-group><funding-source id=\"cn004\">European Commission<named-content content-type=\"fundref-id\">10.13039/501100000780</named-content></funding-source><award-id rid=\"cn004\">874827</award-id></award-group></funding-group><counts><fig-count count=\"3\"/><table-count count=\"3\"/><equation-count count=\"0\"/><ref-count count=\"323\"/><page-count count=\"27\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>A Perspective on Cardiac Disease and Regenerative Medicine</title><p>Organ transplantation is one of the greatest medical achievements of the 20th century. However, its applicability is hampered by donor shortage, life-long immunosuppression and its success rates are linked to the experience of the surgical team. It requires a well-coordinated national effort, which is sometimes hindered by ethical issues (<xref rid=\"B221\" ref-type=\"bibr\">Prabhu, 2019</xref>). The search for novel ways to approach organ repair inspired the field of regenerative medicine, with Stem Cell Therapy as one of the most representative examples. Since this began, stem cells have been discovered even in low turnover adult tissues, such as the central nervous system (<xref rid=\"B59\" ref-type=\"bibr\">Doetsch et al., 1999</xref>), the lung, (<xref rid=\"B230\" ref-type=\"bibr\">Rock et al., 2009</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Barkauskas et al., 2013</xref>) or the heart, (<xref rid=\"B22\" ref-type=\"bibr\">Beltrami et al., 2003</xref>) and have been widely assayed in animal models of disease, quickly reaching clinical trials. This swift progression in general met with rapid failure, but on the bright side, it also enabled specialists to gain immense insights into their mechanisms and ways of action.</p><p>Nothing exemplifies this journey better than the cardiac field. Cardiovascular diseases are well recognised as the leading cause of death worldwide, accounting for almost 1 in 2 deaths in Europe and causing 3.9 million deaths per year (<xref rid=\"B268\" ref-type=\"bibr\">Townsend et al., 2016</xref>). Ischemic heart disease (IHD) is one shade on this spectrum. It is generally caused by the clotting of a coronary vessel, which in turn leads to the death of a portion of the myocardium and the subsequent functional impairment of the organ. Being mostly non-regenerative, the heart is chronically impaired (<xref rid=\"B69\" ref-type=\"bibr\">Eschenhagen et al., 2017</xref>). A real epidemic, IHD is the leading single cause of death globally, responsible for over 15 million deaths in 2016, ranking first in high- and lower-middle-income countries, and third in low-income countries (<xref rid=\"B296\" ref-type=\"bibr\">World Health Organization [WHO], 2018</xref>). As per 2015, over 22 million EU citizens were living with the disease, with approximately 3 million new cases yearly. IHD imposes an enormous burden on society as affected patients must be cared for by health systems, requiring lifelong highly specialised medical attention and multimedication. It also jeopardises the structure of the workforce and puts significant pressure on families. In terms of economic cost, the total burden of IHD for EU economies is estimated at €59 billion/year. Of these, €19 billion is directly related to healthcare costs, while €20 billion is linked to productivity losses and the remaining €20 billion to the cost of indirect care. Estimations sketch out a grim future. In the United States, heart attacks are projected to contribute more than $818 billion to annual healthcare costs and lost productivity by 2030, (<xref rid=\"B199\" ref-type=\"bibr\">Nowbar et al., 2019</xref>) while in the South Asia region, direct medical costs for CVD are estimated to reach US $16.6 billion in 2021 (<xref rid=\"B282\" ref-type=\"bibr\">Walker et al., 2018</xref>). Organ transplant cannot match this overwhelming demand and become a widespread therapeutic option (<xref rid=\"B254\" ref-type=\"bibr\">Stehlik et al., 2011</xref>).</p><p>It was hoped that cell therapy would provide new means to regenerate the scarred myocardium. Remarkable discoveries encouraged rapid progression towards clinical trials, with the first one launched in record time (<xref rid=\"B21\" ref-type=\"bibr\">Behfar et al., 2014</xref>). After the BOOST and REPAIR-AMI trials reported significant benefits in cardiac function, (<xref rid=\"B293\" ref-type=\"bibr\">Wollert et al., 2004</xref>; <xref rid=\"B238\" ref-type=\"bibr\">Schächinger et al., 2006</xref>) hundreds of patients were recruited often in individual efforts mostly based on local experience. This led to the first voicing of concerns. The primary initial objective, improvement of cardiac function, failed to be met in many cases. Although statistically significant benefits in cardiac function were sometimes found, their magnitude was not as great as had been expected (reviewed in <xref rid=\"B93\" ref-type=\"bibr\">Gyöngyösi et al., 2016</xref>; <xref rid=\"B178\" ref-type=\"bibr\">Menasché, 2018</xref>). This reversal of fortunes coincided also with a remarkable twist in our understanding of what basic science and animal models conveyed: no true regeneration of the myocardium was achieved by adult stem cells. The underlying effects were mostly due to the paracrine secretion of beneficial molecules (<xref rid=\"B106\" ref-type=\"bibr\">Hodgkinson et al., 2016</xref>). Although this general perspective is valid for most adult stem cells, use of their embryonic counterpart, although boosted by their capacity to give rise to new tissue once transplanted, was still marred by some common issues such as lack of proper engraftment, and crucially by safety concerns as teratoma formation and need for immune suppression, as well as ethical issues.</p><p>Did stem cell regenerative medicine approaches fail? They obviously did not achieve the initial aims, but looking back, those can no doubt be branded as overambitious (<xref rid=\"B209\" ref-type=\"bibr\">Pagano et al., 2019</xref>). It did succeed in gathering a whole new compendium of knowledge, which has led to renewed and better efforts in the regenerative direction and, importantly, has built a worldwide network of excellence encompassing not only different nationalities, but also very diverse scientific disciplines. One of the greatest and perhaps now evident realisations in the field is the notion that cells are not alone in a tissue, and that the extracellular matrix (ECM) has a predominant role, not only as a passive architectural element, but crucially as a signal transducer and determinant of functionality (<xref rid=\"B166\" ref-type=\"bibr\">Majkut et al., 2013</xref>; <xref rid=\"B52\" ref-type=\"bibr\">Crowder et al., 2016</xref>; <xref rid=\"B148\" ref-type=\"bibr\">Kumar et al., 2017</xref>). Specifically in the myocardium, the ECM is highly dynamic, changing during development and disease. This latter change is bidirectional, as disease induces pathological ECM deposition but an abnormal matrix is able to produce malfunction (<xref rid=\"B79\" ref-type=\"bibr\">Frangogiannis, 2019</xref>). In consequence, it is now recognised that a cell-based cardiac regeneration without an adequate ECM is not viable. Generating new myocardium thus requires the participation of the most promising cells, with a surrounding matrix able to replicate the conditions of the native tissue and the proper 3D architecture. This is precisely one of the main directions of cardiac Tissue Engineering.</p><p>Cardiac Tissue Engineering (cTE) is a highly interdisciplinary scientific discipline, aiming at reproducing as accurately as possible the function and biology of cardiac muscle, during development or maturity, health or disease. Although its first objective was focused on meeting the needs of cardiac regenerative Medicine, as knowledge and experience on how the ECM influences cardiac cell biology increased and the fabrication capacities widened, its scope has greatly expanded into areas such as disease modelling, drug testing and personalised medicine amongst others (<xref rid=\"B73\" ref-type=\"bibr\">Feric et al., 2019</xref>; <xref rid=\"B198\" ref-type=\"bibr\">Noor et al., 2019</xref>; <xref rid=\"B171\" ref-type=\"bibr\">Mastikhina et al., 2020</xref>). This review aims at presenting the reader with an overview of the specific characteristics of the myocardium that determine the needs regenerative cTE has to meet, as well as providing a non-exhaustive revision of what the field has delivered to attain this end, with a focus on human myocardium and human pluripotent stem cells.</p></sec><sec id=\"S2\"><title>The Heart</title><p>The mammalian heart is an incredible organ. Its main role is to provide a continuous unidirectional supply of blood to the organism. This comes at a stringent metabolic cost, consuming the equivalent of 6 kg of ATP per day, with a complete renewal of its ATP pool every 10 seconds. Most of this energy is obtained through the oxidation of fatty acids in adulthood, though cardiac metabolism is dependent on glucose during embryonic stages, being able to employ lactate as a metabolic substrate (<xref rid=\"B196\" ref-type=\"bibr\">Neejy, 1974</xref>). Correct function is achieved through a specialised organ architecture, dividing the heart into 4 chambers: atria, which are smaller in size and muscular mass, receive blood and push it out into the ventricles, which in turn pump either towards the lungs or the body. In consequence, the left ventricle is larger and has a thicker muscular wall than its right counterpart. Chambers, inlets and outlets are separated by valves impeding back flow. The heart is the first organ to function, around day 8 in mice and the 4<sup>th</sup> gestational week in humans (<xref rid=\"B30\" ref-type=\"bibr\">Brand, 2003</xref>). It pumps continuously throughout life, efficiently ejecting blood through an exquisite 3D structure, (<xref rid=\"B34\" ref-type=\"bibr\">Buckberg, 2002</xref>) established by a complex set of embryonic movements, cellular growth and incorporation (<xref rid=\"B91\" ref-type=\"bibr\">Günthel et al., 2018</xref>). Disruption of this structure is seen in disease and can be in itself the cause of organ malfunction: cardiac congenital defects and malformation are a main cause of perinatal death, (<xref rid=\"B31\" ref-type=\"bibr\">Bressan et al., 2013</xref>) but also give rise to many cardiomyopathies (<xref rid=\"B174\" ref-type=\"bibr\">McKenna et al., 2017</xref>).</p><sec id=\"S2.SS1\"><title>Cardiac Embryonic Development: A Brief Overview</title><p>The formation of the mammalian four-chambered heart encompasses a series of tightly coordinated morphological, cellular and molecular events (reviewed in <xref rid=\"B277\" ref-type=\"bibr\">Vincent and Buckingham, 2010</xref>; <xref rid=\"B176\" ref-type=\"bibr\">Meilhac et al., 2014</xref>; <xref rid=\"B234\" ref-type=\"bibr\">Ruiz-Villalba et al., 2016</xref>). Different pools of cardiac and extracardiac progenitors are involved, including the mesoderm-derived First, Second and Third Heart Fields (FHF, SHF and THF respectively) and the Cardiac Neural Crest Cells (CNCCs). Cells of the FHF contribute primarily to the left ventricle (LV) but there is also a small contribution to the atria; SHF will form the right ventricle (RV), outflow tract (OFT), atria and part of inflow tract (IFT); (<xref rid=\"B37\" ref-type=\"bibr\">Buckingham et al., 2005</xref>). THF cells contribute to the sinus node, some regions in the caval myocardium, and the Pro-Epicardial Organ (PEO) (<xref rid=\"B188\" ref-type=\"bibr\">Mommersteeg et al., 2010</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Bressan et al., 2013</xref>). CNCCs arise from the dorsal neural tube, contribute to the parasympathetic innervation of the heart, valves and play a pivotal role in OFT patterning and optimal septation (<xref rid=\"B135\" ref-type=\"bibr\">Keyte et al., 2014</xref>).</p><p>Early precardiac progenitors from the lateral mesoderm have been mapped into the mid-anterior region of the primitive streak, characterised by the presence of both anterior Nodal/Activin and posterior bone morphogenetic protein (BMP) signalling at low levels, (<xref rid=\"B314\" ref-type=\"bibr\">Zhang et al., 2008</xref>; <xref rid=\"B275\" ref-type=\"bibr\">Vallier et al., 2009</xref>; <xref rid=\"B299\" ref-type=\"bibr\">Yamauchi et al., 2010</xref>; <xref rid=\"B305\" ref-type=\"bibr\">Yu et al., 2011</xref>) promoting the emergence of cardiogenic mesodermal MIXL1 + KDR + cells. As a result of these signalling gradients set in gastrulation, multipotent cardiovascular progenitor (M) expressing the cardiac master regulator MESP1 move in an anterior-lateral direction, forming a horseshoe-like region termed the cardiac crescent or FHF (<xref rid=\"B29\" ref-type=\"bibr\">Bondue et al., 2008</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Chan S. S. K. et al., 2013</xref>). At a molecular level, MESP1 induces the expression of the minimal core of the essential cardiogenic transcription factors including ISL1, TBX5, NKX2.5, and GATA4, in combination with the chromatin remodeller SMARCD3 (BAF60C), which further drive cardiomyogenesis (<xref rid=\"B277\" ref-type=\"bibr\">Vincent and Buckingham, 2010</xref>; <xref rid=\"B176\" ref-type=\"bibr\">Meilhac et al., 2014</xref>; <xref rid=\"B175\" ref-type=\"bibr\">Meilhac and Buckingham, 2018</xref>). The cardiac crescent fuses at the midline, forming the linear heart tube, which consists of an interior layer of endocardial cells and an exterior layer of myocardial cells separated by an acellular, ECM-rich space, the cardiac jelly. Located central and dorsal to the FHF, SHF cells remain in contact with the pharyngeal endoderm and in a proliferative state as undifferentiated ISL1 + MEF2C + cells. As development proceeds, SHF cells are added to the poles of the heart tube, with the tube looping to position the different regions into place. Chambers balloon out as a result of the differential proliferation rates of CMs (<xref rid=\"B123\" ref-type=\"bibr\">Jong et al., 1997</xref>; <xref rid=\"B50\" ref-type=\"bibr\">Christoffels et al., 2004</xref>). As already mentioned, THF cells (TBX18 + NKX2.5-) contribute to the sinus node, caval myocardial cells and the PEO. PEO-cells give rise to the epicardium, and some cells of this layer undergo epithelial-to-mesenchymal transition to form epicardial derived cells (EPDC), which will differentiate into vascular cells (including the coronaries) as well as interstitial fibroblasts and valvular cells, being essential for compaction (<xref rid=\"B217\" ref-type=\"bibr\">Pérez-Pomares et al., 2002</xref>; <xref rid=\"B288\" ref-type=\"bibr\">Weeke-Klimp et al., 2010</xref>; <xref rid=\"B131\" ref-type=\"bibr\">Katz et al., 2012</xref>). Lastly, CNCCs originate by delamination from the neuroectoderm, (<xref rid=\"B102\" ref-type=\"bibr\">Hildreth et al., 2008</xref>) initially contributing to smooth muscle cells and CMs, (<xref rid=\"B185\" ref-type=\"bibr\">Mjaatvedt et al., 2001</xref>) and making a significant contribution to the innervation of the organ and to the OFT (<xref rid=\"B102\" ref-type=\"bibr\">Hildreth et al., 2008</xref>; <xref rid=\"B249\" ref-type=\"bibr\">Sizarov et al., 2012</xref>). We refer the reader to <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> for a full description of the mentioned gene abbreviations.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Full description of genes names.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Abbreviation</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Description</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MIXL1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mix Paired-Like Homeobox</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">KDR</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Kinase Insert Domain Receptor</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MESP1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mesoderm Posterior BHLH Transcription Factor 1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ISL1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Islet-1 LIM Homeobox</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TBX5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">T-Box Transcription Factor 5</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NKX2.5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NK2 homeobox 5</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GATA4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GATA Binding Protein 4</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SMARCD3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily D, Member 3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BX18</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">T-Box Transcription Factor 18</td></tr></tbody></table></table-wrap></sec><sec id=\"S2.SS2\"><title>Post-birth Cardiac Development: Foetal CMs vs. Adult CMs</title><p>Aside from the formation of the mammalian heart, CMs continue to develop postnatally (<xref rid=\"B92\" ref-type=\"bibr\">Guo and Pu, 2020</xref>). Embryonic CMs can beat spontaneously, express sarcomeric proteins and ion channels, and exhibit action potentials and calcium transients which are significantly distinctive from their adult counterpart (<xref rid=\"B277\" ref-type=\"bibr\">Vincent and Buckingham, 2010</xref>; <xref rid=\"B176\" ref-type=\"bibr\">Meilhac et al., 2014</xref>). Human and rodent embryonic CMs are around 30-40 fold less in size and feature an irregular shape, in comparison with adult CMs (<xref rid=\"B301\" ref-type=\"bibr\">Yang et al., 2014</xref>). These are characterised by an ultrastructural organisation with a large mitochondrial volume and specific mitochondria positioning between myofibrils. Sarcomeres in postnatal CMs are long and well-aligned, in contrast to shorter and disarrayed ones found in foetal CM. At a metabolic level, embryonic CMs rely on glycolysis, whereas adult myocytes preferentially consume fatty acids, a much more efficient energy source. Myofibrillar protein isoform undergoes switching, being myosin heavy chain 7 (MYH7), myosin light chain 2 ventricular isoform (MLC2v), cardiac troponin I3 (TNNI3) and a shorter and stiffer Titin isoform, preferentially expressed in adult CMs, in contrast to myosin heavy chain 6 (MYH6), myosin light chain 2 atrial isoform (MLC2a), and slow skeletal-type troponin I1 (TNNI1) on foetal CMs (<xref rid=\"B20\" ref-type=\"bibr\">Bedada et al., 2014</xref>). All these differences directly correlate with contractile capacity, with adult CMs able to generate more force than embryonic ones (<xref rid=\"B277\" ref-type=\"bibr\">Vincent and Buckingham, 2010</xref>; <xref rid=\"B176\" ref-type=\"bibr\">Meilhac et al., 2014</xref>; <xref rid=\"B261\" ref-type=\"bibr\">Tan and Ye, 2018</xref>). For example, strips of adult rat myocardium have been reported to produce a peak twitch tension of 56.4 ± 44 mN/mm2, (<xref rid=\"B97\" ref-type=\"bibr\">Hasenfuss et al., 1991</xref>) whereas collagen constructs with neonatal rat CMs generated 0.4-0.8 mN/mm2 (<xref rid=\"B322\" ref-type=\"bibr\">Zimmermann et al., 2002</xref>). The same difference in magnitude is believed to exist for human cells, as comparisons with primary foetal human CMs are rare (<xref rid=\"B301\" ref-type=\"bibr\">Yang et al., 2014</xref>).</p><p>The cardiac action potential and associated channels and currents also distinguishes adult and foetal CMs. In immature CMs, the expression of channels involved in repolarisation, including potassium transient outward channels, L-type calcium currents and the rectifying K + current (encoded mainly by KCNJ2), is lower than in adult cells resulting in a less negative resting membrane potential (−50mv ∼−60 mv in embryonic CMs) compared to normal (–85mv ∼ –90 mv in adult CMs) (<xref rid=\"B311\" ref-type=\"bibr\">Zhang et al., 2009</xref>). Also, the pacemaker current I<sub>f</sub> is present in embryonic CMs but does not occur in adult myocytes (<xref rid=\"B235\" ref-type=\"bibr\">Sartiani et al., 2007</xref>). The distribution of the gap junction protein connexin 43 (Cx43) also plays an important role in regulating electrical activity. While Cx43 concentrates at the intercalated disc of adult CMs, it is circumferentially distributed in immature CMs, which is not optimal for longitudinal electrical propagation (<xref rid=\"B279\" ref-type=\"bibr\">Vreeker et al., 2014</xref>; <xref rid=\"B121\" ref-type=\"bibr\">Jiang et al., 2018</xref>). Adult CMs have a well-developed sarcoplasmic reticulum (SR) with a high level of SR-specific proteins like ryanodine receptor 2 (RYR2) and sarcoplasmic/endoplasmic reticulum Ca2 + ATPase 2a (SERCA2), (<xref rid=\"B111\" ref-type=\"bibr\">Ivashchenko et al., 2013</xref>) which, coupled with the presence of transverse tubules (t-tubules), leads to a highly coordinated Ca-induced-Ca-release and hence faster Ca transient kinetics and amplitude when compared to foetal CMs (<xref rid=\"B160\" ref-type=\"bibr\">Louch et al., 2015</xref>). Finally, where embryonic CMs are diploid, adult CMs present different degrees of polyploidy, achieved through DNA-synthesis without karyokinesis (<xref rid=\"B2\" ref-type=\"bibr\">Adler and Costabel, 1975</xref>; <xref rid=\"B99\" ref-type=\"bibr\">Herget et al., 1997</xref>). Understanding how an embryonic CM evolves into a mature cell is already proving fundamental in human cTE. As the bioartificial tissues developed so far resemble more their foetal counterpart, this insight is being incorporated into the effort of driving engineered tissues towards an adult-like functionality (<xref rid=\"B128\" ref-type=\"bibr\">Karbassi et al., 2020</xref>).</p></sec><sec id=\"S2.SS3\"><title>Heart Characteristics: What We Aim to Engineer</title><p>Generating human myocardial surrogates in the laboratory requires knowing what the natural composition and properties of the organ are. The following paragraphs provide an overview of what nature has achieved, specifically, what the main cellular and extracellular components of the heart are, how they are arranged in space, and importantly, what this means regarding the resulting material properties (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Main components of the mammalian heart. The two main constituents of the myocardium, cardiac cells and the surrounding ECM, both contribute to and modulate the specific material properties of the tissue.</p></caption><graphic xlink:href=\"fbioe-08-00955-g001\"/></fig><sec id=\"S2.SS3.SSS1\"><title>Cellular Composition</title><p>As already explained, most cells forming the structure of the heart are of mesodermal origin: CMs, vascular (endothelial and smooth muscle) cells and fibroblasts. Others reside in the tissue but are formed elsewhere, as immune cells, which play a significant role in organ surveillance and disease. Deciphering the cellular composition of the heart has been a very controversial subject, be it in rodents or humans (<xref rid=\"B318\" ref-type=\"bibr\">Zhou and Pu, 2016</xref>). Histology can determine that CMs are the largest fraction by volume. However, numbers vary, with reports of murine myocytes being the largest population by number [56/27/7 for CMs/fibroblasts/endothelial cells respectively (<xref rid=\"B11\" ref-type=\"bibr\">Banerjee et al., 2007</xref>)] and others attributing greater numbers to endothelial cells [43.6% vs 31% of CMs (<xref rid=\"B218\" ref-type=\"bibr\">Pinto et al., 2016</xref>)]. Human proportions are similarly contradictory, with some publications showing non-CM/endothelial cells are the most abundant (<xref rid=\"B24\" ref-type=\"bibr\">Bergmann et al., 2015</xref>) and others endothelial cells (<xref rid=\"B6\" ref-type=\"bibr\">Anversa et al., 1978</xref>). Furthermore, several studies have reported varying cell proportions throughout the anatomical regions of the organ (<xref rid=\"B257\" ref-type=\"bibr\">Sussman et al., 2002</xref>; <xref rid=\"B83\" ref-type=\"bibr\">Gaudesius et al., 2003</xref>; <xref rid=\"B44\" ref-type=\"bibr\">Camelliti et al., 2004</xref>, <xref rid=\"B43\" ref-type=\"bibr\">2005</xref>; <xref rid=\"B145\" ref-type=\"bibr\">Kohl, 2004</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Baudino et al., 2006</xref>). Things become more complicated if we take into account the age of the individual, as some claim the final number of myocytes is reached by one month, remaining constant over the lifetime of the individual (<xref rid=\"B24\" ref-type=\"bibr\">Bergmann et al., 2015</xref>) whereas others have reported a 3.4-fold increase in CM number between 1 and 20 years of age (<xref rid=\"B187\" ref-type=\"bibr\">Mollova et al., 2013</xref>). Other cell type numbers change dynamically over time, with a reported 6.5-fold increase in endothelial cells and an 8.2-fold increase for mesenchymal cells (including fibroblasts) during heart growth. Interactions between these cells are multidirectional and exert great influence over crucial aspects of cardiac biology. Both endothelial cells and fibroblasts are key for tissue function and homeostasis. Aside from delivering oxygen and nutrients to the metabolically demanding CMs, the endothelium is fundamental for tissue hypertrophy and post-disease remodelling, (<xref rid=\"B108\" ref-type=\"bibr\">Holopainen et al., 2015</xref>) and maturation, (<xref rid=\"B86\" ref-type=\"bibr\">Giacomelli et al., 2020</xref>) displaying a strong paracrine influence (reviewed in <xref rid=\"B154\" ref-type=\"bibr\">Leucker and Jones, 2014</xref>). Fibroblasts also affect organ function and cell maturity, (<xref rid=\"B294\" ref-type=\"bibr\">Woodall et al., 2016</xref>; <xref rid=\"B283\" ref-type=\"bibr\">Wang Y. et al., 2020</xref>) whilst other cell types, such as immune cells, have been reported to display direct and significant interactions with CMs, as is the case with the electrical coupling of macrophages with atrioventricular node cells (<xref rid=\"B110\" ref-type=\"bibr\">Hulsmans et al., 2017</xref>). All in all, the general consensus supported by unambiguous histological evidence is that CMs are the largest fraction by volume, each nurtured by a median of 3 capillaries, where fibroblasts constantly keep the ECM through a degradation-deposition equilibrium. This brings us to our next player: the cardiac ECM.</p></sec><sec id=\"S2.SS3.SSS2\"><title>ECM Composition</title><p>The cardiac cell types discussed above are arranged within a glycoprotein matrix which supports and provides them with a structure. Moreover, the cardiac ECM also has an active role in transmitting contraction and avoiding hyper-stretching of CMs. Its principal component is collagen, which accounts for 2–5% of the total weight of the heart, mainly types I (89%) and III (11%). Collagen type IV is present in the basement membranes, and collagen type V is located in the pericellular space (<xref rid=\"B286\" ref-type=\"bibr\">Weber, 1989</xref>; <xref rid=\"B66\" ref-type=\"bibr\">Eghbali and Weber, 1990</xref>; <xref rid=\"B251\" ref-type=\"bibr\">Sommer et al., 2015a</xref>). The collagen matrix has classically been categorised depending on which elements it tethers together into endomysium (binds adjoining CMs), perimysium (aggregates myocytes into myofibrils) and epimysium (present at the epicardial and endocardial surfaces). Cardiac fibroblasts have been identified as the main cell type responsible for secreting and remodelling the collagen matrix, although CMs seem to contribute to collagen type IV deposition (<xref rid=\"B65\" ref-type=\"bibr\">Eghbali et al., 1988</xref>). Apart from the structural function, the collagen network makes an important contribution to the whole myocardial tensile properties (<xref rid=\"B77\" ref-type=\"bibr\">Fomovsky et al., 2010</xref>).</p><p>Another key element of the cardiac extracellular matrix is elastic fibres. These are composites, made of an elastin core surrounded by a myriad of microfibrils. They provide elastic properties, by stretching upon mechanical demand and going back to their original length once the load is removed. Hence the importance of elastic fibres in tissues which have to accommodate their structure, such as skin, arteries or lungs or the heart. However, although elastic fibres are paramount for the heart’s elasticity, other factors are known to have an influence upon it, namely the proportion of muscle bundles to fibrotic tissue, and the density of collagen crosslinking (<xref rid=\"B78\" ref-type=\"bibr\">Forrest and Jackson, 1971</xref>; <xref rid=\"B213\" ref-type=\"bibr\">Parmley et al., 1973</xref>). In fact, elastic fibres are found in most cases close to the collagenous network and in intimate association with it (<xref rid=\"B236\" ref-type=\"bibr\">Sato et al., 1983</xref>). Of note, mature elastic fibres show slight architectural differences depending on the tissue (<xref rid=\"B139\" ref-type=\"bibr\">Kielty et al., 2002</xref>). As aforementioned, elastin forms the core of elastic fibres. Unlike most matrix proteins, which undergo a constant/continuous deposition and turnover, in healthy conditions elastin is synthesised only until adolescence (<xref rid=\"B63\" ref-type=\"bibr\">Dubick et al., 1981</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Burnett et al., 1982</xref>; <xref rid=\"B57\" ref-type=\"bibr\">Davidson et al., 1982</xref>; <xref rid=\"B193\" ref-type=\"bibr\">Myers et al., 1985</xref>; <xref rid=\"B241\" ref-type=\"bibr\">Sephel et al., 1987</xref>; <xref rid=\"B212\" ref-type=\"bibr\">Parks et al., 1988</xref>; <xref rid=\"B219\" ref-type=\"bibr\">Pollock et al., 1990</xref>; <xref rid=\"B227\" ref-type=\"bibr\">Ritz-Timme et al., 2003</xref>). Other fundamental components of the cardiac matrix include, to a lesser extent, laminin, fibronectin, proteoglycans and glycoproteins (<xref rid=\"B71\" ref-type=\"bibr\">Fan et al., 2012</xref>). Laminin molecules are part of the basement membrane and are thus in close contact with the cell, playing an active role in modulating cell behaviour, including migration, differentiation and phenotype stabilisation (<xref rid=\"B302\" ref-type=\"bibr\">Yap et al., 2019</xref>). Fibronectin, besides promoting cell attachment, acts as an ECM organiser and is involved in collagen deposition (<xref rid=\"B274\" ref-type=\"bibr\">Valiente-Alandi et al., 2018</xref>). However, all components are crucial for tissue integrity and function.</p><p>Many, if not all, cardiovascular diseases have repercussions for the cardiac ECM. The reverse is also true. For instance, infarction studies in pigs show that the collagenous network starts to become disarranged after just 20 min of coronary occlusion, whilst elastin begins to disappear after 40 min, and both components appear detached from the basement membrane after 120 min. The balance of collagens I and III has been widely studied, revealing a significant increase in type III collagen after myocardial infarction (MI) (<xref rid=\"B236\" ref-type=\"bibr\">Sato et al., 1983</xref>). Dilated cardiomyopathies, in which the shape of the cardiac cavity is abnormal, are at least partly related to aberrant collagen remodelling, with less thick collagen and thinner fibres, which results in weaker tensile properties, more muscle slippage and wall thinning (<xref rid=\"B287\" ref-type=\"bibr\">Weber et al., 1988</xref>; <xref rid=\"B286\" ref-type=\"bibr\">Weber, 1989</xref>). Ventricular hypertrophy consists of the thickening of the ventricular wall associated with some conditions like hypertension, and it is found together with overexpression of collagen in the form of interstitial fibrosis (<xref rid=\"B66\" ref-type=\"bibr\">Eghbali and Weber, 1990</xref>).</p></sec><sec id=\"S2.SS3.SSS3\"><title>Cardiac Architecture</title><p>In most tissues, structure and function are closely intertwined, and the heart is no exception. However, certain aspects of this relationship are still under debate. The overall manner in which the heart contracts and pumps blood is known, as is the arrangement of the tissue microstructure. The gap lies in providing a theory that explains how the different architectural elements interact to produce the global behaviour. For example, there is an ongoing debate about whether the myocardium forms a single myocardial band, (<xref rid=\"B35\" ref-type=\"bibr\">Buckberg et al., 2015a</xref>, <xref rid=\"B36\" ref-type=\"bibr\">b</xref>) or the so-called myocardial mesh model is more accurate (<xref rid=\"B162\" ref-type=\"bibr\">MacIver et al., 2018a</xref>, <xref rid=\"B163\" ref-type=\"bibr\">b</xref>). The controversy has two aspects. On the one hand, there is no consensus on whether the basic functional unit is the CM, or groupings of this into bundles (groups of CMs), sheetlets (groups of bundles), sheets (groups of sheetlets) or even laminae (groups of sheets). On the other hand, although imaging techniques allow us to visualise phenomena across the whole myocardium, it is not feasible to ascertain the distinct contribution of the individual functional units to producing the global outcome. According to Buckberg et al., the CM can undergo six functional events: shortening, lengthening, narrowing, widening, twisting and uncoiling (<xref rid=\"B35\" ref-type=\"bibr\">Buckberg et al., 2015a</xref>, <xref rid=\"B33\" ref-type=\"bibr\">2018</xref>). There are an estimated 2.5-10 billion cells (<xref rid=\"B24\" ref-type=\"bibr\">Bergmann et al., 2015</xref>) in the heart, each of them performing one or more of these six actions in the same or a different direction, and all we are able to see is the macroscopic effect: a torsion-contraction movement of the organ. Still under controversy, there are at least 7 proposed models to accurately describe cardiac architecture, (<xref rid=\"B88\" ref-type=\"bibr\">Gilbert et al., 2007</xref>) which is widely recognised to have a profound effect, whether at a mechanical (<xref rid=\"B153\" ref-type=\"bibr\">LeGrice et al., 1995</xref>; <xref rid=\"B323\" ref-type=\"bibr\">Zócalo et al., 2008</xref>) or electrical (<xref rid=\"B228\" ref-type=\"bibr\">Roberts et al., 1979</xref>; <xref rid=\"B258\" ref-type=\"bibr\">Taccardi et al., 2008</xref>) level. During disease, myocardial architecture is severely disarranged, leading to inefficient contraction (<xref rid=\"B229\" ref-type=\"bibr\">Roberts et al., 1987</xref>; <xref rid=\"B291\" ref-type=\"bibr\">Wickline et al., 1992</xref>). It is expected that the application of advanced technology like diffusion tensor MRI (DT-MRI), which can obtain highly detailed information on fibre architecture, will soon shed light on this debate (<xref rid=\"B239\" ref-type=\"bibr\">Scollan et al., 1998</xref>; <xref rid=\"B220\" ref-type=\"bibr\">Poveda et al., 2013</xref>).</p><p>At a simpler histological level, CMs (and CM bundles/sheetlets) are arranged in different orientations depending on their location in the organ, which in turn determines the direction of the stress produced. Myocytes are always in intimate contact with capillaries, which no doubt stems from the high metabolic demand of an ever-working muscular tissue: capillaries are located within 20 μm of CMs. Each CM is surrounded by a basement membrane containing laminin and collagen type IV, amongst others, and embedded in a highly structured ECM where collagen type I, as already discussed, is the main component (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). CMs connect to each other mainly by intercalated disks at their ends, but also through side branches, coupled to at least 2 CMs on the long axis and 1 laterally (<xref rid=\"B253\" ref-type=\"bibr\">Spach and Heidlage, 1995</xref>). Intercalated disks contain gap junctions, allowing fast current flow between neighbouring cells (<xref rid=\"B143\" ref-type=\"bibr\">Klabunde, 2012</xref>). As mentioned above, both individual CMs and groupings of these are surrounded by enveloping collagen. Fibroblasts do not participate in the electrical syncytium formed by the CMs, but rather lie in the interstitial space.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Cardiac structure. The endocardial-to-pericardial structure is outlined, with the main cellular and extracellular components.</p></caption><graphic xlink:href=\"fbioe-08-00955-g002\"/></fig></sec><sec id=\"S2.SS3.SSS4\"><title>Cardiac Biophysical Properties</title><p>When attempting to engineer a tissue, it is essential to carefully recapitulate not only the cellular-extracellular components and their architecture, but also the resulting biophysical properties. These must reliably mimic those of their natural counterpart. In the heart, material properties are very complex: not only are they direction-dependent, but they also vary within the anatomical regions and the stage of the cardiac cycle. As an example, the literature reports variations in stiffness between the beginning and the end of diastole higher than an order of magnitude. For human LV, the reported values are 10-20 kPa at the beginning of the diastole, and 200-500 kPa at the end (<xref rid=\"B49\" ref-type=\"bibr\">Chen et al., 2008</xref>). Contraction itself results in a significant stiffening: from 0.5 to > 10 kPa. This magnitude is species-dependent, with a reported 3-fold increase in mouse, (<xref rid=\"B116\" ref-type=\"bibr\">Jacot et al., 2010</xref>) 2-fold in rat, (<xref rid=\"B222\" ref-type=\"bibr\">Prakash et al., 1999</xref>) and over 20 times in zebrafish (<xref rid=\"B147\" ref-type=\"bibr\">Krieg et al., 2008</xref>). Development also leads to a stiffening in the tissue, which arises from a relatively soft mesodermal layer (<xref rid=\"B147\" ref-type=\"bibr\">Krieg et al., 2008</xref>). Disease severely stiffens the organ, mostly due to the excessive deposition of collagen (scar for MI, interstitial in hypertension or other conditions), with values of over 50-100 kPa (<xref rid=\"B68\" ref-type=\"bibr\">Engler et al., 2008</xref>). Stiffness itself has a fundamental influence on how efficient CM contraction is, with CMs on softer- or stiffer-than-normal substrates doing little work or overstraining themselves, respectively (<xref rid=\"B68\" ref-type=\"bibr\">Engler et al., 2008</xref>). Reports on cardiac mechanical properties are extremely variable. This stems from a mix-up of animal vs human, fresh vs fixed, and healthy vs diseased data. In general, it is now accepted that most of the heart’s passive mechanical properties are due to the collagen in the matrix, (<xref rid=\"B251\" ref-type=\"bibr\">Sommer et al., 2015a</xref>) but at short sarcomere lengths the protein titin is the predominant contributor (<xref rid=\"B197\" ref-type=\"bibr\">Nguyen-Truong and Wang, 2018</xref>). Quoting Sommer et al., ‘results suggest that the passive human LV myocardium under quasi-static and dynamic multiaxial loadings is a non-linear, anisotropic (orthotropic), viscoelastic and history-dependent soft biological material undergoing large deformations’ (<xref rid=\"B252\" ref-type=\"bibr\">Sommer et al., 2015b</xref>). Or in simpler words: it is very complex and with multiple contributions from cellular/extracellular components. Added to this, scales differ, depending whether the tissue is macroscopically characterised using biaxial mechanical tests, (<xref rid=\"B252\" ref-type=\"bibr\">Sommer et al., 2015b</xref>) or whether isolated CMs are probed at a cell-relevant scale with Atomic Force Microscopy (AFM) (<xref rid=\"B5\" ref-type=\"bibr\">Andreu et al., 2014</xref>). Furthermore, some conflicting results have been reported, from reports showing force production from CMs to be increased with increasing stiffness, (<xref rid=\"B26\" ref-type=\"bibr\">Bhana et al., 2010</xref>) to stiffness having no influence at all (<xref rid=\"B117\" ref-type=\"bibr\">Jacot et al., 2008</xref>). In fact and as explained by <xref rid=\"B60\" ref-type=\"bibr\">Domian et al. (2017)</xref>, it might even be the case that the material properties of cardiac tissue are not the main actor in the myocardial scenario, but this role is rather played by chamber pressure. More experimental and theoretical work needs to be done in this area before we reach a definitive conclusion.</p><p>Adding another layer of complexity, the heart has constant, potent and highly relevant electrical activity. Sparking at a small and specialised region called the sinoatrial node, the electrical wave travels through the auricles, reaching the atrioventricular node where it is delayed (allowing for the filling of the ventricles), and then spreads apex-to-base through the ventricles in a coordinated manner. All this process is controlled by a singular CM type, termed pacemaker cell, displaying disarrayed sarcomeres and low work generation capacity, but able to autonomously start the cardiac action potential. Ultimately, the action potential results in the entry of Ca<sup>+2</sup> ions into the CM, releasing the sarcoplasmic stores of Ca<sup>+2</sup> and freeing myosin of the inhibitory action of troponin I. CMs are also electrically connected through connexins, which form bridges between the cytoplasm of adjacent myocytes, effectively making the myocardium an electrical syncytium. However, it is an anisotropic one, with faster propagation in the direction of the fibres as opposed to the transverse direction (<xref rid=\"B51\" ref-type=\"bibr\">Chung et al., 2007</xref>). Conduction velocity is the speed with which the cardiac impulse travels from one point in the tissue to another. In adulthood, it lies in the range of 0.3-1 m/s, but developmental stage and disease will affect it (<xref rid=\"B301\" ref-type=\"bibr\">Yang et al., 2014</xref>). Achieving a similar value in any cardiac engineered tissue is paramount, given the fact that a mismatch between conduction velocities may give rise to potentially fatal electrical abnormalities such as arrhythmias (<xref rid=\"B125\" ref-type=\"bibr\">Kadota et al., 2013</xref>; <xref rid=\"B312\" ref-type=\"bibr\">Zhang et al., 2018</xref>). It is interesting that one of the foci in cTE is towards providing material-based electronic conductivity, although cardiac cells do not function by transmitting electrons but ions.</p><p>As mentioned already, both the mechanical and electrical properties exert a strong influence upon myocardial biology and function, in both health and disease. For example, increased fibrosis due to pathological conditions like MI or hypertension significantly stiffens cardiac muscle and affects CM contraction (<xref rid=\"B244\" ref-type=\"bibr\">Sessions and Engler, 2016</xref>). Ventricle loading induces CM elongation which, as explained by the Frank-Starling law, renders higher stroke with increase diastolic filling (<xref rid=\"B250\" ref-type=\"bibr\">Solaro, 2007</xref>). The coordinated conduction of the depolarisation wave throughout the organ, including the atrioventricular delay and the apex-to-base transmission, all contribute to optimal functionality and must be taken into account when engineering a human myocardium.</p></sec></sec></sec><sec id=\"S3\"><title>Engineering Cardiac Tissue: The Building Blocks</title><p>cTE aims to generate tissue surrogates, either micro or macro, for various purposes, from developmental biology, (<xref rid=\"B303\" ref-type=\"bibr\">Young and Engler, 2011</xref>) to therapy (<xref rid=\"B184\" ref-type=\"bibr\">Miyagawa et al., 2018</xref>). In the following paragraphs, we will outline the main cells and materials, as well as the different fabrication technologies assayed in the field and the procedures for their maturation. <xref rid=\"T2\" ref-type=\"table\">Table 2</xref> summarises some of the most relevant engineered myocardium examples, with a focus on human cardiac tissue.</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Summary of materials, cells and methods employed to engineer cardiac tissues, their biomimicry and resulting outcome.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">REF</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Materials</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Fabrication</td><td valign=\"top\" align=\"center\" colspan=\"4\" rowspan=\"1\">Cellular mimicry</td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Material mimicry</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Maturation</td><td valign=\"top\" align=\"center\" colspan=\"4\" rowspan=\"1\">Benefit of the selected approach?</td></tr><tr><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"4\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" colspan=\"4\" rowspan=\"1\"><hr/></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Hydrogel</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Fibres</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">CM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">EC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SMC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">CF</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mech.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Elect.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Align</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mech</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Elec</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">vs.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Gene exp.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Structure</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Function</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B89\" ref-type=\"bibr\">Godier-Furnémont et al., 2015</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Col I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"center\" colspan=\"4\" rowspan=\"1\">Rat neonatal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B104\" ref-type=\"bibr\">Hirt et al., 2014</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Fibrin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"center\" colspan=\"4\" rowspan=\"1\">Rat neonatal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">EHT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B115\" ref-type=\"bibr\">Jackman et al., 2018</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">FGN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"center\" colspan=\"4\" rowspan=\"1\">Rat neonatal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B4\" ref-type=\"bibr\">Amdursky et al., 2018</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Albumin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"center\" colspan=\"4\" rowspan=\"1\">Rat neonatal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2D</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B200\" ref-type=\"bibr\">Nunes et al., 2013</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Col I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B233\" ref-type=\"bibr\">Ruan et al., 2016</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Col I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">EHT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B266\" ref-type=\"bibr\">Tiburcy et al., 2017</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Col I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">FK</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2D</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B276\" ref-type=\"bibr\">Valls-Margarit et al., 2019</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Col I + ELN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">FK</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2D</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B308\" ref-type=\"bibr\">Zhang et al., 2013</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Fibrin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2D</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B104\" ref-type=\"bibr\">Hirt et al., 2014</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Fibrin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">EHT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B289\" ref-type=\"bibr\">Weinberger et al., 2016</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Fibrin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B271\" ref-type=\"bibr\">Ulmer et al., 2018</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Fibrin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2D</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B231\" ref-type=\"bibr\">Ronaldson-Bouchard et al., 2018</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Fibrin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">DF</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2D</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B114\" ref-type=\"bibr\">Jackman et al., 2016</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">FGN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B245\" ref-type=\"bibr\">Shadrin et al., 2017</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">FGN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B56\" ref-type=\"bibr\">Dattola et al., 2019</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PVA*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Foaming + FD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2D</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B95\" ref-type=\"bibr\">Han et al., 2016</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PCL</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SE</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2D</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B122\" ref-type=\"bibr\">Joanne et al., 2016</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Col I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SE</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B248\" ref-type=\"bibr\">Sireesha et al., 2015</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">POCS–FGN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SE</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hCM</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2D</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B136\" ref-type=\"bibr\">Khan et al., 2015</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PLGA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SE</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2D</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B232\" ref-type=\"bibr\">Roshanbinfar et al., 2020</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Col I/HA/PANi</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SE</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">EHT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B164\" ref-type=\"bibr\">Macqueen et al., 2018</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PCL/Gelat</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Pull spinning</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2D</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B46\" ref-type=\"bibr\">Castilho et al., 2018</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Col I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PCL</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MEW</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2D</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B273\" ref-type=\"bibr\">Vaithilingam et al., 2019</xref></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">PETra + MWCNT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3DP–SLA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2D</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B152\" ref-type=\"bibr\">Lee et al., 2019</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Col I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3DbioP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">CF</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B165\" ref-type=\"bibr\">Maiullari et al., 2018</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">FGN/PEG</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3DbioP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HUVEC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">EHT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B198\" ref-type=\"bibr\">Noor et al., 2019</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">dECM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3DbioP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B7\" ref-type=\"bibr\">Arai et al., 2018</xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3DbioP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">hPSC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HUVEC</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">DF</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">EHT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">nd</td></tr></tbody></table><table-wrap-foot><attrib><italic>REF, Reference; CM, Cardiomyocyte; EC, Endothelial cell; SMC, Smooth muscle cell; CF, Cardiac fibroblast; Mech, mechanical; Elec, electrical; Gene exp., Gene expression; Col I, collagen type I; FGN, Fibrinogen; hPSC, human pluripotent stem cell; FK, Foreskin fibroblast; DF, Dermal fibroblast; PVA, poly-vinil-alcohol; FD, Freeze-drying; <sup>∗</sup>, foam; SE, Solution electrospinning; POCS, poly[1,8-octanediol-co-(citric acid)-co-(sebacic acid)]; hCM, primary human CM; PLGA, polylactide-co-glycolide; PCL, polycaprolactone; Gelat, Gelatin; PETrA, pentaerythritol triacrylate; MWCNT, Multi-walled carbon nanotubes; 3DP,3D Printing; SLA, Stereolithography; PEG, polyethylene glycol; ALG, Alginate; 3DbioP, 3D bioprinting; nd, not described; dECM, decellularised extracellular matrix; ELN, elastin; HA, Hyaluronic acid; PANi, polyaniline; NM, native myocardium; EHT, engineered heart tissue.</italic></attrib></table-wrap-foot></table-wrap><sec id=\"S3.SS1\"><title>Cells</title><p>The capacity to obtain human cardiac cell phenotypes in the laboratory began with the derivation of human embryonic stem cells (hESC) by Thomson and colleagues in 1998, (<xref rid=\"B264\" ref-type=\"bibr\">Thomson et al., 1998</xref>) which was soon followed by the first protocols for differentiation towards CMs (<xref rid=\"B192\" ref-type=\"bibr\">Mummery et al., 2003</xref>; <xref rid=\"B129\" ref-type=\"bibr\">Kattman et al., 2006</xref>). In 2006, thanks to the breakthrough of the reprogramming technology (<xref rid=\"B259\" ref-type=\"bibr\">Takahashi and Yamanaka, 2006</xref>; <xref rid=\"B304\" ref-type=\"bibr\">Yu et al., 2007</xref>) it became possible to relieve the field of some of its most notorious encumbrances, including the ethical ones. Both, hESCs and human induced pluripotent stem cells (hiPSC) fall within the wider category of human pluripotent stem cells (hPSC). Current methods, including scaling up protocols, (<xref rid=\"B243\" ref-type=\"bibr\">Serra et al., 2011</xref>) have paved the way for their widespread use. In general direct, efficient and reproducible hPSCs differentiation methods try to recapitulate embryonic development, from the induction of cardiac mesoderm, to CM, endothelial cells (ECs), cardiac fibroblast (CFs) or smooth muscle cells (SMCs) <italic>in vitro</italic> specification and maturation (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>) (<xref rid=\"B42\" ref-type=\"bibr\">Burridge et al., 2015</xref>).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Cardiac differentiation of hPSC. hPSC differentiation <italic>in vitro</italic> mimics embryonic development. Induction signals, main molecular pathways and lineage markers are outlined.</p></caption><graphic xlink:href=\"fbioe-08-00955-g003\"/></fig><p>According to the culture format employed, derivation of CMs, CFs, ECs and pericytes/SMCs from hPSCs can be categorised into 3 main approaches: (i) inductive co-culture with visceral endodermal-like cells, (ii) suspension aggregates such as three dimensional (3D) embryoid bodies (EBs) and (iii) two-dimensional (2D) cell monolayer differentiation (<xref rid=\"B192\" ref-type=\"bibr\">Mummery et al., 2003</xref>; <xref rid=\"B129\" ref-type=\"bibr\">Kattman et al., 2006</xref>; <xref rid=\"B149\" ref-type=\"bibr\">Laflamme et al., 2007</xref>; <xref rid=\"B190\" ref-type=\"bibr\">Moretti et al., 2010</xref>). Early reports showed that co-culturing hPSCs with the mouse endodermal cell line END2 was able to induce beating foci (<xref rid=\"B162\" ref-type=\"bibr\">MacIver et al., 2018a</xref>). The low efficiency of this method, as well as the need for xenogenic co-culture, precluded its widespread application. EBs are formed by culturing dissociated hPSC in non-adherent plastic dishes and partially recapitulate the 3D structure and interactions of a developing embryo. hESC-EBs differentiate to derivatives of the three primary germ layers, resulting in spontaneously contracting outgrowths of human CM (<xref rid=\"B134\" ref-type=\"bibr\">Kehat et al., 2001</xref>). Based on EB differentiation protocols, CM from a variety of hESC and hiPSC lines have been generated, usually with a purity of < 10% (<xref rid=\"B311\" ref-type=\"bibr\">Zhang et al., 2009</xref>). ECs can also be isolated from spontaneously differentiating EBs, at a similarly low yield (≈2%) (<xref rid=\"B155\" ref-type=\"bibr\">Levenberg et al., 2002</xref>). In both cases, early reports explored the addition of cardiac mesoderm-inducing growth factors, including FGF2, VEGF BMP4, Activin A, Wnt agonists (WNT3A) or antagonists (DKK1), amongst others (<xref rid=\"B306\" ref-type=\"bibr\">Yuasa et al., 2005</xref>; <xref rid=\"B129\" ref-type=\"bibr\">Kattman et al., 2006</xref>, <xref rid=\"B130\" ref-type=\"bibr\">2011</xref>; <xref rid=\"B300\" ref-type=\"bibr\">Yang et al., 2008</xref>; <xref rid=\"B269\" ref-type=\"bibr\">Tran et al., 2009</xref>; <xref rid=\"B118\" ref-type=\"bibr\">James et al., 2010</xref>). In general, however, EB-based differentiations have lost ground to more advanced and defined procedures, as the former are generally inefficient and render a mixture of cardiac cells with other non-cardiac phenotypes, requiring additional purification.</p><p>Monolayer-based differentiation is nowadays the most usually applied method. Cytokine-based protocols were developed first (<xref rid=\"B258\" ref-type=\"bibr\">Taccardi et al., 2008</xref>). These have been progressively modified by the discovery of Wnt signals playing a biphasic role in cardiac differentiation <italic>in vivo</italic>, (<xref rid=\"B168\" ref-type=\"bibr\">Marvin et al., 2001</xref>; <xref rid=\"B270\" ref-type=\"bibr\">Ueno et al., 2007</xref>) with early signals directing hPSCs towards cardiac fate, whilst later inhibition of those signals is a prerequisite for CM specification. Almost 10 years ago, this concept was incorporated into the CM differentiation from hPSCs, (<xref rid=\"B156\" ref-type=\"bibr\">Lian et al., 2012</xref>) paving the way for the grounding of a chemically defined procedure (<xref rid=\"B41\" ref-type=\"bibr\">Burridge et al., 2014</xref>). Based on small molecules rather than cytokines, and thus less costly, this protocol is now widely applied, providing highly pure yields of hPSC-derived CMs when in combination with a metabolic-based selection (<xref rid=\"B267\" ref-type=\"bibr\">Tohyama et al., 2013</xref>). This means that complicated and inefficient EB-forming procedures or expensive and time-consuming immune-selection protocols have now been discarded (<xref rid=\"B40\" ref-type=\"bibr\">Burridge et al., 2007</xref>; <xref rid=\"B98\" ref-type=\"bibr\">Hattori et al., 2010</xref>; <xref rid=\"B67\" ref-type=\"bibr\">Elliott et al., 2011</xref>; <xref rid=\"B272\" ref-type=\"bibr\">Uosaki et al., 2011</xref>). However, even this latest protocol still requires a degree of set up to avoid inconsistent efficiencies amongst cell lines and experimental repeats, mostly related to different patterns of endogenous early canonical Wnt expression (<xref rid=\"B210\" ref-type=\"bibr\">Paige et al., 2010</xref>). In general, CMs obtained from these protocols consist of a mixture of pacemaker, atrial and ventricular myocytes, though some researchers consider that this is open to question, as hPSC-CMs are immature and intrinsically plastic (<xref rid=\"B61\" ref-type=\"bibr\">Du et al., 2015</xref>).</p><p>The derivation of other cardiac phenotypes has been also achieved, with a variety of protocols now available. Palpant et al. reported the generation of CMs, cardiac- or hemogenic-derived ECs as well as blood cells by finely dosing BMP4 and Activin A in order to pattern hPSC towards different mesodermal fates (<xref rid=\"B211\" ref-type=\"bibr\">Palpant et al., 2017</xref>). Others have employed a mixture of small molecules and cytokines to derive vascular cells from hPSCs in monolayer culture with high efficiency (<xref rid=\"B206\" ref-type=\"bibr\">Orlova et al., 2014</xref>; <xref rid=\"B215\" ref-type=\"bibr\">Patsch et al., 2015</xref>). Global gene transcription analysis has demonstrated low variability between ECs differentiated via cytokine-based methods from multiple lines of hPSCs (<xref rid=\"B290\" ref-type=\"bibr\">White et al., 2013</xref>). CFs, have been increasingly recognised as major players in cardiac development and homeostasis, having a similarly significant effect upon the capacity to build cardiac tissues in the lab. Recently, two independent groups have reported the generation of hPSC-derived CFs, giving also proof of their capacity to affect hPSC-CM function (<xref rid=\"B309\" ref-type=\"bibr\">Zhang H. et al., 2019</xref>; <xref rid=\"B310\" ref-type=\"bibr\">Zhang J. et al., 2019</xref>). Epicardial cells have similarly been derived, (<xref rid=\"B292\" ref-type=\"bibr\">Witty et al., 2014</xref>) demonstrating their ability to increase the therapeutic capacity of hPSC-CMs <italic>in vivo</italic> (<xref rid=\"B13\" ref-type=\"bibr\">Bargehr et al., 2019</xref>). Finally, sinoatrial node pacemaker CMs have been obtained from hPSC, and their capacity to pace tissues <italic>in vivo</italic> has been reported (<xref rid=\"B223\" ref-type=\"bibr\">Protze et al., 2017</xref>). Other approaches to the differentiation of cardiac lineages include the generation of CVPs, (<xref rid=\"B28\" ref-type=\"bibr\">Blin et al., 2010</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Birket et al., 2015</xref>; <xref rid=\"B315\" ref-type=\"bibr\">Zhang Y. et al., 2016</xref>) or direct reprogramming strategies, (<xref rid=\"B186\" ref-type=\"bibr\">Mohamed et al., 2017</xref>) but they have rarely been explored in cTE.</p></sec><sec id=\"S3.SS2\"><title>Materials</title><p>In parallel to the way differentiation of hPSC mimics the natural embryonic development, the current view is that the more a material replicates the properties of cardiac tissue, the higher the chances of success. Development over the last 15 years has yielded a wide portfolio of materials and biomaterials. Classifications are numerous, be it by origin (natural, synthetic or hybrid), crosslinking (chemical vs physical), size (macro, micro or nano), polymerisation mechanism (enzymatic, light-triggered or pH-responsive) or whether they are or not reinforced with other structures like fibres. For specific insight into these classifications, we direct the reader towards some of the excellent latest papers (<xref rid=\"B216\" ref-type=\"bibr\">Peña et al., 2018</xref>; <xref rid=\"B159\" ref-type=\"bibr\">Liu et al., 2019</xref>; <xref rid=\"B298\" ref-type=\"bibr\">Xu et al., 2019</xref>). One of the most relevant classifications is, however, on the physical consistency of the applied material, where we can differentiate (i) injectable materials and hydrogels, (ii) solid or fibrous scaffolds and (iii) composite systems.</p><p>Hydrogels are probably the most widely explored type of material in cTE. Collagen, being the main component of the cardiac ECM, has been widely employed (see the following sections). It can be readily isolated from animal or even human tissues in sufficient quantities, extracted and solubilised, although it requires acidic pH for this. Therefore, a careful control over pH is needed for optimal polymerisation and embedded cell survival. Gelatin, being denatured/digested collagen, has also been intensely explored and the basis for the generation of some of the most applied semi-synthetic materials, such as gelatin methacryloyl (GelMA)(<xref rid=\"B307\" ref-type=\"bibr\">Yue et al., 2015</xref>) and several biorthogonal derivatives (<xref rid=\"B146\" ref-type=\"bibr\">Koshy et al., 2016</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Bertlein et al., 2017</xref>). Alginate, a sugar-based natural hydrogel obtained from algae, (<xref rid=\"B205\" ref-type=\"bibr\">Orive et al., 2006</xref>) has been employed due to their tailorable mechanical properties and simple polymerisation, mediated by cations such as Ca or Mg, albeit lacking biological binding motifs. Silk and its derivatives have also been processed into hydrogels, (<xref rid=\"B107\" ref-type=\"bibr\">Holland et al., 2019</xref>) and modified to incorporate electrically active particles (<xref rid=\"B15\" ref-type=\"bibr\">Barreiro et al., 2019</xref>) or photocrosslinkable chemical groups (<xref rid=\"B53\" ref-type=\"bibr\">Cui et al., 2020</xref>). Allergic and anti-inflammatory side reactions have been reported with some silk derivatives, so care should be taken when incorporating them into an engineered tissue. Amongst natural hydrogels, decellularised ECM (dECM) has attracted significant interest since the breakthrough discovery of the process, as applied to the building of tissues (<xref rid=\"B207\" ref-type=\"bibr\">Ott et al., 2008</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Belviso et al., 2020</xref>). In principle, dECM retains all components of the ECM of origin, thus creating a more complex and biomimetic environment. Garreta et al. obtained human dECM slices on which they cultured hPSC-CMs. The human CMs demonstrated enhanced conduction velocity and gene expression of related genes (SERCA, KCNJ2, CACNA1C or SCN5A amongst others) (<xref rid=\"B82\" ref-type=\"bibr\">Garreta et al., 2016</xref>). The group of Lior Gepstein employed dECM-chitosan mixtures in combination with hiPSC-CMs. The resulting engineered myocardium displayed enhanced maturity as compared with cells cultured in 2D, showing tissue-like drug responses. Their work also provided proof-of-concept of the capacity of this system to model human cardiac diseases (Long QT syndrome) and arrhythmias (<xref rid=\"B90\" ref-type=\"bibr\">Goldfracht et al., 2019</xref>). On the synthetic side, polyethylene glycol (PEG) and its several modifications have been extensively studied in the TE field (<xref rid=\"B112\" ref-type=\"bibr\">Iyer et al., 2009</xref>).</p><p>Most of the materials so far outlined in this section can be processed into fibres employing strategies explained in the next section. Thermoplastics have also been applied to cTE, mostly as fibrous scaffolds. Examples include poly-ε-caprolactone (PCL), (<xref rid=\"B295\" ref-type=\"bibr\">Woodruff and Hutmacher, 2010</xref>) or elastomers like poly (glycerol-sebacate) (PGS) (<xref rid=\"B137\" ref-type=\"bibr\">Kharaziha et al., 2013</xref>). Conductive polymers have only recently began to be applied to the field, though some of the most remarkable examples have not incorporated the use of cells and would therefore not qualify as engineered tissues (<xref rid=\"B172\" ref-type=\"bibr\">Mawad et al., 2016</xref>; <xref rid=\"B127\" ref-type=\"bibr\">Kapnisi et al., 2018</xref>). Finally, given the low mechanical properties displayed by most hydrogels, composite fibre-reinforced materials are also being developed, (<xref rid=\"B18\" ref-type=\"bibr\">Bas et al., 2015</xref>) with some examples explained in the following section.</p></sec><sec id=\"S3.SS3\"><title>Maturation Stimuli</title><p>Cells derived from hPSC are immature (see <xref rid=\"B128\" ref-type=\"bibr\">Karbassi et al., 2020</xref> for a review). Although not the direct focus of this work, neonatal myocytes, which are another cell source commonly employed, also suffer from this drawback. cTE has long been aware of this limitation and has applied three main stimuli, namely physical, mechanical and electrical, and combinations thereof <xref rid=\"B214\" ref-type=\"bibr\">Parsa et al. (2016)</xref> and <xref rid=\"B256\" ref-type=\"bibr\">Stoppel et al. (2016)</xref>. Perfusion is able to improve engineered tissues’ properties, as it will boost nutrient access and renewal, as has been shown for neonatal cells (<xref rid=\"B225\" ref-type=\"bibr\">Radisic et al., 2004b</xref>) as well as hiPSC-cardiac derivatives (<xref rid=\"B276\" ref-type=\"bibr\">Valls-Margarit et al., 2019</xref>). Materials’ physical properties (e.g., stiffness) are able to induce maturation features in these CMs, or at least preserve primary CMs from dedifferentiation (<xref rid=\"B4\" ref-type=\"bibr\">Amdursky et al., 2018</xref>). In general, most hydrogels are able to replicate the right myocardial-like properties. For example, Feaster and colleagues found that plating hiPSC-CMs on thick Matrigel induced a certain degree of molecular and functional maturation, in comparison to thin, diluted hydrogel coating, which essentially transmits the rigidity of the underlying plastic (<xref rid=\"B72\" ref-type=\"bibr\">Feaster et al., 2015</xref>). Herron et al. employed soft (albeit supra-cardiac) substrates and compared them with glass in their capacity to influence hiPSC-CMs, with a significant effect on maturation, showing improvement in the expression of Na and K channels, as well as on the degree of binucleation, cell cycle exit and hypertrophy (<xref rid=\"B101\" ref-type=\"bibr\">Herron et al., 2016</xref>). Although not purely based on engineered tissues, they and others give proof of the crucial role stiffness plays in cardiac maturation.</p><p>Mechanical stimulation has a leading role in cardiac development and aging (<xref rid=\"B96\" ref-type=\"bibr\">Happe and Engler, 2016</xref>; <xref rid=\"B244\" ref-type=\"bibr\">Sessions and Engler, 2016</xref>). It can be isometric, isotonic or auxotonic (<xref rid=\"B158\" ref-type=\"bibr\">Liaw and Zimmermann, 2016</xref>). In isometric stimulation, the construct is preloaded and must exert force against a static load. In isotonic stimulation, a device will cyclically exert active elongation on the engineered tissue. Finally, auxotonic stimulation occurs when the myocardial tissue has to contract against a resilient load. In principle, the auxotonic mode confers a more physiological stimulation, although all three are reported to deliver maturation upon engineered myocardium (<xref rid=\"B89\" ref-type=\"bibr\">Godier-Furnémont et al., 2015</xref>; <xref rid=\"B161\" ref-type=\"bibr\">Lux et al., 2016</xref>; <xref rid=\"B233\" ref-type=\"bibr\">Ruan et al., 2016</xref>; <xref rid=\"B271\" ref-type=\"bibr\">Ulmer et al., 2018</xref>). Although the exact mechanisms by which mechanical stimulation matures the cardiac engineered tissue are not known in depth, it is presumed that they will operate by the same ones occurring during cardiac development or physiologic hypertrophy (<xref rid=\"B194\" ref-type=\"bibr\">Nakamura and Sadoshima, 2018</xref>). After all, being a striated muscle, the myocardium can undergo hypertrophy if exercised (<xref rid=\"B140\" ref-type=\"bibr\">Kim et al., 2008</xref>).</p><p>As an electro-sensitive organ, the heart can be stimulated by electrical pulses, which can help maintain its function <italic>ex vivo</italic> (<xref rid=\"B285\" ref-type=\"bibr\">Watson et al., 2019</xref>). Electrical stimulation is known to have a relevant effect on hPSC-CM differentiation and maturation. Over 20 years ago, Sauer et al. established a relationship between this stimulation and mouse ESC differentiation to CM (EB method). They showed the effect was at least partly mediated by reactive oxygen species (ROS) generation and NF-κB, and could be replicated by ROS from H<sub>2</sub>O<sub>2</sub> incubation (<xref rid=\"B237\" ref-type=\"bibr\">Sauer et al., 1999</xref>). Serena et al. analysed the effects of the type of electrode and stimulation length on CM differentiation via the EB method from hESC, finding a role for ROS, though the effect on the efficiency of differentiation was not determined (<xref rid=\"B242\" ref-type=\"bibr\">Serena et al., 2009</xref>). Hernández et al. employed brief (5 min) electrical stimulation, of hiPSC-EBs, finding an increase in the percentage of cardiac differentiation (% of beating EBs) after 14 days (<xref rid=\"B100\" ref-type=\"bibr\">Hernández et al., 2016</xref>). However, the use of the EB method complicates findings, as the effect could also be mediated by other cell types within the EB. Electrical stimulation has also been shown to enhance hPSC-CM maturation at the gene expression and functional levels (Ca transients), as well as promoting the ventricular phenotype (<xref rid=\"B48\" ref-type=\"bibr\">Chan Y. C. et al., 2013</xref>). Reasoning that exogenous electrical stimulation would act as an artificial pacemaker, Richards and coworkers evaluated the implementation of electrically conductive silicon nanowires in hiPSC-derived cardiac spheroids, showing that it was able to improve CM-to-CM communication (measured by staining for Connexin 43 and N-cadherin) and structural quality, though some of these quantifications might nowadays be regarded as debatable (<xref rid=\"B226\" ref-type=\"bibr\">Richards et al., 2016</xref>). The cTE field has implemented electrical stimulation to cardiac constructs with success. The group of Gordana Vunjak-Novakovic pioneered work in this area, showing the enhanced of contraction (synchronicity) and structure (alignment, ultrastructure) on neonatal rat CMs seeded on Ultrafoam collagen sponges and Matrigel (<xref rid=\"B224\" ref-type=\"bibr\">Radisic et al., 2004a</xref>). The group developed stimulation protocols as well as bioreactors, (<xref rid=\"B263\" ref-type=\"bibr\">Tandon et al., 2008</xref>, <xref rid=\"B262\" ref-type=\"bibr\">2009</xref>; <xref rid=\"B170\" ref-type=\"bibr\">Massai et al., 2013</xref>) which have influenced the whole field. The group of Milica Rasidic built hPSC-based cardiac tissues by embedding hPSC-dissociated EBs in collagen type I and Matrigel. After electrical stimulation, they showed an increased myofibril ultrastructural organisation and improved function (conduction velocity and Ca handling properties) as compared to non-stimulated controls (<xref rid=\"B200\" ref-type=\"bibr\">Nunes et al., 2013</xref>).</p><p>Finally, some remarkable advances have been made when combining electrical and mechanical stimulation. Ruan et al. generated collagen-based cardiac engineered tissues containing hPSC-CMs, which were subjected to electromechanical stimulation and compared to static stretch or no stimulus. Results showed a positive Frank-Starling effect (increased force production with increased preload), a less negative force-frequency relationship (increased force production with increased pacing frequency) and maximum stress generation for the electromechanical stimulation group, which was correlated with increased expression of RYR2 and SERCA2, thus supporting the use of combined stimulation for enhanced maturation (<xref rid=\"B233\" ref-type=\"bibr\">Ruan et al., 2016</xref>). The group of Dr. Zimmermann employed auxotonic stimulation delivered through stretchers in combination with electrical pacing at 0, 2, 4, or 6 Hz (<xref rid=\"B89\" ref-type=\"bibr\">Godier-Furnémont et al., 2015</xref>). Results on tissues generated with collagen and neonatal rat cells showed that the 4 Hz regime was able to generate tissues with a physiological and positive force-frequency and enhanced functionality. Also, the presence of T-tubules was demonstrated. Finally, the group of Prof Vunjak-Novakovic generated hiPSC-CM-based collagen tissues on flexible stretchers (auxotonic mechanical stimulation) and supplied no stimulation (control), 2 Hz and 0.33 Hz/day progressive increase over 2 weeks (‘intensity training’). Their results demonstrated the effectiveness of this strategy, as shown by a physiological sarcomere length, increased density of mitochondria, T-tubules, a more mature metabolism and functional improvements at the level of Ca cycling and a positive force-frequency relationship (<xref rid=\"B231\" ref-type=\"bibr\">Ronaldson-Bouchard et al., 2018</xref>).</p></sec><sec id=\"S3.SS4\"><title>Fabrication Strategies</title><p>Materials confer cTE with significant options, not only due to the available range, but also through the application of different fabrication modalities, which can deliver different properties out of the same starting material. For example, collagen can be mould-casted, (<xref rid=\"B89\" ref-type=\"bibr\">Godier-Furnémont et al., 2015</xref>) extruded, (<xref rid=\"B8\" ref-type=\"bibr\">Araña et al., 2014</xref>) or bioprinted, (<xref rid=\"B152\" ref-type=\"bibr\">Lee et al., 2019</xref>) and the resulting properties will vary widely, with casted and bioprinted collagen having a stiffness in the range of kPa, whilst the extruded film will be significantly stiffer (MPa). In addition, structure will also differ. 3D printing and bioprinting have no doubt revolutionised our capacity to engineer cardiac tissues, however, other technologies can provide relevant features. The following paragraphs outline some of the most employed strategies, classified depending on their capacity to produce a controlled architecture (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>).</p><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>Summary of fabrication methods, advantages and disadvantages.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fabrication technique</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Advantages</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Disadvantages</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">No true architecture control</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mould casting</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mould casting (<xref rid=\"B120\" ref-type=\"bibr\">Janik and Marzec, 2015</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– Simplicity and cost <break/>– High cell survival <break/>– Range of compatible materials <break/>– Mechanical properties <break/>– Scale up feasible</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– Lowest architectural control <break/>– Limited thickness</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pore-forming</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Solvent Casting, Particle Leaching, Cryogelation (<xref rid=\"B120\" ref-type=\"bibr\">Janik and Marzec, 2015</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– Control of pore size <break/>– No specialised equipment <break/>– Cost</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– Use of organic solvents <break/>– Limited scaffold thickness <break/>– Mechanical properties <break/>– Time consuming leaching <break/>– Limited pore architecture</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Electrically produced</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SE (<xref rid=\"B157\" ref-type=\"bibr\">Liang et al., 2007</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– Nanometer features <break/>– Range of compatible materials <break/>– Scaling up feasible <break/></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– Use of organic solvents <break/>– Limited scaffold thickness <break/>– Usually produces high stiffness substrates <break/></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Textile- <break/>based</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weaving, Braiding, Knitting (<xref rid=\"B3\" ref-type=\"bibr\">Akbari et al., 2016</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– Simple <break/>– Scaling up feasible <break/></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– Specialised equipment (cost) <break/>– Not widespread <break/>– Limited porosity</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">True architecture control</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Build & seed</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SLA (<xref rid=\"B177\" ref-type=\"bibr\">Melchels et al., 2010</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– High architectural control <break/>– Self-supporting process <break/></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– Only photosensitive polymers <break/>– Remove supporting materials <break/>– Use of UV light <break/>– Toxicity of photoinitiator <break/>– Specialised equipment (cost)</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SLS (<xref rid=\"B173\" ref-type=\"bibr\">Mazzoli, 2013</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– No solvents required <break/>– High architectural control <break/>– Self-supporting process <break/>– Range of compatible materials</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– High temperature <break/>– Materials in powder form <break/>– Rough surface <break/>– Specialised equipment (cost)</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MEW (<xref rid=\"B32\" ref-type=\"bibr\">Brown et al., 2011</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– No solvents required <break/>– Control over porosity, pore size and fibre diameter <break/>– High architectural control</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– Limited thickness <break/>– Range of available material <break/>– Specialised equipment (cost) <break/></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FDM (<xref rid=\"B191\" ref-type=\"bibr\">Moroni et al., 2006</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– No solvents required <break/>– Speed of printing <break/>– Good reproducibility</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– Restricted to materials with good melt viscosity properties <break/>– High temperature <break/>– Filament required <break/>– Limited resolution</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bioprinting</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">LGDW, LIFT, BioLP (<xref rid=\"B144\" ref-type=\"bibr\">Koch et al., 2010</xref>; <xref rid=\"B80\" ref-type=\"bibr\">Gaebel et al., 2011</xref>; <xref rid=\"B109\" ref-type=\"bibr\">Hu et al., 2017</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– Range of cells/biomaterials <break/>– Single cell resolution <break/>– Precise cell printing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– Low cell viability <break/>– Limited 3D structure <break/>– Time consuming</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3DbioP (<xref rid=\"B313\" ref-type=\"bibr\">Zhang et al., 2015</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– Range of cells/biomaterials <break/>– High cell viability <break/>– Process at room temperature</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">– Weak structural support <break/>– Specialised equipment <break/>– Multimaterial printing expensive (multinozzle)</td></tr></tbody></table><table-wrap-foot><attrib><italic>SE, Solution electrospinning; LGDW, Laser-guided direct writing; LIFT, Laser Induced Forward Transfer, BioLP, Bio-laser Printing; 3DbioP, 3D bioprinting; SLA, Stereolithography; SLS, Selective laser sintering; MEW, Melt Electrospinning Writing; FDM, Fused deposition modelling.</italic></attrib></table-wrap-foot></table-wrap><sec id=\"S3.SS4.SSS1\"><title>Techniques With No True Architecture Control</title><sec id=\"S3.SS4.SSS1.Px1\"><title>Mould casting</title><p>Probably the simplest and most widely employed fabrication mode, requires the generation of a mould of the desired shape, and is the fabrication technique of choice in many biomedical-based laboratories, where other methods could not be implemented due to lack of expertise or specific equipment. It does not provide much control over the resulting architecture, but can be combined with others, like porogen leaching, to add specifically selected features to the resulting tissue. The first reports on engineered cardiac tissues by Thomas Eschenhagen and coworkers in the late 1990s were developed by mould-casting a mixture of chick embryonic CMs embedded in a collagen solution. This was allowed to gel between two Velcro-coated glass tubes. The resulting tissues, later termed engineered heart tissues (EHTs), could be cultured <italic>in vitro</italic> and maintained over several days, responded to electrical stimulation, displayed a positive Frank-Starling relationship, were sensitive to levels of extracellular calcium, and could be modified with viral vectors (<xref rid=\"B70\" ref-type=\"bibr\">Eschenhagen et al., 1997</xref>; <xref rid=\"B320\" ref-type=\"bibr\">Zimmermann et al., 2000</xref>).</p><p>Since then, the technology has evolved enormously. It was expanded to neonatal rat cells, (<xref rid=\"B322\" ref-type=\"bibr\">Zimmermann et al., 2002</xref>) and refined, with the possibility of fabricating more complex and thicker EHTs, which functioned synchronously (<xref rid=\"B321\" ref-type=\"bibr\">Zimmermann et al., 2006</xref>). These stacked EHTs also showed promise as a therapy when transplanted in a rat model of infarction. EHTs have been employed by the groups of Eschenhagen and Zimmermann, either fibrin- or collagen type I-based, to study the effect of chronic stretch on CM hyperthrophy, (<xref rid=\"B74\" ref-type=\"bibr\">Fink et al., 2000</xref>) as a disease model of hypertrophic cardiomyopathy, (<xref rid=\"B255\" ref-type=\"bibr\">Stöhr et al., 2013</xref>) or to analyze the effect of electrical stimulation (<xref rid=\"B104\" ref-type=\"bibr\">Hirt et al., 2014</xref>). EHT technology has greatly benefited from the implementation of hPSC-derived cells, as the findings, models and application have gained greater impact, be it as a drug testing platform (<xref rid=\"B64\" ref-type=\"bibr\">Eder et al., 2016</xref>), an <italic>in vitro</italic> tool to study cardiac stimulation, (<xref rid=\"B89\" ref-type=\"bibr\">Godier-Furnémont et al., 2015</xref>) or as a potential myocardial regenerative therapeutic (<xref rid=\"B289\" ref-type=\"bibr\">Weinberger et al., 2016</xref>; <xref rid=\"B266\" ref-type=\"bibr\">Tiburcy et al., 2017</xref>). The Bursac group has also employed mould casting of rat and human (hiPSC-derived) cells to engineer cardiac tissues and explore different strategies to mature them <italic>in vitro</italic> (<xref rid=\"B114\" ref-type=\"bibr\">Jackman et al., 2016</xref>). They applied this strategy to fabricate tissues up to human scale, though thickness was limited by nutrient and oxygen diffusion. When assayed in a rat model of disease, their cardiopatches retained integrity after 3 weeks, showing extensive vascularisation by host-derived vessels and maintaining electrical activity, although they did not functionally integrate with the endogenous myocardium (<xref rid=\"B245\" ref-type=\"bibr\">Shadrin et al., 2017</xref>). The team of Milica Radisic employed PDMS moulds to form engineered cardiac tissues around a surgical suture, composed of hPSC-derived cardiac cells, collagen type I and 10% Matrigel (<xref rid=\"B200\" ref-type=\"bibr\">Nunes et al., 2013</xref>). By employing electrical stimulation, through carbon rods immersed in the culture medium, they were able to drive the hPSC-CMs towards a more mature phenotype and functionality. This system was later applied also to drug testing (<xref rid=\"B73\" ref-type=\"bibr\">Feric et al., 2019</xref>). Finally, one of the most advanced pieces of evidence for hPSC-CM maturation <italic>in vitro</italic> was provided by the group of Gordana Vunjak-Novakovic, as explained in the previous section (<xref rid=\"B231\" ref-type=\"bibr\">Ronaldson-Bouchard et al., 2018</xref>). They employed a 3:1 mixture of hPSC-CMs and dermal fibroblasts embedded in a fibrin matrix, cast into wells where flexible PDMS posts were also included. These conferred pre-tension on the generated engineered myocardium, thus adding physiological-like auxotonic stimulation (<xref rid=\"B167\" ref-type=\"bibr\">Mannhardt et al., 2019</xref>). A set of custom-made carbon electrodes delivered electrical stimulation. The resulting bioartifical myocardium displayed adult-like gene expression profiles, ultrastructural features such as M-bands and T-tubules, as well as oxidative metabolism and mature functionality. In summary, although mould casting cannot generate fine features, it is one of the most widely applied and highly evolved methods to obtain human mature cardiac tissue, and the one closest to translation.</p></sec><sec id=\"S3.SS4.SSS1.Px2\"><title>Macro-to-micro pore-forming strategies</title><p>Cryogelation, based on freeze drying, is a common preservation strategy that is also applied to TE. The pre-crosslinked material is subjected to freeze-thawing cycles, which induces ice crystal formation. These crystals act as porogens, to be removed when pressure is decreased and the solvent sublimated. In general, pores are well inter-connected, as demonstrated early on by O’Brien and colleagues for collagen-glycosaminoglycans scaffolds (<xref rid=\"B201\" ref-type=\"bibr\">O’Brien et al., 2004</xref>). In addition, varying conditions make it possible to modulate material properties, as shown by <xref rid=\"B141\" ref-type=\"bibr\">Kim et al. (2015)</xref>. In combination with gas foaming, Dattola et al. fabricated a poly(vinyl) alcohol scaffold with pores within the size of CMs, and Young’s Modulus similar to that of the native cardiac ECM. These substrates are able to support the growth and cardiac differentiation of hiPSC, albeit at apparently low rates as demonstrated by the immunostaining for cardiac proteins (<xref rid=\"B56\" ref-type=\"bibr\">Dattola et al., 2019</xref>).</p><p>Gas foaming has also been extensively applied to TE, though its use within the cardiac field is not extensive. This technique relies on the formation of bubbles, either by adding exogenous agents such as sodium bicarbonate or through the inclusion of enzymatic-driven reaction in the fabrication process (<xref rid=\"B189\" ref-type=\"bibr\">Mooney et al., 1996</xref>). As the solution polymerises, these bubbles are trapped inside. In consequence, the choice of foaming agent is crucial for the later survival of cells. Although relatively simple to implement, this technique provides no control over the degree of interconnection between the formed pores or their directionality.</p><p>Porogen templating is based on the inclusion of salt crystals in the pre-polymer solution. Similar to freeze drying, these crystals will act as templates, creating an empty space (pore) when the particles are leached out, in most cases by dissolving in aqueous solution, weak bases or heating, (<xref rid=\"B265\" ref-type=\"bibr\">Thomson et al., 1995</xref>) with sodium chloride being one of the most frequently employed porogens. On the down side, limitations are related to low architectural control and suboptimal processing of porogens, which might compromise biocompatibility as well as mechanical properties. Salt leaching has not been as widely employed in cTE as in other areas. Ganji et al. used table salt as a simple yet efficient way to generate defined pores in polyurethane-based scaffolds where gold nanotubes and nanowires were incorporated, thus combining 3-dimensionality with the additional benefit of electrical conductivity (<xref rid=\"B81\" ref-type=\"bibr\">Ganji et al., 2016</xref>). Biocompatibility tested with H9c2 rat cardiomyoblasts showed a positive effect on cell growth, though no hPSC-derived cells were tested. Other examples have employed porogen templating for the fabrication of porous polysaccharide-based vascular scaffolds and engineered heart valves (<xref rid=\"B150\" ref-type=\"bibr\">Lavergne et al., 2012</xref>; <xref rid=\"B169\" ref-type=\"bibr\">Masoumi et al., 2014</xref>).</p><p>Thermally induced phase separation is based on the use of temperature to induce the de-mixing of a homogeneous polymer solution. The controlled change in temperature prompts the formation of a polymer-rich and a polymer-poor phase, which can be used to obtain an interconnected porous structure (<xref rid=\"B195\" ref-type=\"bibr\">Nam and Park, 1999</xref>). Vozzi et al. synthesised the elastomer polyesterurethane, and used thermal phase separation to create a porous and biocompatible structure, which they functionalised with fibronectin by NHS-EDC chemistry. Although material properties were off the cardiac optimal range (in the order of > 0.25 MPa), neonatal rat cardiac cells attached and grew on the scaffolds, with specific modulation of gene expression. However, no use of human cells was reported (<xref rid=\"B278\" ref-type=\"bibr\">Vozzi et al., 2018</xref>).</p></sec><sec id=\"S3.SS4.SSS1.Px3\"><title>Electrically produced</title><p>Solution electrospinning (SE), on the other hand, was one of the first and most employed fabrication modalities in the field. In this technique, a polymer solution is subjected to a relatively high electric field while it travels towards a collector plate. This electric field causes the polymeric jet to whip, producing very thin fibres of even sub-micron size, that can be collected randomly or in an aligned fashion. For a review see <xref rid=\"B240\" ref-type=\"bibr\">Sensini and Cristofolini (2018)</xref>. In general, most SE applications produce low thickness mats of (semi-)randomly arranged fibres of different materials, though use of appliances such as a rotating mandrel can increase alignment (<xref rid=\"B95\" ref-type=\"bibr\">Han et al., 2016</xref>). Although relying on the use of potentially toxic solvents, SE has the advantage of being open to a wide range of materials, both natural and synthetic (<xref rid=\"B142\" ref-type=\"bibr\">Kitsara et al., 2017</xref>).</p><p>The concurrent use of SE with hPSC-derived cardiac cells has not been widely explored but some remarkable examples can be found. The group of Onnik Agbulut tested different crosslinking times of a clinical-grade collagen electrospun scaffold for the most suitable conditions for hiPSC-CM culture. The resulting mats had fibres of 0.6-2.2 μm, with pores in the range of 2-3 μm. After no deleterious effects were found after mat implantation in animals, the mats were seeded with hiPSC-CMs (10<sup>6</sup> per scaffold), and produced a significant benefit when transplanted in a dilated cardiomyopathy model. Importantly, scaffolds were compliant enough to allow the generation of macroscopic contractions by the hiPSC-CMs (<xref rid=\"B122\" ref-type=\"bibr\">Joanne et al., 2016</xref>). Sireesha et al. used a combination of the elastomer poly[1,8-octanediol-co-(citric acid)-co-(sebacic acid)] and fibrinogen to produce scaffolds with a sub-micron fibre diameter, whose mechanical properties laid on the upper end of cardiac elasticity (hundreds of kPa) and which showed good biocompatibility with human CMs. However, no further tests in animal models were performed (<xref rid=\"B248\" ref-type=\"bibr\">Sireesha et al., 2015</xref>). Khan and co-workers electrospun polylactide-co-glycolide (PLGA) as 50 μm-thick aligned nanofibrous scaffolds, seeded with hiPSC-CM, and compared the outcome versus conventional tissue culture plastic surfaces. Results showed the scaffolds were able to align the CMs, as well as inducing changes at the level of functionality (Ca transients) and gene expression, though the high stiffness of the mats (1-2 orders of magnitude above the cardiac tissue) could have a negative impact (<xref rid=\"B136\" ref-type=\"bibr\">Khan et al., 2015</xref>). On the other side of the coin, Han et al. assayed the capacity of aligned electrospun mats made of PCL coated with matrigel to induce hiPSC-CM maturation, showing a limited effect at the functional level, but with some differences in gene expression (<xref rid=\"B95\" ref-type=\"bibr\">Han et al., 2016</xref>).</p><p>Aside from employing different materials or combinations of natural and synthetic polymers, SE offers increased possibilities by modifying the properties of the resulting engineered tissue by combination with nanoparticles or other fabrication technologies. For example, the group of Tal Dvir built on his previous work on the electrospinning of albumin (<xref rid=\"B76\" ref-type=\"bibr\">Fleischer et al., 2014</xref>) to increase the anisotropy of the fabricated engineered tissue. To do this, they employed a double strategy: they used laser patterning to create micro-holes and unidirectional grooves, in order to increase mass transport and cell alignment respectively, and they stacked several layers of patterned mats, inspired by the anisotropy found across the ventricular wall. In this fashion, they also modulated the mechanical properties of the layers, getting closer to human myocardial values. They did not, however, test the stiffness of the resulting stacked construct, nor populate it with human cells, using rat neonatal cardiac cells instead (<xref rid=\"B75\" ref-type=\"bibr\">Fleischer et al., 2017</xref>). The group of Ali Khademhosseini used poly(glycerol sebacate):gelatin (PG) solution where gelatin-methacryloyl (GelMA)-coated carbon nanotubes had been incorporated to electrospun aligned nanofibrous mats with improved electrical properties, though at the cost of increased stiffness (<xref rid=\"B138\" ref-type=\"bibr\">Kharaziha et al., 2014</xref>). This is similar to their previous work with GelMA-embedded carbon nanotubes, (<xref rid=\"B247\" ref-type=\"bibr\">Shin et al., 2013</xref>) with a significant effect on gene expression, structure and functionality, but again no human cells were employed. Walker et al. employed bio-ionic liquids to modulate the electrical properties and adhesion strength of electrospun GelMA scaffolds, assaying their <italic>in vitro</italic> and <italic>in vivo</italic> regenerative potential, though no tissue was constructed (<xref rid=\"B281\" ref-type=\"bibr\">Walker et al., 2019</xref>). Again, mechanical properties were affected. Recently, the group of Felix Engel used SE to generate fibres combining the conductive polymer polyaniline (PANi) with collagen/hyaluronic acid, producing mats of suitable mechanical and electrical properties. Scaffolds supported the attachment of hiPSC-CMs, which displayed typical striations and contractions. Substrates incorporating PANi induced a faster beating rate on hiPSC-CMs, though this was not further explored (<xref rid=\"B232\" ref-type=\"bibr\">Roshanbinfar et al., 2020</xref>). All in all, SE is a very versatile fabrication technique, and this is also increased by the possibility of combining it with other materials and technologies. However, it faces limitations related to the thickness and dimensions of the final construct, as well as long-range alignment capacity.</p></sec><sec id=\"S3.SS4.SSS1.Px4\"><title>Textile-based fabrication</title><p>Historically speaking, weaving is one of the oldest fabrication techniques and many (bio)materials can be processed by knitting, weaving or braiding, (<xref rid=\"B3\" ref-type=\"bibr\">Akbari et al., 2016</xref>) giving rise to new architectures and importantly, mechanical properties unattainable for individual fibres. Curiously, these have not been mainstream in the cTE field, with few studies combining these fabrication methods with hPSC-derived cells. Though their work cannot fully be categorised as textile-based, the lab of Kevin K. Parker employed pull spinning to generate nanofibrous scaffolds composed of PCL/Gelatin over an ellipsoidal mandrel, mimicking the shape of an idealised ventricle. After additional coating with the ECM protein fibronectin, they seeded either neonatal cardiac rat cells or hiPSC-CMs, generating scale-models of the heart with striking functional properties (<xref rid=\"B164\" ref-type=\"bibr\">Macqueen et al., 2018</xref>).</p></sec></sec><sec id=\"S3.SS4.SSS2\"><title>Techniques With True Architecture Control</title><p>Additive manufacturing, also known as 3D printing, is one of the technological revolutions of this century. It is based on the building of a volumetric object from a computer aided design (CAD), often in a layer-by-layer basis. Broadly speaking, we can distinguish between bioprinting, where cells are also printed, from the build & seed strategies, where the scaffold is first printed and the cells incorporated in a subsequent step, either as a standalone element or embedded in a hydrogel matrix. In any case, the degree of control over the resulting structure is orders of magnitude above what is achievable with the above-mentioned technologies, with the exception of filament dimensions, with SE being able to go into sub-micron diameter, albeit at the expense of thickness and architecture.</p><sec id=\"S3.SS4.SSS2.Px1\"><title>3D Build and Seed</title><p>In this category we have gathered those additive manufacturing technologies that do not allow the concurrent printing of materials and cells, be it because they employ high temperature, damaging lasers, toxic solvents or for other reasons. This section includes selective laser sintering (SLS), consisting of iteratively spreading layers of powdered materials and fusing it together to achieve the programmed shape, (<xref rid=\"B62\" ref-type=\"bibr\">Duan et al., 2010</xref>) stereolithography, where a bath with photosensitive resin is selectively cured layer by layer with a UV laser or similar power source, (<xref rid=\"B84\" ref-type=\"bibr\">Gauvin et al., 2012</xref>) or fused deposition modelling (FDM), by which the printed polymer is melted and deposited in layers in order to acquire the desired 3D architecture (<xref rid=\"B191\" ref-type=\"bibr\">Moroni et al., 2006</xref>). All these technologies have in common the need to first generate the architecture and in a second step add the biologicals, with the only exception of laser-curable materials in SLS. Although these technologies are in increased demand for the building of prosthetics, personalised solid implants or educational/surgery planning models, (<xref rid=\"B87\" ref-type=\"bibr\">Giannopoulos et al., 2016</xref>) their application to cTE is not widespread, especially in light of the increasing importance of bioprinting techniques (next section).</p><p>A special note however must be dedicated to Melt Electrowriting (MEW), a highly specialised additive manufacturing technology (<xref rid=\"B32\" ref-type=\"bibr\">Brown et al., 2011</xref>). It works on the same basis as SE, where a high voltage is applied between a syringe tip and a grounded collector. In this case however, the polymer is not dissolved, but melted. The superior stability of the jet allows a highly controlled deposition of fibres, which solidify in-flight or shortly upon collection. These, although bigger in diameter than those from SE, are at least an order of magnitude below what is usually attainable with 3D printing or bioprinting (tens of μm in diameter). The polymer of choice must be melted within the capacities of the printer, which limits the range of the available polymers. Another significant limitation is the achievable 3-dimensionality, which is usually below the mm-range. The groups of Paul Dalton and Dietmar Hutmacher have spearheaded its use (<xref rid=\"B18\" ref-type=\"bibr\">Bas et al., 2015</xref>, <xref rid=\"B17\" ref-type=\"bibr\">2017</xref>; <xref rid=\"B105\" ref-type=\"bibr\">Hochleitner et al., 2018</xref>) and have developed methods to increase the thickness of the MEW scaffolds to almost one cm, but again these are far from mainstream (<xref rid=\"B297\" ref-type=\"bibr\">Wunner et al., 2018</xref>). The groups of Jos Malda and Joost Sluijter have been the first ones to apply MEW to cTE. In their first approach, they MEW-printed the hydroxyl-functionalised polyester, (poly(hydroxymethylglycolide-co-ε-caprolactone) in orthogonal patterns, and seeded them with human cardiac progenitor cells (CPC) embedded in a collagen type I hydrogel. CPCs survived well, with the scaffolds producing a significant alignment (<xref rid=\"B45\" ref-type=\"bibr\">Castilho et al., 2017</xref>). In a further optimisation of the system, Castilho et al. fabricated hexagonally patterned MEW scaffolds with superior compliance, and seeded them with hiPSC-CMs. Their results showed the superior biaxial mechanical properties of the hexagonal designs, and their significant, positive effect on cell alignment and gene expression. Moreover, these scaffolds could be transplanted in a large animal (pig) after passing through a catheter-like tube, paving the way for their future percutaneous transplantation (<xref rid=\"B46\" ref-type=\"bibr\">Castilho et al., 2018</xref>). Finally, an auxetic patch, that is, featuring a negative Poisson ratio, was recently fabricated with MEW, and the conductive polymer polypyrrole <italic>in situ</italic> polymerised on it, with electroconductive properties close to those of the human myocardium (<xref rid=\"B203\" ref-type=\"bibr\">Olvera et al., 2020</xref>).</p></sec><sec id=\"S3.SS4.SSS2.Px2\"><title>Bioprinting</title><p>The capacity to print human tissues on-demand has roused the expectations of scientists, clinicians and patients alike, with a range of modalities available. Laser-assisted bioprinting (LAB) for example, features several methodologies. Laser-induced forward transfer of material/ink (LIFT)(<xref rid=\"B144\" ref-type=\"bibr\">Koch et al., 2010</xref>) allows printing with a single-cell resolution at high densities (up to 10<sup>8</sup> cells/ml) (<xref rid=\"B80\" ref-type=\"bibr\">Gaebel et al., 2011</xref>). However, it requires selected materials to gel relatively fast, which in practice restricts the range of available materials, and can be technically challenging. 2 photon polymerisation is a type of LAB with high spatial resolution (as low as 70 nm) without the need for support structures (<xref rid=\"B109\" ref-type=\"bibr\">Hu et al., 2017</xref>). Vaithilingam et al. employed this technique to fabricate electroactive 3D architectures by incorporation of multi-walled carbon nanotubes (CNTs). The mechanical properties were however highly deviated from what has been described for human cardiac tissue (GPa range). hiPSC-CMs were able to survive and, after the application of electrical stimulation, a significant effect on sarcomere length was found (<xref rid=\"B273\" ref-type=\"bibr\">Vaithilingam et al., 2019</xref>). There are other printing technologies based on LAB, such as Biological Laser Printing and Laser Guided Direct Writing, (<xref rid=\"B202\" ref-type=\"bibr\">Odde and Renn, 2000</xref>; <xref rid=\"B16\" ref-type=\"bibr\">Barron et al., 2004</xref>) but no reports on these include hiPSC-derived cardiac cells.</p><p>Extrusion-based bioprinting is probably the best-known bioprinting method: a bioink containing both cells and the biomaterial is extruded through a nozzle onto a motorised collector. The coordination between the gelation of the bioink and its deposition permits the generation of thick tissues. A range of nozzles, including pneumatic, screw- or plunger-based, as well as inks, is available. However, the material properties of the latter are fundamental, as shear stress generated upon printing highly impacts cell viability (<xref rid=\"B124\" ref-type=\"bibr\">Jungst et al., 2016</xref>). Some strategies, like <italic>in situ</italic> crosslinking of the bioink immediately prior to its deposition, (<xref rid=\"B208\" ref-type=\"bibr\">Ouyang et al., 2017</xref>) are being developed to overcome these and other limitations. Extrusion-based bioprinting has been extensively researched in the cTE field. Jang et al. employed a multi-nozzle printer to generate tissues with 2 decellularised cardiac ECM-based bioinks reinforced with a thermoplastic (PCL) backbone. One of the bioinks contained human CPCs (c-kit-positive), and the other mesenchymal stem cells (MSC) and pro-angiogenic factors (VEGF). The application of this patch in a rat model of disease demonstrated a rapid vascularisation as well as a significant benefit at the functional and histological levels. However, no new contractile tissue was formed (<xref rid=\"B119\" ref-type=\"bibr\">Jang et al., 2017</xref>). Zhang and coworkers employed a more advanced strategy, where they used coaxial printing to pattern human umbilical vein endothelial cells (HUVECs) in a ink containing alginate, GelMA and the UV-curable photoinitiator Irgacure. The mixture was quickly crosslinked by CaCl<sub>2</sub> from the outer sheath, and further gelled by exposure to UV light (for alginate and GelMA respectively) (<xref rid=\"B316\" ref-type=\"bibr\">Zhang Y. S. et al., 2016</xref>). HUVECs migrated to the outer border of the printed structures, forming 3D vessel-like structures over a period of 15 days, after which neonatal rat CMs were added to complete the tissue. Izadifar et al. explored varying the electrical properties of the generated tissues by incorporating alginate-coated carbon nanotubes (CNT) to methacrylated collagen. The system displayed as expected enhanced electrical properties, once more at the expense of becoming stiffer, though within acceptable values, and was able to support the growth of human coronary artery endothelial cells. No CMs were employed (<xref rid=\"B113\" ref-type=\"bibr\">Izadifar et al., 2018</xref>). Maiullari and coworkers applied a different version of extrusion printing termed microfluidic printing, to generate vascularised engineered tissues combining HUVECs and hiPSC-CMs (<xref rid=\"B165\" ref-type=\"bibr\">Maiullari et al., 2018</xref>). The bioartificial myocardium was fabricated with a bioink combining alginate for a rapid crosslinking, and polyethylene glycol (PEG) monoacrylate-fibrinogen, for UV-curing and cell adhesion. These tissues were tested <italic>in vivo</italic>, were the presence of vascular cells was able to promote integration with the recipient vascular system. Zhu et al. formulated a bioink with capacity to electrically influence neonatal rat CMs by incorporating gold nanorods in GelMA. Thick constructs were generated, although cell viability was below 80% in most cases. This is extremely relevant if we take into account that CMs are non-proliferative cells, so no re-growth is likely to occur (<xref rid=\"B319\" ref-type=\"bibr\">Zhu et al., 2017</xref>).</p><p>Freeform embedding of suspended hydrogels (FRESH) is one of most exciting 3D bioprinting methods (<xref rid=\"B103\" ref-type=\"bibr\">Hinton et al., 2015</xref>). Here, the biomaterial ink or bioink of choice is printed within the support of another hydrogel serving as a temporary support. Noor and co-workers developed a personalised hydrogel ink from human decellularised omental tissue, which could be printed into complex structures using FRESH. They used it in combination with hiPSC which they differentiated within the matrix into beating, perfused cardiac mini-tissues (<xref rid=\"B198\" ref-type=\"bibr\">Noor et al., 2019</xref>). Lastly, the Feinberg group used FRESH to accurately (20 μm resolution) 3D print different components of the human heart with collagen, including a model of the left ventricle on the scale of the neonatal organ, incorporating hESC-CMs (<xref rid=\"B152\" ref-type=\"bibr\">Lee et al., 2019</xref>). These engineered tissues displayed synchronised functional activity.</p><p>Finally, some the applications build tissues without the use of biomaterials. This includes the additive manufacturing with spheroids, as demonstrated by Arai et al., where they combined commercially available hPSC-CMs with HUVECs and dermal fibroblasts to generate the spheroids, organised in a tubular 3D structure with a bioprinter (<xref rid=\"B7\" ref-type=\"bibr\">Arai et al., 2018</xref>). However, the authors used a needle array to provide initial transient support for their structures, which in the end played the role of a scaffolding material. Recently, Ayan et al. reported a novel strategy, termed aspiration-assisted bioprinting, relying on the aspiration of 3D spheroids and their precise deposition in 3D. This could also be combined with FRESH, but no use of hiPSC-derived cardiac cells was reported (<xref rid=\"B9\" ref-type=\"bibr\">Ayan et al., 2020</xref>).</p></sec></sec></sec></sec><sec id=\"S4\"><title>Clinical Translation of hPSC-Based cTE Strategies</title><p>Survival post-cardiac ischemia has raised with advances in the cardiology and cardiovascular surgery areas, as well as through the development of new drugs in the last decades. This includes the application of reperfusion by percutaneous coronary intervention, anticoagulants and antithrombotics, amongst others. Though cTE has potential to address different cardiac conditions, its main activity has been directed towards the loss of viable myocardium by supplying new one, engineered in the lab.</p><p>In stark contrast to the short time elapsing between the first reports on regenerative medicine with adult stem cells and their first in-human application (reviewed in <xref rid=\"B12\" ref-type=\"bibr\">Banerjee et al., 2018</xref>) cTE with hPSCs has scarcely reached the clinical arena. Be it because both scientists and clinicians have learnt from past experiences or because translation presents additional complications, (<xref rid=\"B58\" ref-type=\"bibr\">Desgres and Menasché, 2019</xref>; <xref rid=\"B85\" ref-type=\"bibr\">Ghaemi et al., 2019</xref>; <xref rid=\"B133\" ref-type=\"bibr\">Ke and Murphy, 2019</xref>) clinical application is yet to become a reality. The technology faces challenges in the area of regulation, where reprogrammed cells under GMP-conditions are not widely derived, in logistics, with the size of engineered tissues in most instances requiring open chest surgery, and in economic terms. However, progress is being made. Tiburcy et al., created an engineered human myocardium with advanced structural and functional maturation by combining hPSC-derived cells with collagen type I (<xref rid=\"B266\" ref-type=\"bibr\">Tiburcy et al., 2017</xref>). Their work, standing on the shoulders of decade-long progress, (<xref rid=\"B322\" ref-type=\"bibr\">Zimmermann et al., 2002</xref>, <xref rid=\"B321\" ref-type=\"bibr\">2006</xref>) demonstrated the building of human-scale relevant tissues (3.5 × 3.4 cm, containing 40 million CMs) under defined conditions and their pre-clinical safety assessment in rats, though no large animal testing was performed. The group of Prof Bursac applied dynamic culture conditions to improve the maturation of a patch formed from hiPSC-derived cardiac cells in a fibrin matrix. Although they achieved human adult-like electrical properties (e.g., conduction velocity) and formed large grafts when transplanted into rodents, the use of matrigel in the forming mixture precludes further translation (<xref rid=\"B245\" ref-type=\"bibr\">Shadrin et al., 2017</xref>; <xref rid=\"B115\" ref-type=\"bibr\">Jackman et al., 2018</xref>). In addition, myocytes were mostly randomly arrayed, which added to the low thickness of the grafts precludes any efficient and substantial force generation, and therefore makes providing contractile support to diseased hearts difficult.</p><p>Menasche et al. performed the first clinical case of an hPSC-based patch in 2015, transplanting a 20 cm<sup>2</sup> fibrin-based tissue. This patch contained 4 million hESC-derived Isl<sup>+</sup> SSEA1<sup>+</sup> CPCs and was implanted epicardially in a 68-year-old patient with advanced heart failure. Concomitant to the implantation of the patch, a coronary artery bypass was also performed. After 3 months, the patient had better symptoms, and there was improved evidence in the echocardiogram too (form akinetic to moderately hypokinetic). Moreover, no adverse effect was observed (<xref rid=\"B180\" ref-type=\"bibr\">Menasché et al., 2015</xref>). This was part of the first clinical trial carried out with six patients suffering from advanced IHD (NCT02057900), called ESCORT. The aim of the trial was to demonstrate the safety of the patch at 1 year. All patients improved symptomatically, with four showing an increased systolic motion. One died early in the post-operative period from treatment-unrelated comorbidities and another after in 22 months due to heart failure (<xref rid=\"B181\" ref-type=\"bibr\">Menasché et al., 2018</xref>).</p><p>Japan has made huge efforts to spearhead research in the hiPSC field, and has one of the most streamlined regulatory frameworks (see <xref rid=\"B55\" ref-type=\"bibr\">Cyranoski, 2019</xref> for a comment). It has recently started the first clinical trial of hiPSC-CM transplant in ischemic heart dysfunction. The study will deliver 100 million hiPSC-CMs in the form of a cell sheet of 4 × 5 × 0.1 cm (<xref rid=\"B132\" ref-type=\"bibr\">Kawamura et al., 2017</xref>). The aim of the study is to evaluate efficacy and safety of the treatment in a total of 10 patients (<xref rid=\"B54\" ref-type=\"bibr\">Cyranoski, 2018</xref>).</p><p>Overall, clinical applications of hPSC-based cardiac engineered tissues, though scarce, are starting to emerge. However, the scientific and logistic/economic concerns mentioned above need addressing further before any of these technologies reach the clinical arena.</p></sec><sec id=\"S5\"><title>Conclusion and Outlook</title><p>The capacity to fabricate human myocardium in the laboratory is no longer restricted to science fiction. Recent breakthroughs such as cell reprogramming and 3D (bio)printing have shifted the question from ‘Can we do it?’ to ‘How do we want to do it?’. However, lessons from its predecessor in the area of regenerative medicine should teach us the value of caution. Crucial questions remain, some of which include the following:</p><p>-<italic>What are the most suitable components for building an engineered myocardium?</italic> Of course, this absolutely depends on the application/pathology we are targeting. If the objective is to reconstruct the post-disease myocardium, especially in cases when muscular mass has been loss (as IHD), this will require new CMs, alongside new vessels and support cells. Although not supplying new CMs might still be beneficial, it will not regenerate the tissue to pre-disease levels. So far, only hPSCs can produce the required cells in sufficiently large amounts, and whether it is better to use fully differentiated cells or progenitors is still an open question. Regarding the ECM-mimics, the current state can seem confusing. All reported (bio)material formulations seem to provide a benefit (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). It is plausible that the most important factor is for the material to recapitulate the mechanical environment of cardiac tissue long enough for delivered cells to build their own structure. Also, although collagen type I has repeatedly been positioned as being the main component of the cardiac ECM, this does not account for all its mechanical capacities, nor it is as abundant as the levels supplemented in engineered tissues. Probably, cost and translation capacity will have much to say here. Lastly, regarding the fabrication mode, though 3D bioprinting has captivated both scientists and the wider public, its actual ability to deliver tissues able to withstand the mechanical forces in play in the human myocardium is far from optimal in most cases. Fibre reinforcement might be a solution to this, but remains to be fully tested in a human scale. In addition, recent technical developments such as 4D printing or cyborganics will certainly widen the scope of possibilities at hand (<xref rid=\"B260\" ref-type=\"bibr\">Tamay et al., 2019</xref>; <xref rid=\"B204\" ref-type=\"bibr\">Orive et al., 2020</xref>).</p><p>– <italic>How is translation going to happen?</italic> The hurdles are significant, (<xref rid=\"B58\" ref-type=\"bibr\">Desgres and Menasché, 2019</xref>; <xref rid=\"B85\" ref-type=\"bibr\">Ghaemi et al., 2019</xref>) not only due to the use of genetically modified cells (hiPSCs), exogenous materials and complex equipment, but also because in order to attain sufficient functional capacity, it is likely that some lengthy period of <italic>in vitro</italic> electromechanical maturation must be implemented. Also, no standard equipment currently exists to apply this to a human-scale tissue. In this area, it is crucial that biomedical researchers work alongside lawmakers. Cell therapy mostly relied on an autologous application of a non-cultured material, which smoothened its approval. Tissue engineering applications based on hiPSC products are highly promising but are impeded by stringent regulations. Japan has spearheaded hiPSC translation as a national flagship (<xref rid=\"B10\" ref-type=\"bibr\">Azuma and Yamanaka, 2016</xref>). Although some consider this too loose, it is clear that unless scientists provide a clear direction and get themselves involved in depth in the regulatory process, advances will be limited.</p><p>– <italic>Will costs be sensible?</italic> hiPSC culture and differentiation are expensive. Additive manufacturing is expensive. GMP-grade materials are expensive. hiPSC line derivation costs between €4000-8000 depending on the provider, multi-nozzle 3D bioprinters are usually budgeted above €30000, and some medical-grade materials as human collagen type I are prohibitive (> €200 per 100 μg). Highly trained personnel costs need to be added on top of this, as well as the cost of hiPSC expansion and differentiation. The cost of a heart transplant in the United States is above $150000 (including 120-day medical care), with Left Ventricular Assist Devices (LVADs), employed for patients with end stage heart failure as a bridge to transplant (and now trialled as destination therapy) are estimated to cost even more (see <xref rid=\"B183\" ref-type=\"bibr\">Miller et al., 2013</xref> for a concise review). If economic issues are not addressed from day one, we run the risk of ending up with a highly effective therapy only that very few people can pay for. Large-scale production of hiPSC-derived cells has been demonstrated and will no doubt help decrease costs (<xref rid=\"B1\" ref-type=\"bibr\">Abecasis et al., 2017</xref>; <xref rid=\"B94\" ref-type=\"bibr\">Halloin et al., 2019</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Buikema et al., 2020</xref>). Strategies to induce hiPSC-CM division would significantly improve the economics.</p><p>– <italic>Will the new therapy be safe?</italic> hPSCs have been marred by concerns related to persistence of pluripotent cells after differentiation and the subsequent risk of teratoma formation. But also, the new tissue must conform to the host’s electrical activity. Again, lessons from Cell Therapy call for caution (<xref rid=\"B179\" ref-type=\"bibr\">Menasché et al., 2008</xref>). Advanced genomics and other analysis tools not available 10 years ago will significantly enhance the safety analysis and help researchers and clinicians along the way (<xref rid=\"B284\" ref-type=\"bibr\">Wang Z. et al., 2020</xref>).</p><p>– <italic>Are the aims achievable?</italic> The objective of cTE is to generate new human myocardium whose properties and functionality are similar to its natural counterpart, but that might not be achievable within a sensible timeframe. Human cardiac maturation advances significantly after birth but continues throughout the first decades of life. Current advanced methods for maturation, like those based on electromechanical stimulation, do not achieve anything even close to adult properties. However, it is plausible that only a minimal maturity is needed in order to avoid lethal arrhythmias, as hPSC-CMs are reported to undergo maturation after transplantation in the heart (<xref rid=\"B126\" ref-type=\"bibr\">Kadota et al., 2017</xref>).</p><p>– <italic>What is the state of non-therapeutic applications?</italic> Though the aim of cTE within regenerative medicine is directed towards treatment, bioartificial human myocardium is becoming increasingly relevant with respect to other applications such as disease modelling and drug testing (<xref rid=\"B280\" ref-type=\"bibr\">Vunjak-Novakovic et al., 2014</xref>; <xref rid=\"B246\" ref-type=\"bibr\">Sharma et al., 2020</xref>), where the scale of the fabricated tissues is not an issue. In drug testing, the CiPA initiative has already pointed to the relevance of hiPSC-CMs, (<xref rid=\"B182\" ref-type=\"bibr\">Millard et al., 2018</xref>) especially given cardiac side effects are one of the main reasons for drug withdrawal, and there is no primary source of human CMs available (<xref rid=\"B151\" ref-type=\"bibr\">Laverty et al., 2011</xref>). Current efforts have already proven the capacity of cTE-based systems to better recapitulate physiological human drug response [reviewed in <xref rid=\"B126\" ref-type=\"bibr\">Kadota et al. (2017)</xref> and <xref rid=\"B182\" ref-type=\"bibr\">Millard et al. (2018)</xref>, or to generate chamber-specific microtissues (<xref rid=\"B317\" ref-type=\"bibr\">Zhao et al., 2019</xref>)]. Translation towards industrial and clinical testing is envisioned to be more straightforward and immediate.</p><p>As can be seen, the challenges ahead are not minor. However, humankind has never been closer to achieving success in tissue fabrication. Whole functional heart generation in the laboratory, though frequently dreamt of, is still not achievable, as severe physiological and technical roadblocks remain in place. Challenges include ensuring adequate vascularisation, electromechanical coupling over long distances, atrioventricular delay, autonomous nervous system innervation or designing materials both biocompatible but capable of supporting the strong mechanics of a whole heart, amongst others. At this extraordinary moment, it is crucial for scientists, clinicians, and lay people to stay focused and deliver.</p></sec><sec id=\"S6\"><title>Author Contributions</title><p>PM, MF-I, SM, and MP revised the literature, wrote the initial draft of the manuscript and figures. DP and JG revised the text and generated the tables. CS, GO, and FP supervised the development and revised the text. MM wrote the final revised version. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by funds from the ISCIII Red TERCEL RETIC RD16/0011/0005, PI 19/01350, ERANET II (Nanoreheart) and Gobierno de Navarra Departamento de Salud GN<sup>a</sup>8/2019, co-funded by FEDER funds, MINECO (Program RETOS Cardiomesh RTC-2016-4911-1), Gobierno de Navarra 0011-1383-2019-000006 and 0011-1383-2018-000011, and European Union’s H2020 Program under grant agreement No. 874827 (BRAV∃).</p></fn></fn-group><ack><p>The authors acknowledge Medical Arts and <ext-link ext-link-type=\"uri\" xlink:href=\"https://biorender.com/\">BioRender</ext-link> (registered user) for the building of the figures in the manuscript.</p></ack><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abecasis</surname><given-names>B.</given-names></name><name><surname>Aguiar</surname><given-names>T.</given-names></name><name><surname>Arnault</surname><given-names>É</given-names></name><name><surname>Costa</surname><given-names>R.</given-names></name><name><surname>Gomes-Alves</surname><given-names>P.</given-names></name><name><surname>Aspegren</surname><given-names>A.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: bioprocess intensification and scaling-up approaches.</article-title>\n<source><italic>J. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Physiol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Physiol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Physiol.</journal-id><journal-title-group><journal-title>Frontiers in Physiology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-042X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32848895</article-id><article-id pub-id-type=\"pmc\">PMC7431659</article-id><article-id pub-id-type=\"doi\">10.3389/fphys.2020.00993</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Physiology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Communication Among Photoreceptors and the Central Clock Affects Sleep Profile</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Damulewicz</surname><given-names>Milena</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/229916/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ispizua</surname><given-names>Juan I.</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/998484/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ceriani</surname><given-names>Maria F.</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/55150/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Pyza</surname><given-names>Elzbieta M.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/50674/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Cell Biology and Imaging, Jagiellonian University</institution>, <addr-line>Kraków</addr-line>, <country>Poland</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Laboratorio de Genética del Comportamiento, Fundación Instituto Leloir, IIBBA-CONICET</institution>, <addr-line>Buenos Aires</addr-line>, <country>Argentina</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Ezio Rosato, University of Leicester, United Kingdom</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Pamela Menegazzi, Julius Maximilian University of Würzburg, Germany; Eran Tauber, University of Haifa, Israel</p></fn><corresp id=\"c001\">*Correspondence: Milena Damulewicz, <email>milena.damulewicz@uj.edu.pl</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Chronobiology, a section of the journal Frontiers in Physiology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>993</elocation-id><history><date date-type=\"received\"><day>02</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>22</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Damulewicz, Ispizua, Ceriani and Pyza.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Damulewicz, Ispizua, Ceriani and Pyza</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Light is one of the most important factors regulating rhythmical behavior of <italic>Drosophila melanogaster</italic>. It is received by different photoreceptors and entrains the circadian clock, which controls sleep. The retina is known to be essential for light perception, as it is composed of specialized light-sensitive cells which transmit signal to deeper parts of the brain. In this study we examined the role of specific photoreceptor types and peripheral oscillators located in these cells in the regulation of sleep pattern. We showed that sleep is controlled by the visual system in a very complex way. Photoreceptors expressing Rh1, Rh3 are involved in night-time sleep regulation, while cells expressing Rh5 and Rh6 affect sleep both during the day and night. Moreover, Hofbauer-Buchner (HB) eyelets which can directly contact with s-LN<sub><italic>v</italic></sub>s and l-LN<sub><italic>v</italic></sub>s play a wake-promoting function during the day. In addition, we showed that L2 interneurons, which receive signal from R1-6, form direct synaptic contacts with l-LN<sub><italic>v</italic></sub>s, which provides new light input to the clock network.</p></abstract><kwd-group><kwd>sleep</kwd><kwd>photoreceptors</kwd><kwd>Hofbauer-Buchner eyelets</kwd><kwd><italic>Drosophila</italic></kwd><kwd>peripheral clock</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Narodowe Centrum Nauki<named-content content-type=\"fundref-id\">10.13039/501100004281</named-content></funding-source></award-group><award-group><funding-source id=\"cn002\">Ministerstwo Nauki i Szkolnictwa Wyższego<named-content content-type=\"fundref-id\">10.13039/501100004569</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"11\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"75\"/><page-count count=\"17\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Circadian rhythms in <italic>Drosophila</italic> are regulated by a system of oscillators, which includes the pacemaker located in the central brain and peripheral oscillators located in various cells, tissues, and organs. Peripheral oscillators such as the ones in glial cells, compound eyes, antennae, gustatory receptor neurons, or Malpighian tubules express clock genes and show circadian rhythms in their structure and physiological processes (<xref rid=\"B62\" ref-type=\"bibr\">Siwicki et al., 1988</xref>; <xref rid=\"B74\" ref-type=\"bibr\">Zerr et al., 1990</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Hege et al., 1997</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Krishnan et al., 1999</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Chatterjee and Hardin, 2010</xref>; <xref rid=\"B2\" ref-type=\"bibr\">Chatterjee et al., 2010</xref>).</p><p>The compound eye consists of ommatidia and each of them contains eight photoreceptors. Six of them, R1–R6, are located in the distal retina and express rhodopsin 1 (Rh1), sensitive to a broad spectrum of light wavelengths (<xref rid=\"B42\" ref-type=\"bibr\">O’Tousa et al., 1985</xref>; <xref rid=\"B16\" ref-type=\"bibr\">Hardie, 1987</xref>). R1–R6 terminate in the lamina, where they form tetrad synaptic contacts with L1, L2, L3, and amacrine cells (<xref rid=\"B33\" ref-type=\"bibr\">Meinertzhagen and O’Neil, 1991</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Meinertzhagen and Sorra, 2001</xref>). The R1–R6 photoreceptors are involved in motion detection and image formation (<xref rid=\"B50\" ref-type=\"bibr\">Rister et al., 2007</xref>; <xref rid=\"B71\" ref-type=\"bibr\">Yamaguchi et al., 2008</xref>). Two other photoreceptors of each ommatidium, R7 and R8, are involved in color vision and detection of polarized light. They terminate in the second optic neuropil (medulla), where they contact transmedulla neurons (Tm5, Tm9, and Tm20; <xref rid=\"B10\" ref-type=\"bibr\">Gao et al., 2008</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Melnattur et al., 2014</xref>). Tm5, Tm20 neurons receive also indirect input from R1–R6, through L3 (<xref rid=\"B10\" ref-type=\"bibr\">Gao et al., 2008</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Melnattur et al., 2014</xref>). R7 forms additional synaptic contacts with amacrine cell Dm8 (<xref rid=\"B10\" ref-type=\"bibr\">Gao et al., 2008</xref>), and moreover, R7 and R8 contact each other in the medulla through direct synaptic contacts (<xref rid=\"B65\" ref-type=\"bibr\">Takemura et al., 2015</xref>), most probably using histamine as a neurotransmitter (<xref rid=\"B17\" ref-type=\"bibr\">Hardie and Raghu, 2001</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Pantazis et al., 2008</xref>; <xref rid=\"B57\" ref-type=\"bibr\">Schnaitmann et al., 2018</xref>). R7 photoreceptors express UV-sensitive rhodopsin 3 (Rh3) or blue light – absorbing Rh4, while R8 cells express Rh3, blue light-sensitive Rh5 or green light-absorbing Rh6 (<xref rid=\"B52\" ref-type=\"bibr\">Salcedo et al., 1999</xref>).</p><p>According to the rhodopsin type expressed in R7 and R8, three different subpopulations of photoreceptors have been described: “pale” ommatidia consist of Rh3-expressing R7 and Rh5-expressing R8, the “yellow” type is composed of Rh4 expressing R7 and Rh6-expressing R8, and “DRA” (dorsal rim area) expresses Rh3 in both R7 and R8 and is involved in the polarized light detection (<xref rid=\"B70\" ref-type=\"bibr\">Wernet et al., 2006</xref>). An additional photoreceptive structure in the visual system is the Hofbauer–Buchner (HB) eyelets (<xref rid=\"B21\" ref-type=\"bibr\">Hofbauer and Buchner, 1989</xref>; <xref rid=\"B72\" ref-type=\"bibr\">Yasuyama and Meinertzhagen, 1999</xref>) composed of 4 cells expressing Rh6, terminating in the accessory medulla (<xref rid=\"B19\" ref-type=\"bibr\">Helfrich-Förster et al., 2002</xref>).</p><p>Different photoreceptor types and photopigments seem to play different roles in the circadian rhythm and behavior regulation. R1–R6, expressing Rh1, play a role in dim light detection in motion vision and phototaxis; they are also important for nocturnal activity which increases in response to raising day-light activity (<xref rid=\"B54\" ref-type=\"bibr\">Schlichting et al., 2014</xref>). Rhodopsin 6, expressed in a population of R8 photoreceptors, plays a role in the integration of light signals received by the other photoreceptors (<xref rid=\"B51\" ref-type=\"bibr\">Saint-Charles et al., 2016</xref>), while Rh5-expressing cells seems to be involved in light entrainment, that is the clock ability to adapt to light cycles and phase shifts of the rhythm, which suggests that Rh5 may use NorpA-independent pathway (<xref rid=\"B51\" ref-type=\"bibr\">Saint-Charles et al., 2016</xref>). Finally, HB eyelets play a role in regulating evening onset under high intensity light conditions, as well as the length of the siesta (<xref rid=\"B56\" ref-type=\"bibr\">Schlichting et al., 2019</xref>).</p><p>The role of the visual system in circadian entrainment has already been studied (<xref rid=\"B38\" ref-type=\"bibr\">Nippe et al., 2017</xref>), however, the effect of different photoreceptors on sleep is still not fully recognized. In this study we examined the role of different photoreceptors and their postsynaptic targets in the regulation of sleep and locomotor activity. We have shown that Rh1 and Rh3-expressing photoreceptors affect sleep during the night, while Rh5 and Rh6-expressing cells, both during the day and night. Moreover, we have presented that photoreceptors influence the pace of the molecular clock in pacemaker cells and that retinal oscillators are as much important for maintaining sleep as synaptic transmission from photoreceptors to target cells. Finally, we have described connections between the visual system and clock cells.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Fly Strains</title><p>The following strains of <italic>Drosophila melanogaster</italic> were used in the present study: GMR-Gal4, <italic>Rh1</italic>-Gal4, <italic>Rh3</italic>-Gal4, <italic>sp/Cy0; Rh5</italic>-Gal4, <italic>yw; Sp/Cy0; Rh6</italic>-Gal4/TM6B, R82F12AD/Cy0; R75H08DBD/TM6B (herein called <italic>L2</italic>-Gal4), <italic>yw; Pdf</italic>-Gal4, <italic>w</italic>; UAS-Δ<italic>cyc24, w</italic>; UAS-<italic>TeTxLC</italic> (herein called UAS-<italic>TeTx</italic>), <italic>w; Pdf-</italic>LexA,AS<italic>-GFP<sub>1–10</sub>/Cy0;</italic> LexAop<italic>-GFP<sub>11</sub></italic> (for GRASP experiment), <italic>yw</italic>,UAS<italic>-myrGFP</italic>,QUAS<italic>-HA::RFP; transTANGO</italic> (herein called <italic>transTANGO</italic>), <italic>w</italic>; UAS-<italic>GCaMP6f</italic>, w; <italic>Pdf</italic>-LexA,LexAopGFP<sub>11</sub>; UAS-Nrx::GFP<sub>1–10</sub> (for Nrx GRASP experiment).</p><p>L2-Gal4 strain was obtained from Janelia Research Campus, the others from Bloomington Drosophila Stock Center.</p><p>Flies were maintained under 12 h of light and 12 h of darkness (LD12:12) conditions and at a constant temperature of 25°C, unless the procedure required constant darkness (DD).</p></sec><sec id=\"S2.SS2\"><title>Recording Locomotor Activity and Sleep</title><p>The locomotor activity was recorded using a monitoring system (TriKinetics) composed of monitors equipped with infrared light-emitting diodes and detectors, connected to a computer. Each monitor houses 32 glass-tubes of a diameter just sufficient to maintain a single fly. Tubes are sealed at both ends: one by food and the other by a foam stopper. When the fly passes the emitter/detector pair, the infrared beam is interrupted resulting in a signal transmitted to the computer. To analyze circadian rhythms in locomotor activity, flies were maintained for 7 days under LD12:12 and next under constant darkness (DD) for next 7 days.</p><p>Activity was counted every 5 min (1 bin) and analyzed in Excel by using “Befly!” software (Department of Genetics, University of Leicester). Lomb–Scargle normalized periodogram was used to determine rhythmic flies; flies with period value lower than 10 (confidence level 0.05) were regarded as arrhythmic. Flies which did not survive until the end of experiments were removed from analyses. Every experiment was repeated three times, at least 60 flies in total were used.</p><p>The Anticipation Index (Morning and Evening) was calculated for individual flies under LD12:12, by determining the proportion of activity counts during the 3 h preceding the phase transition over the activity within 6 h preceding phase transition.</p><p>To study sleep pattern, activity of flies was analyzed in the second day in LD12:12. Sleep was measured as intervals of at least 5 min of inactivity.</p><p>Heterozygous parental strains were used as control. In case of gene silencing, progeny of driver line crossed with UAS-VALIUM10-GFP was used as additional control. This strain has expression of empty VALIUM10-GFP vector in targeted cells.</p><p>Statistical analysis was performed using ANOVA with a Tukey’s multiple comparison test for normally distributed data. To analyze rhythmicity of flies we used non-parametric Kruskal–Wallis test to compare percentage from three repetitions. GraphPad Software was used to performed statistical analysis.</p></sec><sec id=\"S2.SS3\"><title>Immunohistochemistry</title><p>Flies were decapitated and their heads were fixed in 4% paraformaldehyde in phosphate buffer saline (PBS; pH 7.4) for 4 h, then they were washed in PBS twice and cryoprotected by incubation in 12.5% sucrose for 10 min and in 25% sucrose at 4°C overnight. Material was embedded in Tissue Tek, frozen in liquid nitrogen, and cryostat 20 μm sections were cut. The sections were washed in PBS for 30 min and five times in phosphate buffer with an addition of 0.2% Triton X100 (PBT). After that, sections were incubated in 5% normal goat serum (NGS) with an addition of 0.5% bovine serum albumin (BSA) for 30 min at room temperature. Next they were incubated with primary antibodies for 24 h. Afterwards, sections were washed six times in PBT/BSA, blocked in 5% NGS for 45 min and secondary antibodies were applied for overnight incubation at 4°C. Finally, sections were washed twice in BSA, six times in PBT, and twice in PBS. Then, cryosections were mounted in Vectashield medium (Vector) and examined with a Zeiss Meta 510 Laser Scanning Microscope.</p><p>In addition to sections of the brain, whole brains were also used for immunohistochemistry. They were isolated after 1 h of head fixation in 4% PFA, washed in PBS and fixed again for the next 45 min. The next steps of immunostaining were done according to the protocol described above for cryosections.</p><p>For immunohistochemistry the following antibodies were used: nc82 (against the presynaptic protein Bruchpilot) (1:20, Developmental Studies Hybridoma Bank), PDF C7 (against Pigment Dispersing Factor) (1:500, Developmental Studies Hybridoma Bank), anti-GFP (rabbit, 1:1000, Novus Biological), anti-GFP (mouse, 1:20, Sigma Aldrich), goat anti-mouse conjugated with Cy3 (1:500, Jackson ImmunoResearch Laboratories, Inc.), goat anti-rabbit conjugated with Alexa 488 (1:1000, Molecular Probes), goat anti-mouse conjugated with Cy2 (1:500, Abcam).</p></sec><sec id=\"S2.SS4\"><title>Calcium Imaging</title><p>L2><italic>GCaMP6f</italic> flies were dissected on ice at specific time points, and brains were placed in PBS. Images of the brain were collected immediately with a confocal microscope. The fluorescence intensity was measured using ImageJ software. The ratio of fluorescence per area was calculated using ImageJ macro. Data obtained at different time points were compared.</p></sec><sec id=\"S2.SS5\"><title>TransTANGO</title><p>L2-Gal4 or GMR-Gal4 crossed with <italic>transTANGO</italic> flies were raised at 18°C, adult males were separated and aged for 15 days at 18°C. The ICCs procedure was the same as described above, except for the length of the incubation with primary antibody, which was extended to 5 days at 4°C. The following primary antibodies were used: rabbit anti-DsRed (1:250, Rockland), chicken anti-GFP (1:250, Aves Labs) and rat anti-PDF (1:250; <xref rid=\"B7\" ref-type=\"bibr\">Depetris-Chauvin et al., 2011</xref>). The following secondary antibodies were used: Cy2-conjugated anti-chicken, Cy5-conjugated anti-rat, and Cy3-conjugated anti-rabbit (1:250, Jackson ImmunoResearch Laboratories, Inc). Images were acquired with a ZEISS LSM 880 Confocal Laser Scanning Microscope.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Retina Photoreceptors Are Involved in Sleep Regulation</title><p>To analyze the contribution of specific photoreceptors to the regulation of rhythmic behavior we first examined the impact of blocking light input through the retina. In order to do so we took advantage of the GMR><italic>TeTx</italic> strain, in which neurotransmission from cells expressing <italic>glass</italic> was totally blocked by expression of tetanus toxin light chain, which cleaves synaptobrevin protein and blocks neurotransmitter release. GMR><italic>TeTx</italic> flies showed no defects in rhythmicity or periodicity, but the evening anticipation was increased (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Interestingly, these flies exhibited an altered sleep profile (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>) with increased sleep time during the day (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>), resulting in lower levels of total activity (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1A</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Locomotor activity of flies with disrupted synaptic transmission (TeTx) or molecular clock (Δ<italic>cyc24</italic>) in the specific type of cells in the visual system.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Genotype</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Period [h]</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">% Rhythmic</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MAI</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">EAI</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Number of flies</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UAS-<italic>cycΔ24</italic>/+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">93</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.63</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.76</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">88</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UAS-<italic>TeTx</italic>/+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">94</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.56</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.79</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">84</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UAS-<italic>ChATRNAi</italic>/+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.69</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.65</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">58</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GMR-Gal4/+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.54</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.74</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GMR>Δcyc24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>22.9****</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>45*</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.9****</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GMR><italic>TeTx</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.52</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.91****</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh1</italic>-Gal4/+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh1</italic>>Δcyc24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">77</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.79</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh1</italic>><italic>TeTx</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.79****</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh3</italic>-Gal4/+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">97</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.71</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.72</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">124</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh3</italic>>Δ<italic>cyc24</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">83</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.57</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.72</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">71</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh3</italic>><italic>TeTx</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">75</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.73</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">84</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh5</italic>-Gal4/+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.63</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.81</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">95</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh5</italic>>Δcyc</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.59</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh5</italic>><italic>TeTx</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">97</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.56</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.71</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">89</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh6</italic>-Gal4/+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.63</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">78</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rh6>Δcyc</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.59</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.91****</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">74</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh6</italic>><italic>TeTx</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">66</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.72</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">74</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh6</italic>><italic>Val10</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">96</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">55</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh6</italic>><italic>ChatRNAi</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">86</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.52</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.88*</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">73</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">L2><italic>TeTx</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">96</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.65</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.87****</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">L2-Gal4/+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">90</td></tr></tbody></table><table-wrap-foot><attrib><italic>Columns present: genotype, period of locomotor activity, % of rhythmic flies, morning anticipation index (MAI), evening anticipation index (EAI), and total number of flies used for the experiments. Experimental flies which showed differences with both controls are highlighted with bold. Statistically significant differences marked with asterisks (*<italic>p</italic> ≤ 0.05; ****<italic>p</italic> ≤ 0.0001). Detailed statistics are presented in <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Tables S1</xref>, <xref ref-type=\"supplementary-material\" rid=\"TS2\">S2</xref>.</italic></attrib></table-wrap-foot></table-wrap><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Effects of <italic>glass</italic>-expressing cells on sleep. <bold>(A)</bold> Sleep pattern of flies with blocked neurotransmission from photoreceptors (GMR><italic>TeTx</italic>) measured as number of sleep bins per hour. <bold>(B)</bold> Total sleep time of GMR><italic>TeTx</italic> flies measured as minutes per 12 h, separately for day and night-time. <bold>(C)</bold> Sleep pattern of flies with clock disruption in photoreceptors (GMR>Δ<italic>cyc24</italic>). <bold>(D)</bold> Total sleep time of GMR>Δ<italic>cyc24</italic> flies. Heterozygous parental strains were used as control. Statistically significant differences marked with asterisk *<italic>p</italic> ≤ 0.05; **<italic>p</italic> ≤ 0.01; ****<italic>p</italic> ≤ 0.0001. Detailed statistics are presented in <xref ref-type=\"supplementary-material\" rid=\"TS3\">Supplementary Tables S3</xref>, <xref ref-type=\"supplementary-material\" rid=\"TS4\">S4</xref>.</p></caption><graphic xlink:href=\"fphys-11-00993-g001\"/></fig><p>The retina photoreceptors are peripheral oscillators with rhythmic expression of clock genes. To check whether retinal clocks are involved in the network regulating sleep in flies, we used GMR>Δ<italic>cyc24</italic> strain, in which expression of the dominant negative form of CYCLE causes disruption of the molecular clock in photoreceptors. We found that this manipulation triggered arrhythmicity in 55% of GMR>Δ<italic>cyc24</italic> flies in constant darkness conditions (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>) and in rhythmic flies, the period was shorter (22.9 h) compared to the control (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Moreover these flies exhibited an increased morning anticipation index (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>), altered sleep pattern and length during the day and night (<xref ref-type=\"fig\" rid=\"F1\">Figures 1C,D</xref>), and a decreased total activity (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1A</xref>). Knowing that <italic>cyc</italic> dominant negative overexpression in <italic>tim</italic>-Gal4 cells is lethal (<xref rid=\"B3\" ref-type=\"bibr\">Chow et al., 2016</xref>), we carefully examined the eye morphology and anti-BRP labeling in the brain to exclude a possibility that the observed results originate from some neuronal degeneration. However, we did not observe any changes in the examined individuals.</p><p>These two experiments showed that <italic>glass</italic>-expressing cells, which are mostly retinal photoreceptors, are involved in the regulation of sleep, while they are not necessary to maintain the locomotor activity rhythm, as it has already been shown (<xref rid=\"B13\" ref-type=\"bibr\">Grima et al., 2004</xref>).</p></sec><sec id=\"S3.SS2\"><title>The Clock Located in the Retina Photoreceptors Regulates Their Own Circadian Output</title><p>To investigate how oscillators located in the retina transmit rhythmic signals to the deep brain, we looked at the effect of peripheral clock disruption on the presynaptic protein Bruchpilot (BRP) cycling in the photoreceptor terminals in the lamina. Under LD12:12 BRP levels oscillate daily with two maxima observed at the beginning of the day (ZT1) and at the beginning of the night (ZT13) (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Górska-Andrzejak et al., 2013</xref>). Interestingly, in our study in GMR>Δ<italic>cyc24</italic> flies the expression was changed, the BRP level at ZT1 was not significantly different than at ZT4 and ZT16, and only one peak at the beginning of the night (ZT13) was observed (<xref ref-type=\"fig\" rid=\"F2\">Figures 2B,C</xref>). This result is in accordance to what was already described for DD conditions, reinforcing the notion that morning BRP peak is controlled by light (<xref rid=\"B11\" ref-type=\"bibr\">Górska-Andrzejak et al., 2013</xref>). However, the fluorescence intensity level at ZT1 in the experimental and control flies was similar, and enhanced at ZT4 and ZT16, which may suggest that BRP degradation rather than expression is affected.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Effects of peripheral clocks located in the photoreceptors on the presynaptic protein Bruchpilot (BRP) expression. The immunofluorescence signal intensity was measured in the distal lamina on cryosections of control [Canton S, <bold>(A)</bold>] and experimental [GMR>Δ<italic>cyc24</italic>, <bold>(B)</bold>] flies at four time points (ZT1, ZT4, ZT13, and ZT16). Statistically significant differences were marked with letters, where different letters above the bar means confirmed changes between time points. <bold>(C)</bold> Confocal images of BRP immunostaining in the lamina of GMR>Δ<italic>cyc24</italic> at different time points.</p></caption><graphic xlink:href=\"fphys-11-00993-g002\"/></fig><p>We then explored the effect of peripheral clock located in the photoreceptors on the pace of the main oscillator, by blocking neurotransmission from the retina photoreceptors (GMR><italic>TeTx</italic>) or disrupting the clock in these cells (GMR>Δ<italic>cyc24)</italic>. Strikingly, we found that GMR><italic>TeTx</italic> and GMR>Δ<italic>cyc24</italic> flies display a clear dampening of PER oscillations in the small and large LN<sub><italic>v</italic></sub>s (<xref ref-type=\"fig\" rid=\"F3\">Figures 3A–D</xref>, respectively). Thus, blocking neurotransmission from the retina photoreceptors and disruption of the clock in <italic>glass</italic>-expressing cells decreased the amplitude in PER cycling in essential pacemaker neurons (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Effects of peripheral clock located in the photoreceptors on <italic>per</italic> expression in the pacemaker. The immunofluorescence signal intensity was measured in s-LN<sub><italic>v</italic></sub>s and l-LN<sub><italic>v</italic></sub>s marked with anti-PDF staining for GMR><italic>TeTx</italic>\n<bold>(A,B)</bold>, and GMR>Δ<italic>cyc24</italic>\n<bold>(C,D)</bold> experimental flies. Asterisks (*<italic>p</italic> ≤ 0.05; **<italic>p</italic> ≤ 0.01; ****<italic>p</italic> ≤ 0.0001) show statistically significant differences between experimental flies and controls at specific time point. Detailed statistics are presented in <xref ref-type=\"supplementary-material\" rid=\"TS5\">Supplementary Table S5</xref>.</p></caption><graphic xlink:href=\"fphys-11-00993-g003\"/></fig></sec><sec id=\"S3.SS3\"><title>Specific Photoreceptor Types Regulate Sleep at Different Ways</title><p>Because our experiments with GMR strain showed changes in sleep pattern and level, we focused on this behavior in the next experiments. GMR expression is not limited to the retina, however. In fact, GMR is expressed in some clock neurons (DN1p; <xref rid=\"B24\" ref-type=\"bibr\">Klarsfeld et al., 2004</xref>), which are involved in the sleep regulation (<xref rid=\"B14\" ref-type=\"bibr\">Guo et al., 2018</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Lamaze and Stanewsky, 2020</xref>). To investigate which cell types triggered the observed responses, we expressed tetanus toxin in different types of photoreceptors, using various rhodopsin drivers, which allowed us to exclude the effect of DN1p. Surprisingly, we obtained different effects depending on photoreceptor types. In general, blocking input from different photoreceptor types resulted in decreased total activity, but their effects on sleep and morning/evening peaks were different. No changes in the period of locomotor activity were observed (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><p>After blocking light transmission pathway from R1–R6 cells (<italic>Rh1</italic>-expressing; <xref ref-type=\"fig\" rid=\"F4\">Figures 4A,B</xref>) we observed clear changes in the sleep pattern (<xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>) with significant longer sleep time during the night, no effect on day-time sleep (<xref ref-type=\"fig\" rid=\"F4\">Figure 4D</xref>), and a subtly reduced evening peak (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS2\">Supplementary Figure S2A</xref>). These effects were opposite to those observed in case of GMR; nevertheless, in this case a decrease of total activity was also observed (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1B</xref>).</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>R1-6 photoreceptors control sleep during the night. <bold>(A)</bold>\n<italic>Rh1</italic>-expressing cells are R1-6 photoreceptors, with terminals in the lamina (cryosection of <italic>Rh1</italic>>GFP brain). <bold>(B)</bold> Graphical presentation of pathways which are blocked after TeTx expression in <italic>Rh1</italic>-expressing cells (a – amacrine cells, L1-3 – lamina monopolar cells). <bold>(C)</bold> Sleep pattern of <italic>Rh1>TeTx</italic> flies. <bold>(D)</bold> Total sleep amount during the day and night after blocking of synaptic transmission from R1-6 (<italic>Rh1>TeTx</italic>). <bold>(E)</bold> Sleep pattern for <italic>Rh1>Δcyc24</italic> flies. <bold>(F)</bold> Total sleep time of <italic>Rh1>Δcyc24</italic> strain. Heterozygous parental strains were used as control. Statistically significant differences marked with asterisks ****<italic>p</italic> ≤ 0.0001. Detailed statistics are presented in <xref ref-type=\"supplementary-material\" rid=\"TS3\">Supplementary Table S3</xref>.</p></caption><graphic xlink:href=\"fphys-11-00993-g004\"/></fig><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Morning and evening peaks of activity of flies with disrupted synaptic signaling (TeTx) or molecular clock in specific type of cells in the visual system compared with parental strains (Gal4/+ and UAS/+, respectively).</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"5\" rowspan=\"1\">Morning peak<hr/></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"5\" rowspan=\"1\">Evening peak<hr/></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GAL4/+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic>-value</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">UAS/+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic>-value</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F</italic> (DFn, dFD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Gal4/+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic>-value</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">UAS/+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic>-value</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F</italic> (DFn, dFD)</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh1</italic>><italic>TeTx</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">83.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">76.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.271</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">84.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.9985</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.080(2,384)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>100.2</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">183.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.0001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">134.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0007</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">45.68(2,384)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh1</italic>><italic>cycΔ24</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">69.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">76.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.3044</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">85.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.015</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.93(2,281)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">112.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">183.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.0001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">129.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.1753</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">37.43(2,360)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh3</italic>><italic>TeTx</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>98.4</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">79.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0011</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">84.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0171</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.576(2,341)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>77.0</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">111.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0002</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">134.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.0001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24.51(2,341)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh3</italic>><italic>cycΔ24</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>105.2</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">79.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.0001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">85.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0036</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13.38(2,242)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">113.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">111.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.9576</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">129.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.1486</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.077(2,321)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh5</italic>><italic>TeTx</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>64.2</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">83.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0004</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">84.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11.05(2,396)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>81.6</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">116.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.0001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">134.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.0001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.04(2,396)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh5</italic>><italic>cycΔ24</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>58.7</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">83.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0002</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">85.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0007</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.931(2,239)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>86.7</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">116.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">129.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.0001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14.51(2,318)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh6</italic>><italic>Tetx</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">93.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">72.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0004</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">84.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.1361</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.514(2,330)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">101.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">102.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.9928</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">134.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0002</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11.84(2,330)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh6</italic>><italic>cycΔ24</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">84.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">72.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0603</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">85.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.9663</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.904(2,224)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">131.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">102.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0029</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">129.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.9677</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.013(2,303)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>L2</italic>><italic>TeTx</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>59.3</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">75.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0014</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">84.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.0001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.05(2,360)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">113.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">111.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.9647</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">134.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0157</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.635(2,360)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rh6</italic>><italic>ChaTRNAi</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">51.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">87.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.0001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">52.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.9991</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25.27(2,247)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>109.3</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">84.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0011</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">39.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.0001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">49.43(2,247)</td></tr></tbody></table><table-wrap-foot><attrib><italic>Experimental flies which showed statistically significant differences with both controls are highlighted with bold. Each experimental strain was compared with control strains (Gal4 and UAS) using one-way ANOVA and Tukey’s test. Genotypes with <italic>p</italic> < 0.05 with both controls are marked as statistically significant changes with bold. Degrees of freedom [<italic>F</italic> (DFn, DFd)] are listed for every group.</italic></attrib></table-wrap-foot></table-wrap><p>Similar effects were obtained after disrupting the clock in the R1-6 photoreceptors (<xref ref-type=\"fig\" rid=\"F4\">Figure 4E</xref>), despite the impact on night-time sleep was less pronounced (<xref ref-type=\"fig\" rid=\"F4\">Figure 4F</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS3\">Supplementary Figure S3A</xref>).</p><p>We next inquired the relevance of the R7-8 photoreceptors. The block of neurotransmission from the Rh3-expressing cells, reaching the medulla (<xref ref-type=\"fig\" rid=\"F5\">Figures 5A,B</xref>), had strong effect on the activity patterns, with an increased morning peak and a decreased evening peak (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS2\">Supplementary Figure S2B</xref>). The sleep pattern was also affected (<xref ref-type=\"fig\" rid=\"F5\">Figure 5C</xref>), with increased night-time sleep (<xref ref-type=\"fig\" rid=\"F5\">Figure 5D</xref>), and decreased total activity (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1C</xref>). Similarly, the clock disruption in these cells resulted in a decreased total activity, although the impact on night-time sleep was less pronounced (<xref ref-type=\"fig\" rid=\"F5\">Figures 5E,F</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figures S1C</xref>, <xref ref-type=\"supplementary-material\" rid=\"FS3\">S3B</xref>).</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p><italic>Rh3</italic>-expressing cells affect night-time sleep. <bold>(A)</bold> Rh3 is expressed in R7 and R8 cells, which terminate in the medulla (cryosection of <italic>Rh3</italic>>GFP brain). <bold>(B)</bold> Graphical presentation of synaptic connections formed by <italic>Rh3</italic>-expressing cells which are blocked in <italic>Rh3>TeTx</italic> flies (Tm – transmedulla neurons). <bold>(C)</bold> Sleep pattern of <italic>Rh3>TeTx</italic> flies. <bold>(D)</bold> Sleep time during the day and night of <italic>Rh3>TeTx</italic> flies. <bold>(E)</bold> Sleep pattern of <italic>Rh3>Δcyc24.</italic>\n<bold>(F)</bold> Amount of sleep during the day and night of <italic>Rh3>Δcyc24.</italic> Heterozygous parental strains were used as control. Statistically significant differences marked with asterisks **<italic>p</italic> ≤ 0.01; ***<italic>p</italic> ≤ 0.001; ****<italic>p</italic> ≤ 0.0001. Detailed statistics are presented in <xref ref-type=\"supplementary-material\" rid=\"TS3\">Supplementary Table S3</xref>.</p></caption><graphic xlink:href=\"fphys-11-00993-g005\"/></fig><p>Next we examined the contribution of Rh5-expressing R8 “pale” ommatidia (<xref ref-type=\"fig\" rid=\"F6\">Figures 6A,B</xref>). <italic>Rh5</italic>><italic>TeTx</italic> flies exhibited reduced total activity (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1D</xref>) and both morning and evening activity peaks were decreased compared to controls (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS2\">Supplementary Figure S2C</xref>). The sleep pattern changed (<xref ref-type=\"fig\" rid=\"F6\">Figure 6C</xref>) with increased sleep time in both, day and night (<xref ref-type=\"fig\" rid=\"F6\">Figure 6D</xref>). Overexpression of <italic>cycΔ24</italic> in “pale” R8 cells recreated the effects linked to TeTx expression: decreased total activity (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1D</xref>), blunted morning and evening peaks (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS3\">Supplementary Figure S3C</xref>) and changes in the sleep pattern (<xref ref-type=\"fig\" rid=\"F6\">Figure 6E</xref>) and level during the day and night (<xref ref-type=\"fig\" rid=\"F6\">Figure 6F</xref>).</p><fig id=\"F6\" position=\"float\"><label>FIGURE 6</label><caption><p><italic>Rh5</italic>-expressing cells affect sleep both, during the day and night. <bold>(A)</bold> Rh5 is expressed in R8 cell which terminates in the medulla (cryosection of <italic>Rh5</italic>>GFP brain). <bold>(B)</bold> Graphical representation of pathways blocked in <italic>Rh5>TeTx</italic> strain. Solid line represents input to R8 cell coming from R7. Dashed line represents blocked output from R8 to downstream cells. <bold>(C)</bold> Sleep pattern of <italic>Rh5>TeTx.</italic>\n<bold>(D)</bold> Sleep time during the day and night of <italic>Rh5>TeTx</italic> flies. <bold>(E)</bold> Sleep pattern of <italic>Rh5>Δcyc24.</italic>\n<bold>(F)</bold> Amount of sleep during the day and night of <italic>Rh5>Δcyc24.</italic> Heterozygous parental strains were used as control. Statistically significant differences marked with asterisks *<italic>p</italic> ≤ 0.05; ***<italic>p</italic> ≤ 0.001; ****<italic>p</italic> ≤ 0.0001). Detailed statistics are presented in <xref ref-type=\"supplementary-material\" rid=\"TS3\">Supplementary Table S3</xref>.</p></caption><graphic xlink:href=\"fphys-11-00993-g006\"/></fig><p>Rh6 is expressed in the R8 “yellow” ommatidia as well as in the HB eyelets (<xref ref-type=\"fig\" rid=\"F7\">Figures 7A,B</xref>). Blocking the synaptic transmission from these cells did not affect overall activity levels (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figures S1E</xref>, <xref ref-type=\"supplementary-material\" rid=\"FS2\">S2D</xref>) and the sleep was slightly extended at the beginning of the day and during the night (<xref ref-type=\"fig\" rid=\"F7\">Figures 7C,D</xref>). Clock disruption in this type of photoreceptors only mirrored the one triggered during the day (<xref ref-type=\"fig\" rid=\"F7\">Figures 7E,F</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS3\">Supplementary Figure S3D</xref>).</p><fig id=\"F7\" position=\"float\"><label>FIGURE 7</label><caption><p><italic>Rh6</italic>-expressing cells regulate sleep during the day and night. <bold>(A)</bold>\n<italic>Rh6</italic>-expressing photoreceptors (R8) terminate in the medulla. The cryosection of <italic>Rh6</italic>>GFP brain does not show Hofbauer-Buchner (HB) eyelets. <bold>(B)</bold> Graphical presentation of synaptic contacts (dotted line) between <italic>Rh6</italic>-expressing photoreceptors or HB eyelets and their targets. Solid line represent input to R8 coming from R7 cell. <bold>(C)</bold> Sleep pattern of <italic>Rh6>TeTx</italic> flies. <bold>(D)</bold> Sleep time of <italic>Rh6>TeTx</italic> flies is increased during the day and night. <bold>(E)</bold> Sleep pattern of <italic>Rh6>Δcyc24.</italic>\n<bold>(F)</bold> Sleep amount of <italic>Rh6>Δcyc24</italic>. <bold>(G)</bold> Sleep pattern of flies with downregulated acetylcholine synthesis in HB eyelets (<italic>Rh6>ChAT-RNAi</italic>). <bold>(H)</bold> Sleep amount of <italic>Rh6>ChAT-RNAi</italic> is increased during the day only. Heterozygous parental strains were used as control, additional control <italic>Rh6>Valium10-GFP</italic> was used for the last experiment. Statistically significant differences marked with asterisks *<italic>p</italic> ≤ 0.05; ****<italic>p</italic> ≤ 0.0001. Detailed statistics are presented in <xref ref-type=\"supplementary-material\" rid=\"TS3\">Supplementary Table S3</xref>.</p></caption><graphic xlink:href=\"fphys-11-00993-g007\"/></fig><p>To distinguish between the contribution of R8 photoreceptors and the HB eyelets, both expressing Rh6, we knocked down the expression of choline acetyltransferase, necessary for the synthesis of acetylcholine, used as neurotransmitter by the HB eyelets. In <italic>Rh6</italic>><italic>ChATRNAi</italic> flies changes in sleep pattern and increased sleep time became evident only during the day (<xref ref-type=\"fig\" rid=\"F7\">Figures 7G,H</xref>).</p><p>Taking these data together, we can conclude that R1-6 cells, as well as Rh3 and Rh6-expressing retinal photoreceptors affect sleep during the night, while R8 expressing Rh5 is involved in both day- and night-time sleep regulation. Moreover, cholinergic HB eyelets play a wake-promoting role during the day.</p></sec><sec id=\"S3.SS4\"><title>L2 Interneurons as an Additional Peripheral Clock Output</title><p>Since light signals are transmitted to the pacemaker neurons from the retina photoreceptors via the lamina interneurons, we examined the behavior of flies with blocked neurotransmission in L2 interneurons, which receive light input from R1–R6 cells (<xref ref-type=\"fig\" rid=\"F8\">Figure 8A</xref>). We focused on this cell type because it shows rhythmic changes in the size of dendritic trees,’ probably connected with daily changes in signal transmission. Moreover, L2 terminals are located in a close vicinity to l-LN<sub><italic>v</italic></sub>s (<xref ref-type=\"fig\" rid=\"F8\">Figure 8B</xref>). These flies displayed behavioral changes reminiscent of GMR><italic>TeTx</italic> flies, with a smaller morning peak of activity (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). The sleep pattern of these flies was changed (<xref ref-type=\"fig\" rid=\"F8\">Figure 8C</xref>) with increased sleep duration during the day and night (<xref ref-type=\"fig\" rid=\"F8\">Figure 8D</xref>).</p><fig id=\"F8\" position=\"float\"><label>FIGURE 8</label><caption><p>L2 interneurons play important role in the regulation of sleep. <bold>(A)</bold> Graphical presentation of synaptic contacts formed by L2 (dotted line) (Tm –Transmedulla neurons). <bold>(B)</bold> L2 terminals are located in close proximity to PDF-immunoreactive LN<sub><italic>v</italic></sub>s neurons (whole mount immunostaining of L2>GFP flies with anti-GFP and anti-PDF antibodies, blue). <bold>(C)</bold> Sleep pattern of L2><italic>TeTx</italic> flies. <bold>(D)</bold> Sleep amount presented for flies with tetanus toxin expression in L2 cells. Statistically significant differences marked with asterisks ***<italic>p</italic> < 0.001; ****<italic>p</italic> < 0.0001. <bold>(E)</bold> Fluorescence calcium indicator expressed in L2 cells was measured in the terminals in the medulla. <bold>(F)</bold> Calcium level in the L2 terminals measured in flies kept in LD12:12 conditions. <bold>(G)</bold> Calcium level in the L2 terminals of flies in constant darkness (DD). Statistically significant differences were marked with letters, where different letters above the bar means confirmed changes between time points.</p></caption><graphic xlink:href=\"fphys-11-00993-g008\"/></fig><p>To correlate daily changes in the size of dendritic trees’ with activity of L2 cells, we carried out calcium imaging using L2><italic>GCaMP6f</italic> transgenic strain. GCaMP6f is a calcium indicator, which allows to measure Ca<sup>2+</sup> level correlated to the indicator fluorescence intensity. In our experiment we isolated brains at selected time points and measured the fluorescent signal immediately after dissection. The fluorescent intensity in the L2 terminals was measured in the medulla (<xref ref-type=\"fig\" rid=\"F8\">Figure 8E</xref>) and calculated per terminal area, comparing Ca<sup>2+</sup> level/area unit time points. In LD12:12, Ca<sup>2+</sup> level was highest at night (ZT16, ZT20) and lowest in the middle of the day (ZT8; <xref ref-type=\"fig\" rid=\"F8\">Figure 8F</xref>). In DD this pattern subtly changed, with the highest intensity signal at CT16 and CT20, but without the lowest level at ZT8 (<xref ref-type=\"fig\" rid=\"F8\">Figure 8G</xref>).</p><p>The obtained results suggest that L2 interneurons play important wake-promoting role. It is supported by the fact that calcium levels in the terminals are the lowest during siesta.</p></sec><sec id=\"S3.SS5\"><title>Photic Inputs to the Pacemaker May Be Regulated by Synaptic Plasticity</title><p>Some photoreceptor terminals are located next to the clock neurons in a region called the accessory medulla (<xref ref-type=\"fig\" rid=\"F9\">Figures 9A,B</xref>). It was previously shown that the visual system can directly communicate with clock neurons and receive photic information through the HB eyelets (<xref rid=\"B36\" ref-type=\"bibr\">Muraro and Ceriani, 2015</xref>; <xref rid=\"B55\" ref-type=\"bibr\">Schlichting et al., 2016</xref>). Taking advantage of the GFP reconstitution across synaptic partners (GRASP) technique (<xref rid=\"B9\" ref-type=\"bibr\">Feinberg et al., 2008</xref>) we found that the HB eyelets terminals differentially contact the LN<sub><italic>v</italic></sub>s during the day, with stronger contacts during light phase (<xref ref-type=\"fig\" rid=\"F9\">Figure 9C</xref>). To confirm that these results correlate with differential synaptic connectivity, we used Nrx GRASP to visualize only active synaptic contacts (<xref ref-type=\"fig\" rid=\"F9\">Figures 9D,E</xref>). At ZT1 100% of brains showed reconstituted signal (<italic>n</italic> = 20), the proportion of brains decreased at other time points, i.e., 43% at ZT4 (<italic>n</italic> = 21), 59% at ZT13 (<italic>n</italic> = 22), and 35% at ZT16 (<italic>n</italic> = 20), suggesting that the LN<sub><italic>v</italic></sub>s receive direct input from the HB eyelets in a plastic manner, preferentially during the early morning.</p><fig id=\"F9\" position=\"float\"><label>FIGURE 9</label><caption><p>Hofbauer-Buchner eyelets contact with LN<sub><italic>v</italic></sub>s. <bold>(A,B)</bold> Immunostaining of GMR>GFP flies shows that HB terminals are located in aMe area, in close proximity to s-LN<sub><italic>v</italic></sub>s. <bold>(C)</bold> GFP reconstitution across synaptic partners (GRASP) technique allows to visualize synaptic contacts between HB eyelets and PDF-expressing cells, according to location identified as s-LN<sub><italic>v</italic></sub>s. <bold>(D,E)</bold> Nrx GRASP technique confirmed that HB form active synaptic contacts with s-LN<sub><italic>v</italic></sub>s.</p></caption><graphic xlink:href=\"fphys-11-00993-g009\"/></fig><p>The HB eyelets terminals arborize in a close vicinity to the s-LN<sub><italic>v</italic></sub>s cell bodies. Taking into account the localization of the reconstituted signal, it is likely the HB eyelets communicate with the s-LN<sub><italic>v</italic></sub>s rather than the l-LN<sub><italic>v</italic></sub>s.</p><p>To gain insight into how the information from peripheral oscillators is transmitted to the main clock we took advantage of <italic>transTANGO</italic> (<xref rid=\"B66\" ref-type=\"bibr\">Talay et al., 2017</xref>) to uncover postsynaptic cells to photoreceptors (GMR-Gal4) and L2 interneurons (L2-Gal4; <xref ref-type=\"fig\" rid=\"F10\">Figures 10</xref>, <xref ref-type=\"fig\" rid=\"F11\">11</xref>, respectively). This tool uses a modified signaling pathway to express RFP in the postsynaptic cells of a GAL4 of interest, while marking the cells recruited by that driver with GFP. In pursuit of the postsynaptic targets to GMR+ photoreceptors we observed red fluorescence in the lamina and medulla, probably coming from the lamina interneurons and amacrine cells. The signal was also detected in a few cell bodies in the accessory medulla, which project to the dorsal brain and to the contralateral side of the brain. Double staining with antibodies against PDF confirmed that a subset of RFP-expressing cells belong to both the s-LN<sub><italic>v</italic></sub> and l-LN<sub><italic>v</italic></sub> groups and to processes described as the posterior optic tract (POT), which connect the l-LN<sub><italic>v</italic></sub> cluster located on both sides of the brain, and dorsal projections of the s-LN<sub><italic>v</italic></sub>s. Closer inspection of the GFP signal indicated that the cells contacting LN<sub><italic>v</italic></sub>s are those of the HB eyelets (<xref ref-type=\"fig\" rid=\"F10\">Figure 10C</xref>), confirming what is depicted in the recently published connectome (<xref rid=\"B53\" ref-type=\"bibr\">Scheffer et al., 2020</xref>).</p><fig id=\"F10\" position=\"float\"><label>FIGURE 10</label><caption><p><italic>Glass</italic>-expressing cells contact both l-LN<sub><italic>v</italic></sub>s and s-LN<sub><italic>v</italic></sub>s. Immunostaining of GMR><italic>transTANGO</italic> adult male brain. <italic>transTANGO</italic> marks presynaptic <italic>glass</italic>-expressing cells with GFP and the postsynaptic partners of those cells with RFP. Anti-PDF immunostaining confirms that both small and large LN<sub><italic>v</italic></sub>s are postsynaptic to GMR-Gal4 recruited cells. <bold>(A)</bold> Hemibrain. Asterisks were placed where PDF and RFP labeling co-localize: in posterior optic tract (POT) coming from l-LN<sub><italic>v</italic></sub>s, s-LN<sub><italic>v</italic></sub>s, and their terminals in the dorsal brain. <bold>(B)</bold> l-LN<sub><italic>v</italic></sub>s somatas (labeled with PDF, blue) have postsynaptic mark (RFP, magenta), HB eyelets projections can be seen nearby (labeled with GFP, green). <bold>(C,D)</bold> s-LN<sub><italic>v</italic></sub>s somatas and terminals have postsynaptic mark (RFP, magenta). Every brain analyzed (<italic>n</italic> = 9) showed similar staining. The images were acquired from different brains.</p></caption><graphic xlink:href=\"fphys-11-00993-g010\"/></fig><fig id=\"F11\" position=\"float\"><label>FIGURE 11</label><caption><p>L2 interneurons form direct synaptic contacts with large but not small LN<sub><italic>v</italic></sub>s. Immunostaining of L2><italic>transTANGO</italic> adult male brain. <bold>(A)</bold> Hemibrain. Asterisks mark places where PDF and RFP labeling co-localize, which means l-LN<sub><italic>v</italic></sub>s cell bodies and POT. <bold>(B)</bold> l-LN<sub><italic>v</italic></sub>s somatas have postsynaptic labeling, meaning that l-LN<sub><italic>v</italic></sub>s form direct contacts with L2 interneurons. This was seen in 86% of brains (<italic>n</italic> = 7). A L2 projection can be seen in cyan, rounding a somata. <bold>(C,D)</bold> Neither somatas nor projections of the s-LN<sub><italic>v</italic></sub>s have postsynaptic labeling. None of the brains analyzed showed RFP signal in these cells. The images were acquired from different brains.</p></caption><graphic xlink:href=\"fphys-11-00993-g011\"/></fig><p>Despite no direct contact between L2 neurons and LN<sub><italic>v</italic></sub>s was detected through GRASP (<xref rid=\"B36\" ref-type=\"bibr\">Muraro and Ceriani, 2015</xref>), <italic>transTANGO</italic> mapping of L2 postsynaptic targets highlighted the l-LN<sub><italic>v</italic></sub>s, but not the s-LN<sub><italic>v</italic></sub>s, in the majority of brains (86% positive, <italic>n</italic> = 7) (<xref ref-type=\"fig\" rid=\"F11\">Figure 11</xref>). This l-LN<sub><italic>v</italic></sub>s exclusive connection could help explain why disrupting the connectivity or the clock in the retinal peripheral oscillator has a stronger effect on the sleep pattern than in other circadian outputs examined.</p></sec></sec><sec id=\"S4\"><title>Discussion</title><p>The circadian clock relies on a self-sustained molecular mechanism, which is entrained to the daily changes of environmental conditions. The most powerful factor synchronizing circadian clocks is light. The LN<sub><italic>v</italic></sub>s, essential circadian pacemakers, receive light information through the visual system and the deep brain photoreceptor CRY (<xref rid=\"B8\" ref-type=\"bibr\">Emery et al., 2000</xref>; <xref rid=\"B59\" ref-type=\"bibr\">Sheeba et al., 2008a</xref>).</p><p>The involvement of the visual system is still not completely understood. Pacemaker cells are responsible for the temporal organization of rhythmic behavior, such as locomotor activity and sleep but also contribute to other features such as sleep length during the day and night and total activity level (<xref rid=\"B44\" ref-type=\"bibr\">Parisky et al., 2008</xref>; <xref rid=\"B58\" ref-type=\"bibr\">Shang et al., 2008</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Chung et al., 2009</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Potdar and Sheeba, 2018</xref>). In this study we focused on the role of retinal and extra-retinal photoreceptors in the transduction of photic information to modulate sleep patterns. We uncovered that peripheral clocks in the eye contribute to maintaining sleep pattern with little effects on the period of locomotor activity rhythm, as expected (<xref rid=\"B13\" ref-type=\"bibr\">Grima et al., 2004</xref>).</p><p>Clock disruption in <italic>glass</italic>-expressing cells triggered arrhythmicity, and in the remaining rhythmic flies, a shorter period of the locomotor activity pattern. Surprisingly, blocking synaptic transmission from photoreceptors to postsynaptic cells add no effects on either rhythmicity or periodicity, however, observed changes in the sleep pattern and level were similar in both experiments. Subsets of clock neurons employ CRY for light entrainment, while the others use CUL-3 mediated mechanism for molecular light resetting (<xref rid=\"B40\" ref-type=\"bibr\">Ogueta et al., 2020</xref>). However, it seems that they need rhythmic inputs from the visual system, as arrhythmic signaling may affect the molecular mechanism of pacemaker neurons.</p><p>The visual system shows daily changes of sensitivity to light. In addition, the clock-controlled optomotor response in <italic>Drosophila</italic> is higher at night than during the day (<xref rid=\"B31\" ref-type=\"bibr\">Mazzotta et al., 2013</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Damulewicz et al., 2017</xref>). In the first neuropil (lamina) of the optic lobe, circadian rhythms have been observed at the cellular (<xref rid=\"B68\" ref-type=\"bibr\">Weber et al., 2009</xref>) and molecular levels (<xref rid=\"B11\" ref-type=\"bibr\">Górska-Andrzejak et al., 2013</xref>) including the expression of several genes and proteins, for example the alpha subunit of the sodium/potassium pump in the lamina glia (<xref rid=\"B12\" ref-type=\"bibr\">Górska-Andrzejak et al., 2009</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Damulewicz et al., 2013</xref>).</p><p>In the present study, we showed that not only sensitivity of photoreceptors, but also interneurons involved in light transmission changes during the day. L2 monopolar cells, one of the postsynaptic cells in tetrad synapses, which hyperpolarize in response to light, show daily rhythms in calcium levels. The highest Ca<sup>2+</sup> concentration in the terminals was observed at night, similarly to the retina photoreceptors. It seems that the visual system is more sensitive to light during the dark phase to detect low intensity light, such as moonlight, and such sensitivity decreases at the end of the night to be prepared for high intensity of light in the morning (<xref rid=\"B58\" ref-type=\"bibr\">Shang et al., 2008</xref>; <xref rid=\"B60\" ref-type=\"bibr\">Sheeba et al., 2008b</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Nippe et al., 2017</xref>).</p><p>We showed that rhythms observed in the lamina, like the presynaptic protein BRP expression, is controlled by both the pacemaker and the oscillators located in the retina. Moreover, the BRP expression pattern in flies in which the peripheral clock in the retina is disrupted, is unimodal, similar to that observed in constant darkness, when the morning peak of BRP is missing and only the evening peak is observed (<xref rid=\"B11\" ref-type=\"bibr\">Górska-Andrzejak et al., 2013</xref>). However, in our experimental model BRP level at ZT1 was similar to those observed in control, which suggests that clock disruption affects rather degradation of BRP than its expression.</p><p>Despite the strong effect on sleep of genetic manipulations in photoreceptors using the GMR-Gal4 driver, the <italic>glass</italic> gene is also expressed in some DN<sub>1</sub><sub><italic>s</italic></sub> and in a group of cells located next to LN<sub><italic>d</italic></sub>s (<xref rid=\"B67\" ref-type=\"bibr\">Vosshall and Young, 1995</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Klarsfeld et al., 2004</xref>), opening the possibility that the results obtained could not be exclusively eye-specific (<xref rid=\"B30\" ref-type=\"bibr\">Li et al., 2012</xref>; <xref rid=\"B49\" ref-type=\"bibr\">Ray and Lakhotia, 2015</xref>). To avoid multi-cellular effects, we focused on data performed with drivers specific to different photoreceptor types, and we observed various effects on sleep, depending on the photoreceptor type.</p><p>The previous analysis employing the <italic>norpA</italic> mutant, which lacks phospholipase C in the canonical phototransduction pathway (<xref rid=\"B45\" ref-type=\"bibr\">Pearn et al., 1996</xref>), showed that light signals to the clock are transmitted not exclusively from the retina photoreceptors, but also by the circadian photoreceptor CRY and the HB eyelets (<xref rid=\"B8\" ref-type=\"bibr\">Emery et al., 2000</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Helfrich-Förster et al., 2001</xref>). However, <italic>norpA</italic> mutants are not able to entrain to changes in light conditions, because of reduced circadian sensitivity to light (<xref rid=\"B8\" ref-type=\"bibr\">Emery et al., 2000</xref>). Moreover, in the double mutant <italic>norpA; cry<sup><italic>b</italic></sup></italic>, PER cycling was maintained in s-LN<sub><italic>v</italic></sub>s and DN<sub>1</sub>s but abolished in l-LN<sub><italic>v</italic></sub>s and LN<sub><italic>d</italic></sub>s, suggesting that these clusters are photoentrained by an alternative pathway (<xref rid=\"B20\" ref-type=\"bibr\">Helfrich-Förster et al., 2001</xref>). This conclusion is further supported by the fact, that an alternative <italic>norpA</italic>-independent phototransduction pathway occurs in Rh1, Rh5, and Rh6-expressing photoreceptors (<xref rid=\"B61\" ref-type=\"bibr\">Shortridge et al., 1991</xref>; <xref rid=\"B63\" ref-type=\"bibr\">Szular et al., 2012</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Ogueta et al., 2018</xref>). Flies with blocked synaptic transmission using GMR><italic>TeTx</italic> resembled the <italic>norpA</italic> mutant. Since this genetic combination blocks synaptic transmission between photoreceptors, R1–R8, the HB eyelets, and their postsynaptic partners, the effect on sleep pattern was stronger compare to that in <italic>norpA</italic> mutants. More severe changes in PER expression were observed in l-LN<sub><italic>v</italic></sub>s than s-LN<sub><italic>v</italic></sub>s after impairing input from the photoreceptor cells, which is consistent with previous reports (<xref rid=\"B20\" ref-type=\"bibr\">Helfrich-Förster et al., 2001</xref>).</p><p>Lack of photic information from R1–R6 increased sleep duration at night. However, blocking synaptic transmission from L2 interneurons caused more complex effects on both day- and night-time sleep. L2 interneurons are important for front-to-back motion detection at intermediate pattern contrast (<xref rid=\"B50\" ref-type=\"bibr\">Rister et al., 2007</xref>). They are postsynaptic to R1–R6 photoreceptors and to L4 interneurons and presynaptic in feedback synapses, which are formed back to them (both to R1-6 and L4) and use acetylcholine and glutamate as neurotransmitters (<xref rid=\"B33\" ref-type=\"bibr\">Meinertzhagen and O’Neil, 1991</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Kolodziejczyk et al., 2008</xref>; <xref rid=\"B48\" ref-type=\"bibr\">Raghu and Borst, 2011</xref>; <xref rid=\"B64\" ref-type=\"bibr\">Takemura et al., 2011</xref>, <xref rid=\"B65\" ref-type=\"bibr\">2015</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Hu et al., 2015</xref>). These feedback synapses play a role in preventing photoreceptors from saturation and improving signal quality (<xref rid=\"B75\" ref-type=\"bibr\">Zheng et al., 2006</xref>), however, they seem to be extremely important for the functioning of R1–R6 photoreceptors. R1-6 transmit signal to different types of cells, which belongs to pathways specialized to detect contrast increments (ON pathway, L1) or decrements (OFF pathway, L2, L3; <xref rid=\"B23\" ref-type=\"bibr\">Joesch et al., 2010</xref>). In effect, blocking R1-6 signaling affects both pathways, however, L2 cells can still receive signal from L4, and send information to deeper parts of the brain. On the other hand, lack of neurotransmission from L2 cells can disrupt not only downstream OFF pathway, but also proper R1-6 functioning, giving the effect on both, day- and night-time sleep. Moreover, according to our <italic>transTANGO</italic> data, L2 can also directly contact the l-LN<sub><italic>v</italic></sub>s. It is possible, however, that these synapses are formed only in a specific time of the day, or contacts are very weak and conventional GRASP is not strong enough to show positive results (<xref rid=\"B36\" ref-type=\"bibr\">Muraro and Ceriani, 2015</xref>). This aspect needs to be addressed by more detailed experiments in the future.</p><p>Surprisingly, the lack of synaptic transmission from Rh3-expressing cells affected the morning peak of activity in a very specific way, opposite to that observed after blocking signaling from Rh5. Rhodopsin 3 is expressed in R7 in “pale” ommatidia and in both, R7 and R8, in the DRA type. Since the block of transmission from “pale” ommatidia, with R8 expressing Rh5, decreased the morning peak of activity, it is possible that Rh3, which absorbs UV, inhibits morning activity, while blue light, which is absorbed by Rh5, enhances the morning activity. This can also be an effect of the disruption in polarized light detection, which is received by DRA only.</p><p>Siesta or day-time sleep seems to be mostly regulated by R8 “pale” type of photoreceptors. The results obtained in this work may support the idea that blue light inhibits sleep during the day. The exposure to blue light seems to affect lifespan and induce neurodegeneration (<xref rid=\"B37\" ref-type=\"bibr\">Nash et al., 2019</xref>), and short sleep during the day allow avoiding UV and blue light during the day, thus protecting flies against harmful light exposure.</p><p>Rh6-expressing photoreceptors affect sleep in a specific way, since nap was longer after clock disruption but sleep during the night and day was increased after blocking transmission. Rh6, however, is expressed not only in the retina photoreceptors, but also in the HB eyelets. Our data suggest that R8 cells are involved in night-time sleep and HB eyelets in day-time sleep regulation. Acetylcholine expression silenced in Rh6-expressing cells increased sleep time only during the day, while tetanus toxin expression affected both day and night sleep time. As it is known that among Rh6-expressing cells only HB eyelets use acetylcholine as neurotransmitter we can conclude that observed effect is specific for these extracellular photoreceptors. It has been reported that the HB eyelets form direct synaptic contacts with pacemaker cells located in the aMe (<xref rid=\"B36\" ref-type=\"bibr\">Muraro and Ceriani, 2015</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Li et al., 2018</xref>; <xref rid=\"B56\" ref-type=\"bibr\">Schlichting et al., 2019</xref>). Our GRASP results suggest that the s-LN<sub><italic>v</italic></sub>s are main target cells, however, it has been shown that also l-LN<sub><italic>v</italic></sub>s, ITP-expressing LN<sub><italic>d</italic></sub>s, DN<sub>1a</sub> and DN<sub>3a</sub> cells receive signals from the HB eyelets through terminals located in the aMe (<xref rid=\"B29\" ref-type=\"bibr\">Li et al., 2018</xref>), which was confirmed by <italic>transTANGO</italic> data. These contacts are the strongest at the beginning of the day, indicating that signaling between the HB eyelets and LN<sub><italic>v</italic></sub>s takes place in the morning. Down-regulation of histamine receptor expression in the pacemaker PDF-immunoreactive cells (<italic>Pdf</italic>><italic>HisCl</italic>) results in lack of the morning anticipation, which is typical of clock mutants. In turn disruption of histamine signaling from photoreceptors to pacemaker cells affects sleep by increasing its length during the day and night (<xref rid=\"B41\" ref-type=\"bibr\">Oh et al., 2013</xref>). This means that histaminergic neurotransmission from photoreceptors is involved in the regulation of LN<sub><italic>v</italic></sub>s activity. It has been shown that the HB eyelets express histamine and acetylcholine (<xref rid=\"B46\" ref-type=\"bibr\">Pollack and Hofbauer, 1991</xref>; <xref rid=\"B72\" ref-type=\"bibr\">Yasuyama and Meinertzhagen, 1999</xref>; <xref rid=\"B73\" ref-type=\"bibr\">Yasuyama and Salvaterra, 1999</xref>) and LN<sub><italic>v</italic></sub>s have receptors for both neurotransmitters (<xref rid=\"B32\" ref-type=\"bibr\">McCarthy et al., 2011</xref>; <xref rid=\"B28\" ref-type=\"bibr\">Lelito and Shafer, 2012</xref>). According to our Nrx GRASP data the HB eyelets communicate with the s-LN<sub><italic>v</italic></sub>s directly at the beginning of the day. <xref rid=\"B55\" ref-type=\"bibr\">Schlichting et al. (2016)</xref> suggest that HB eyelets may also contact the l-LN<sub><italic>v</italic></sub>s, as histamine bath had inhibitory effect on l-LN<sub><italic>v</italic></sub>s (<xref rid=\"B55\" ref-type=\"bibr\">Schlichting et al., 2016</xref>), nevertheless, there are also other histamine sources in the aMe (<xref rid=\"B15\" ref-type=\"bibr\">Hamasaka and Nassel, 2006</xref>). Light signal received by HB eyelets in the morning is transmitted via acetylcholine and excite s-LN<sub><italic>v</italic></sub>s via nicotinic receptors (<xref rid=\"B69\" ref-type=\"bibr\">Wegener et al., 2004</xref>; <xref rid=\"B32\" ref-type=\"bibr\">McCarthy et al., 2011</xref>; <xref rid=\"B55\" ref-type=\"bibr\">Schlichting et al., 2016</xref>), causing increased cAMP level (<xref rid=\"B28\" ref-type=\"bibr\">Lelito and Shafer, 2012</xref>) and wake-promoting effect. This supports our results, which showed that down-regulation of acetylcholine expression in HB eyelets increases sleep time during the day.</p><p>Taking together, R1-6, R7 (Rh3-expressing photoreceptor), and R8 “yellow” (Rh6-expressing cells) seem to be important for night-time sleep regulation, and HB eyelets for day-time sleep, while the strongest effect, both during the day and night, has neurotransmission from R8 “pale” (Rh5-expressing cells). We observed a similar effect on night-time sleep when synaptic transmission was blocked from all types of photoreceptors, which means that during the night even weak light inputs received by retinal cells are important to keep flies awake. As a result of the decreased photoreception flies are less active and spend more time sleeping during the night. However, it was previously shown that the retinal photoreceptors play a role of peripheral oscillators and regulate daily changes in the visual system, we found that those oscillators also affect behavior. Disruption of the clock in a single type of photoreceptors decreased total activity, and in most cases, it affected sleep time and both morning and evening peaks of activity. These effects were similar to those observed after blocking transmission from specific photoreceptor types. This suggests that synaptic transmission is regulated by the photoreceptor clocks and light. In addition, we showed that HB eyelets, but also L2 interneurons can directly communicate with LN<sub><italic>v</italic></sub>s cells, which provides new light pathway to the clock neurons.</p></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p></sec><sec id=\"S6\"><title>Author Contributions</title><p>MD designed the study and wrote the manuscript with input from all co-authors. MD and JI performed the experiments and analyzed the data. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was funded by grants from the Polish National Science Centre (Narodowe Centrum Nauki, NCN_Grant UMO-2017/27/B/NZ3/00859) and grant Mobility Plus no. 1671/MOB/V/2017/0 funded by Polish Ministry of Science and Higher Education to MD.</p></fn></fn-group><ack><p>We thank Gabriella Mazzotta (Ph.D., University of Padua) for her comments with regard to this manuscript.</p></ack><sec id=\"S9\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fphys.2020.00993/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fphys.2020.00993/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"FS1\"><label>FIGURE S1</label><caption><p>Effects of blocking neurotransmission on activity pattern, performed for: <bold>(A)</bold> R1–R6 photoreceptors (Rh1), <bold>(B)</bold> Rh3-expressing cells, <bold>(C)</bold> Rh5-expressing cells, <bold>(D)</bold> Rh6-expressing cells, <bold>(E)</bold> L2 cells. Red arrows indicate statistically significant changes in morning or evening peak level. Detailed statistics is presented in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>.</p></caption><media xlink:href=\"Image_1.tif\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS2\"><label>FIGURE S2</label><caption><p>Effects of clock disruption on activity pattern, performed for: <bold>(A)</bold> R1–R6 photoreceptors (Rh1), <bold>(B)</bold> Rh3-expressing cells, <bold>(C)</bold> Rh5-expressing cells, <bold>(D)</bold> Rh6-expressing cells. Red arrows indicate statistically significant changes in morning or evening peak level. Detailed statistics is presented in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>.</p></caption><media xlink:href=\"Image_2.tif\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS3\"><label>FIGURE S3</label><caption><p>Effects of neurotransmission block (TeTx) or disruption of the peripheral clock on total activity level, performed for <bold>(A)</bold>\n<italic>glass</italic>-expressing cells (GMR), <bold>(B)</bold> R1–R6 photoreceptors (Rh1), <bold>(C)</bold> Rh3-expressing cells, <bold>(D)</bold> Rh5-expressing cells, <bold>(E)</bold> Rh6-expressing cells, <bold>(F)</bold> L2 interneurons. Heterozygous parental strains were used as control. Statistically significant differences marked with asterisks <sup>∗</sup><italic>p</italic> ≤ 0.05; <sup>∗∗</sup><italic>p</italic> ≤ 0.01; <sup>∗∗∗</sup><italic>p</italic> ≤ 0.001; <sup>****</sup><italic>p</italic> ≤ 0.0001). Detailed statistics is presented in <xref ref-type=\"supplementary-material\" rid=\"TS4\">Supplementary Table S4</xref>.</p></caption><media xlink:href=\"Image_3.tif\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS1\"><label>TABLE S1</label><caption><p>Statistical analysis of the period of locomotor activity rhythm and percent of rhythmic flies. Each experimental strain was compared with control strains (Gal4/+ and UAS/+) using one-way ANOVA and Tukey’s test (for period) or non-parametric Kruskal–Wallis (for % rhythmic). Genotypes with <italic>p</italic> < 0.05 with both controls are marked as statistically significant changes with bold. Degrees of freedom [<italic>F</italic> (DFn, DFd)] are listed for every group.</p></caption><media xlink:href=\"Table_1.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS2\"><label>TABLE S2</label><caption><p>Statistical analysis of morning anticipation index (MAI) and evening anticipation index (EAI). Each experimental strain was compared with control strains (Gal4/+ and UAS/+) using one-way ANOVA and Tukey’s test. Genotypes with <italic>p</italic> < 0.05 with both controls are marked as statistically significant change with bold. Degrees of freedom [<italic>F</italic> (DFn, DFd)] are listed for every group.</p></caption><media xlink:href=\"Table_2.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS3\"><label>TABLE S3</label><caption><p>Statistical analysis of day-time and night-time sleep. Each experimental strain was compared with control strains (Gal4 and UAS) using one-way ANOVA and Tukey’s test. Genotypes with <italic>p</italic> < 0.05 with both controls are marked as statistically significant change with bold. Degrees of freedom [<italic>F</italic> (DFn, DFd)] are listed for every group.</p></caption><media xlink:href=\"Table_3.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS4\"><label>TABLE S4</label><caption><p>Statistical analysis of total activity level. Every experimental strain was compared with control strains (Gal4 and UAS) using one-way ANOVA and Tukey’s test.</p></caption><media xlink:href=\"Table_4.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS5\"><label>TABLE S5</label><caption><p>Statistical analysis of PER expression in the clock neurons s-LN<sub><italic>v</italic></sub>s and l-LN<sub><italic>v</italic></sub>s measured as fluorescence intensity at different time points.</p></caption><media xlink:href=\"Table_5.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chatterjee</surname><given-names>A.</given-names></name><name><surname>Hardin</surname><given-names>P. 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rid=\"c001\"><sup>*</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/22645/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Bannister</surname><given-names>Peter G.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/925307/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Casino</surname><given-names>Gonzalo</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1011273/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Catalani</surname><given-names>Alessia</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Goldman</surname><given-names>Michel</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Morley</surname><given-names>Jessica</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Neunez</surname><given-names>Marie</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/508417/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Prados-Bo</surname><given-names>Andreu</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff7\"><sup>7</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/926635/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Smeesters</surname><given-names>Pierre R.</given-names></name><xref ref-type=\"aff\" rid=\"aff8\"><sup>8</sup></xref><xref ref-type=\"aff\" rid=\"aff9\"><sup>9</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/925421/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Taddeo</surname><given-names>Mariarosaria</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref><xref ref-type=\"aff\" rid=\"aff10\"><sup>10</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Vanzolini</surname><given-names>Tania</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Floridi</surname><given-names>Luciano</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref><xref ref-type=\"aff\" rid=\"aff10\"><sup>10</sup></xref></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Brighton & Sussex Medical School</institution>, <addr-line>Brighton</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Communication Department, Pompeu Fabra University</institution>, <addr-line>Barcelona</addr-line>, <country>Spain</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Iberoamerican Cochrane Center</institution>, <addr-line>Barcelona</addr-line>, <country>Spain</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Biomolecular Sciences, University of Urbino Carlo Bo</institution>, <addr-line>Urbino</addr-line>, <country>Italy</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Institute for Interdisciplinary Innovation in Healthcare (I3h), Université Libre de Bruxelles</institution>, <addr-line>Brussels</addr-line>, <country>Belgium</country></aff><aff id=\"aff6\"><sup>6</sup><institution>Oxford Internet Institute, University of Oxford</institution>, <addr-line>Oxford</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff7\"><sup>7</sup><institution>Blanquerna School of Health Sciences, Ramon Llull University</institution>, <addr-line>Barcelona</addr-line>, <country>Spain</country></aff><aff id=\"aff8\"><sup>8</sup><institution>Molecular Bacteriology Laboratory, Université Libre de Bruxelles</institution>, <addr-line>Brussels</addr-line>, <country>Belgium</country></aff><aff id=\"aff9\"><sup>9</sup><institution>Academic Children Hospital Queen Fabiola, Université libre de Bruxelles</institution>, <addr-line>Brussels</addr-line>, <country>Belgium</country></aff><aff id=\"aff10\"><sup>10</sup><institution>The Alan Turing Institute</institution>, <addr-line>London</addr-line>, <country>United Kingdom</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Bruno Sepodes, University of Lisbon, Portugal</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Frits Lekkerkerker, Consultant, Amsterdam, Netherlands; Segundo Mariz, European Medicines Agency, United Kingdom</p></fn><corresp id=\"c001\">*Correspondence: Pietro Ghezzi <email>p.ghezzi@bsms.ac.uk</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Regulatory Science, a section of the journal Frontiers in Medicine</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†ORCID: Pietro Ghezzi <ext-link ext-link-type=\"uri\" xlink:href=\"https://orcid.org/0000-0003-0911-8358\">orcid.org/0000-0003-0911-8358</ext-link></p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>7</volume><elocation-id>400</elocation-id><history><date date-type=\"received\"><day>06</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>26</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Ghezzi, Bannister, Casino, Catalani, Goldman, Morley, Neunez, Prados-Bo, Smeesters, Taddeo, Vanzolini and Floridi.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Ghezzi, Bannister, Casino, Catalani, Goldman, Morley, Neunez, Prados-Bo, Smeesters, Taddeo, Vanzolini and Floridi</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>The fact that Internet companies may record our personal data and track our online behavior for commercial or political purpose has emphasized aspects related to online privacy. This has also led to the development of search engines that promise no tracking and privacy. Search engines also have a major role in spreading low-quality health information such as that of anti-vaccine websites. This study investigates the relationship between search engines' approach to privacy and the scientific quality of the information they return. We analyzed the first 30 webpages returned searching “vaccines autism” in English, Spanish, Italian, and French. The results show that not only “alternative” search engines (Duckduckgo, Ecosia, Qwant, Swisscows, and Mojeek) but also other commercial engines (Bing, Yahoo) often return more anti-vaccine pages (10–53%) than <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.com\">Google.com</ext-link> (0%). Some localized versions of Google, however, returned more anti-vaccine webpages (up to 10%) than <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.com\">Google.com</ext-link>. Health information returned by search engines has an impact on public health and, specifically, in the acceptance of vaccines. The issue of information quality when seeking information for making health-related decisions also impact the ethical aspect represented by the right to an informed consent. Our study suggests that designing a search engine that is privacy savvy and avoids issues with filter bubbles that can result from user-tracking is necessary but insufficient; instead, mechanisms should be developed to test search engines from the perspective of information quality (particularly for health-related webpages) before they can be deemed trustworthy providers of public health information.</p></abstract><kwd-group><kwd>search engines</kwd><kwd>vaccines</kwd><kwd>health information</kwd><kwd>information quality</kwd><kwd>privacy</kwd><kwd>misinformation</kwd><kwd>fake news</kwd></kwd-group><counts><fig-count count=\"1\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"32\"/><page-count count=\"7\"/><word-count count=\"5310\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>The World Health Organization lists vaccine hesitancy as one of the top 10 threats to global health in 2019 (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>), requiring ongoing global monitoring. Despite the fact that the 1998 study, which incorrectly suggested that the MMR vaccine could cause autism in children and prompted anti-vaccine beliefs (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>), has now been discredited, misinformation and, indeed, disinformation<xref ref-type=\"fn\" rid=\"fn0001\"><sup>1</sup></xref> about vaccines continues to be published on the Internet, perpetuating such beliefs.</p><p>It has been suggested that this misinformation plays a role in the current low uptake of vaccines in developed countries (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>). Understanding whether this is actually the case, and how to address this issue, is crucial as web-based sources of health information may be instrumental to solve the sustainability challenge currently facing health systems across the globe (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>).</p><p>The accuracy of information provided by a website is a key indicator of its overall information quality (IQ). The broader aspects of IQ have been the subject of many studies (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>), but health IQ and trustworthiness of the sources have only partially been characterized (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). Studies looking at the influence of variations in eHealth literacy levels (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>) and trust in different sources of online health information<xref ref-type=\"fn\" rid=\"fn0002\"><sup>2</sup></xref> indicate that the relationship is not linear in all cases, i.e., higher health IQ does not result automatically in higher perceived levels of trustworthiness. For example, in a study by Chen et al. (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>), 618 people were recruited to complete a survey which tested their eHealth literacy level and asked them to identify which of 25 sources of health information they used and how much they trusted each source. The study showed that people with lower eHealth literacy were less likely to trust medical websites (typically higher IQ) and more likely to trust social media, blogs, and celebrity webpages (typically lower IQ) (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>).</p><p>This might seem a spurious result, were it not for the fact that research has shown that those with high eHealth literacy assess more accurately the credibility and relevance of online health information, whereas those with low eHealth literacy often struggle to locate and understand eHealth information (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). This difficulty lowers their self-efficacy (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>), distorts their perception of source credibility, and impacts negatively perceived trustworthiness (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>), ultimately creating a need for individuals with low eHealth literacy to find an alternative means of determining trustworthiness in online sources of information. One such alternative is social endorsement. Visible social endorsement, e.g., “likes,” (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>) enables those with low eHealth literacy to determine trust based on the bandwagon heuristic and assume that, if the source has already been deemed valid by others, then it is safe for them to trust it too (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Traditional medical websites afford those with low eHealth literacy no such alternative means of determining credibility and trust.</p><p>This suggests that those who are more vulnerable to the real-world effects of both disinformation and misinformation (e.g., declining to vaccinate their children) are more likely to rely on online sources with lower health IQ, which are more prone to spread such inflammatory and inaccurate information. Personalization of online search results may favor this phenomenon and lead to a vicious cycle where the more one searches and reads dis- and misinformation about vaccines, the less one finds and reads scientific information on the same topic.</p><p>At the same time, there are increasing concerns about the privacy risks associated with Internet search engines storing potentially sensitive and private health information contained within users search histories, combining it with additional information collected for tracking purposes, and using these data for commercial or other purposes (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>). This creates a public push back against the idea that search engines or public health providers should interfere in the results people see when searching for health information online. This makes it hard to address concerns about health disinformation/misinformation in a way that is at least socially acceptable if not ideally preferable (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). The UK's National Health Service (NHS) discovered this when it announced it would team up with Amazon to use Amazon Alexa as a voice-activated assistant that would automatically search the NHS website and respond with guaranteed high-IQ content from NHS.UK to user voice queries such as “Alexa, how do I treat a migraine.” This resulted in a public outcry over privacy infringement (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>), with advocacy groups raising concerns on handing data from a public healthcare system to a private foreign company. This raises the question whether, in the context of online vaccination information, it is possible at all to balance concerns about user privacy and IQ.</p><p>The objective of this study was to address the research question: “What is the current relationship between search engines' approach to privacy and the scientific quality of the information they return?” For this purpose, we used the example of information returned by different search engines after a search on vaccines and autism. The topic was chosen not only because of its primary importance in public health, as described above, but also because the assessment of IQ is straightforward, based on the wide scientific consensus on vaccine safety and of the lack of a causal relationship between vaccines and autism. The study was performed searching the phrase “vaccines autism” in different languages (English, French, Italian, and Spanish) and comparing a wide range of search engines including the main ones (Google, Bing, Yahoo), those branded as “privacy-savvy” (Duckduckgo, Ecosia, Qwant, Mojeek, Swisscows), and some country-specific ones (Arianna and Virgilio, that do not have a specific no-tracking policy).</p></sec><sec sec-type=\"methods\" id=\"s2\"><title>Methods</title><p>The term “vaccines autism” was used (in French “vaccins autism,” in Italian “vaccini autism,” in Spanish “vacunas autism.” Searches in English were done from Falmer, Sussex, United Kingdom; in Italian from Urbino, Italy; in French from Bruxelles, Belgium; and in Spanish from Barcelona, Spain. Each search was done using a logged-out Chrome browser cleared of cookies and previous search history so that the only identification was the IP address and its geolocalization. Additionally, when available, the local version of each search engine was used (e.g., <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.co.uk\">Google.co.uk</ext-link> and <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.it\">Google.it</ext-link>). For searching <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.com\">Google.com</ext-link>, automatic redirection to <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.co.uk\">Google.co.uk</ext-link> was avoided by using the URL <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.com/ncr\">Google.com/ncr</ext-link> (no-country-redirect).</p><p>The first 30 URL results from each search engine result page (SERP), excluding those marked as advertisements, were transferred to a spreadsheet. Pages that contained no information, aggregators, and indexes were excluded. Websites were then visited and the content of each page was coded as vaccine-positive, -negative or -neutral, depending on the stance taken on the connection between vaccines and autism.</p><p>Webpages recommending vaccination and/or negating the link with autism were coded as “vaccine-positive.” Those promoting vaccine hesitancy, cautioning about the risk of autism or openly anti-vaccine, were coded as “vaccine-negative.” Additionally, webpages that claimed further studies needed to be conducted to clarify the link between vaccines and autism were also coded as “vaccine-negative,” as previous research (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>) has shown that users perceive this as confirmation of the fact that vaccine safety has not been proven. Webpages simply reporting the history of the anti-vaccine movement or a related legal debate were coded as “vaccine-neutral.” Examples of positive, negative, and neutral webpages are provided in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Example of classification of webpages.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Positive stance on vaccines</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.historyofvaccines.org/content/articles/do-vaccines-cause-autism\">https://www.historyofvaccines.org/content/articles/do-vaccines-cause-autism</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.nhs.uk/news/medication/no-link-between-mmr-and-autism-major-study-finds/\">https://www.nhs.uk/news/medication/no-link-between-mmr-and-autism-major-study-finds/</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.nhs.uk/conditions/vaccinations/mmr-vaccine/\">https://www.nhs.uk/conditions/vaccinations/mmr-vaccine/</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.autism.org.uk/get-involved/media-center/position-statements/mmr-vaccine.aspx\">https://www.autism.org.uk/get-involved/media-center/position-statements/mmr-vaccine.aspx</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://kidshealth.org/en/parents/autism-studies.html\">https://kidshealth.org/en/parents/autism-studies.html</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://vaccine-safety-training.org/mmr-vaccine-increases.html\">https://vaccine-safety-training.org/mmr-vaccine-increases.html</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.nejm.org/doi/full/10.1056/NEJMoa021134\">https://www.nejm.org/doi/full/10.1056/NEJMoa021134</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/pubmed/30986133\">https://www.ncbi.nlm.nih.gov/pubmed/30986133</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/pubmed/15366972\">https://www.ncbi.nlm.nih.gov/pubmed/15366972</ext-link></td></tr><tr style=\"border-top: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Negative stance on vaccines</bold></td></tr><tr style=\"border-top: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.thelancet.com/journals/lancet/article/PIIS0140673605756968/fulltext\">https://www.thelancet.com/journals/lancet/article/PIIS0140673605756968/fulltext</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.whale.to/vaccine/vaccine_autism_proven.html\">www.whale.to/vaccine/vaccine_autism_proven.html</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.vaccineriskawareness.com/Infant-Vaccines-Produce-Autism-Symptoms-In-Primates\">www.vaccineriskawareness.com/Infant-Vaccines-Produce-Autism-Symptoms-In-Primates</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://leftbrainrightbrain.co.uk/2014/07/17/more-of-that-vaccineautism-research-that-doesnt-exist/\">https://leftbrainrightbrain.co.uk/2014/07/17/more-of-that-vaccineautism-research-that-doesnt-exist/</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://edition.cnn.com/2011/HEALTH/01/05/autism.vaccines/index.html\">edition.cnn.com/2011/HEALTH/01/05/autism.vaccines/index.html</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.coasttocoastam.com/show/2018/02/21\">https://www.coasttocoastam.com/show/2018/02/21</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.nocompulsoryvaccination.com/2014/08/22/vaccine-autism-cover-up/\">nocompulsoryvaccination.com/2014/08/22/vaccine-autism-cover-up/</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.newswars.com/vaccine-autism-questioned-by-doctor-congressman-elect/\">https://www.newswars.com/vaccine-autism-questioned-by-doctor-congressman-elect/</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.vaxtruth.org/2011/08/vaccines-do-not-cause-autism/\">vaxtruth.org/2011/08/vaccines-do-not-cause-autism/</ext-link></td></tr><tr style=\"border-top: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Neutral stance on vaccines</bold></td></tr><tr style=\"border-top: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.physicsforums.com/threads/vaccine-and-autism-link.880852/\">https://www.physicsforums.com/threads/vaccine-and-autism-link.880852/</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.discovermagazine.com/\">www.discovermagazine.com/</ext-link>2009/jun/06-why-does-vaccine-autism-controversy-live-on<break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ecso.org/news/autism-charity-founder-anti-vaccination-campaigner/\">https://www.ecso.org/news/autism-charity-founder-anti-vaccination-campaigner/</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.theguardian.com/society/2019/jun/01/professor-who-links-vaccines-to-autism-funded-through-university-portal\">https://www.theguardian.com/society/2019/jun/01/professor-who-links-vaccines-to-autism-funded-through-university-portal</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.independent.co.uk/news/world/americas/trump-vaccines-autism-links-anti-vaxxer-us-president-false-vaccine-a8331836.html\">https://www.independent.co.uk/news/world/americas/trump-vaccines-autism-links-anti-vaxxer-us-president-false-vaccine-a8331836.html</ext-link><break/>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2376879/\">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2376879/</ext-link></td></tr></tbody></table></table-wrap><p>Coding was completed by two raters for each language, and inter-rater agreement was calculated with GraphPad, which uses equations 18.16–18.20 from Fleiss (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). On a sample of 59 webpages in English, agreement was 85%, with a Kappa of 0.669 (standard error, 0.077) and a 95% confidence interval from 0.518 to 0.820, a strength of agreement considered to be “good” (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). In Italian, agreement was 90%, with a Kappa of 0.818 (standard error, 0.067) and a 95% confidence interval from 0.687 to 0.950, a strength of agreement considered to be “very good.” In Spanish, agreement was 83%, with a Kappa of 0.609 (standard error, 0.098) and a 95% confidence interval from 0.418 to 0.801, a strength of agreement considered to be “very good.” In French, agreement was 89% with a Kappa of 0.746 (standard error, 0.091) and a 95% confidence interval from 0.568 to 0.924. Disagreements were resolved by further discussion with a third rater to reach an agreement.</p><p>When frequencies of vaccine-negative webpages were compared across different search engines, we used a two-tailed Fisher's test corrected for multiple comparison using the method of Benjamini, Krieger, and Yekutieli and a false discovery rate of 5%. Statistical analysis was performed using GraphPad Prism 8.3.0 for Windows (GraphPad Software, San Diego, CA).</p><p>The list of URLs for all searches and their coding is provided in <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary File 1</xref>.</p></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><p><xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref> shows the ranking of positive (green), neutral (yellow), and negative (red) websites returned by the different search engines in English, French, Italian, and Spanish.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Stance on vaccines in webpages returned by different search engines in four languages. The top 30 webpages returned in the SERPs are shown. Green, vaccine-positive, yellow, vaccine-neutral, red, vaccine-negative.</p></caption><graphic xlink:href=\"fmed-07-00400-g0001\"/></fig><p>Because the purpose of this study was to assess the ranking of misinformation, we compared the frequency of vaccine-negative webpages across the different search engines. <xref rid=\"T2\" ref-type=\"table\">Table 2</xref> shows that Google is consistently returning less misinformation, although the Italian and Spanish versions of Google, as well as its UK English version, returned more vaccine-negative webpages than the English-US version (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.com\">Google.com</ext-link>). Other search engines return more vaccine-negative webpages with some, like Mojeek in English and French or Arianna and Virgilio in Italian, more likely to rank higher webpages with misinformation.</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Vaccine-negative webpages in different SERPs.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>English-UK</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Italian</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Spanish</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>French</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.com\">Google.com</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Local google</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yahoo</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bing</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Duckduckgo</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ecosia</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Qwant</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mojeek</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Swisscows</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Arianna</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Virgilio</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr></tbody></table><table-wrap-foot><p><italic>Data represent the number of vaccine-negative pages (30 webpages for each SERP)</italic>.</p><fn id=\"TN1\"><label>*</label><p><italic>Significantly different from Google in the respective language (or <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.com\">Google.com</ext-link> for English-UK) by a two-tailed Fisher's test corrected for multiple comparison using the method of Benjamini, Krieger, and Yekutieli at a false discovery rate of 5%</italic>.</p></fn></table-wrap-foot></table-wrap><p>In English, the frequency of vaccine-negative webpages in Yahoo, Bing, Duckduckgo, Swisscows, and Mojeek was significantly higher than in <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.com\">Google.com</ext-link>, with Mojeek also significantly higher than <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.co.uk\">Google.co.uk</ext-link>. In Italian, all SERPs had a higher proportion of vaccine-negative webpages compared with <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.it\">Google.it</ext-link>, but this was not statistically significant, although it was if compared with international English <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.google.com\">google.com</ext-link> (5% FDR). The two Italian-only search engines (Virgilio and Arianna) returned the highest number of negative pages in Italian.</p><p>In French, all search engines returned a significantly higher number of vaccine-negative webpages than Google in French. In Spanish, there were, on average, less vaccine-negative results. All the search engines tested had a higher proportion of vaccine-negative results but this was not statistically significant when compared with local Google or with <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.com\">Google.com</ext-link>.</p><p>A common feature was that the SERPs of all search engines providers contained a higher proportion of vaccine-negative results than those obtained from <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.com\">Google.com</ext-link>. However, even the localized versions of Google (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.co.uk\">Google.co.uk</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.it\">Google.it</ext-link> and <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.es\">Google.es</ext-link>) returned more negative pages than the US/international English <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.com\">Google.com</ext-link>.</p></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>The results indicate that currently privacy-enhancing search engines often give more visibility to webpages promoting vaccine hesitancy or with a clear anti-vaccine position than Google. This is in agreement with the findings from a recent study by the Economist which analyzed 175,000 news links returned by Google to demonstrate that the search engine's algorithm favors trustworthy publications (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Reputation and trustworthiness are key factors included in Google's ranking algorithm. In 2019, Google published its search quality evaluation guidelines,<xref ref-type=\"fn\" rid=\"fn0003\"><sup>3</sup></xref> which define webpages containing information that may affect the users' health or financial stability, as “your money your life” (YMYL) pages. These guidelines reveal that when rating YMYL webpages, Google looks at the three criteria of Expertise-Authority-Trustworthiness (E-A-T) and states:</p><p>“High E-A-T information pages on scientific topics should be produced by people or organizations with appropriate scientific expertise and represent well-established scientific consensus on issues where such consensus exists.”</p><p>Thus, in the case of Google, the bandwagon heuristic mechanism of assessing medical information credibility is working in favor of promoting IQ. This is perhaps because those responsible for driving up the “reputation” of specific websites by, for example, linking to them, have higher levels of eHealth literacy and therefore act as pseudo-gatekeepers protecting those with lower eHealth literacy from poor IQ results by ensuring that websites providing inaccurate online health information have a low ranking in the search results.</p><p>In the case of privacy-preserving engines, this appears not to be working—potentially because they do not track “reputation” factors, which could be seen as proxies for IQ and credibility, e.g., clicks and bounce-rate, and so have no gatekeepers (pseudo or otherwise) working to limit the circulation of misinformation. To our knowledge, the algorithms used by these “alternative” search engines are not public—Qwant states that they use their own algorithms<xref ref-type=\"fn\" rid=\"fn0004\"><sup>4</sup></xref>—but despite this, we found a large overlap between the SERP of Qwant and those of Bing and Ecosia. Likewise, Ecosia and Swisscows showed a similar overlap (up to 70%) with Bing, while Ecosia and Qwant often had a complete overlap. This means that it is not possible to check what factors determine the outcome of the decision making processes of the search engines algorithms and, hence, identify those elements that contribute to circulate misinformation.</p><p>It should be mentioned, however, that also non-privacy-savvy search engines, like Bing or Yahoo, often returned more vaccine-negative results than Google, also stressing that IQ is independent on privacy policy. More importantly, even localized versions of Google (UK English, Italian, and Spanish) returned more vaccine-negative results than <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.Google.com\">Google.com</ext-link> or the one in French, indicating that many factors, including the use of non-English language and/or localization can affect the IQ of the results.</p><p>Even without being able to check the exact mechanisms for the overall poorer results, the results suggest that, currently, decisions made by search engines to prioritize privacy preservation may have a negative impact on the IQ of results returned to users in health contexts. The pledge to provide independent and unbiased results or not to promote or hide websites based on political or moral interests can be seen as ethically ambiguous, in view of the potential consequences of pointing to scientifically unsound health information.</p><p>Medical ethics requires that patients give informed consent before treatment and must, therefore, be informed accurately of the risks and benefits associated with treatment, something that is not possible if the search engine provider used by an individual is returning results with low IQ. From this consequentialist perspective, it is inherently unethical choosing not to interfere with a search engine's ranking algorithm to ensure “manually” that results of higher IQ are prioritized, while those of lower IQ are suppressed. This builds on arguments already made in the wider literature about algorithm ethics (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>, <xref rid=\"B21\" ref-type=\"bibr\">21</xref>). For example, the Association for Computing Machinery (ACM) code of ethics and professional conduct includes, as a general principle, “avoid harm” along with that of “respect privacy” (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>), which aligns with the Hippocratic oath. Specifically, the implication that the current design of privacy-preserving search engine algorithms underestimates the need for evaluation of IQ fits into the wider discussion about algorithmic design and how to ensure design decisions are made to protect and incorporate key values such as IQ. It is necessary, but insufficient, to design search engine algorithms that index purely on “relevance,” they must also be designed to index on quality. The challenge lies in the ability to do this in a way that balances the need to accept different perspectives (particularly those that are rooted in different cultural, religious, or social ideals), while also filtering for IQ. Supporters of such arguments, including the authors, note that this requirement necessitates making the workings of search engine algorithms more transparent (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>) to ensure their ethical compliance.</p><p>Providing information on vaccines that is based on misinformation or disinformation (including studies whose data or conclusions have been shown to be wrong) is a deceptive practice, which goes against the basic tenets of medical and business ethics (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). This is in line with those who argue that the promotion of “alternative medicine” is unethical because it lacks the evidence and transparency of clinical efficacy and should be considered “false advertising” (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Online service providers have a moral responsibilities to ensure that users access health information that is scientifically validated (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). Misinformation and disinformation concerning healthcare circulating on the internet can have severe consequences and lead to widespread harm. Consider the example of South African president Mbeki, who delayed introducing anti-retroviral drugs in favor of alternative medicine based on information obtained from HIV-denialist internet websites, a decision estimated to have resulted in over 300,000 deaths (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). More recently, a cancer patient died in China after following an alternative, non-approved therapy they found using a search engine (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). In response, Chinese authorities issued new rules that require search engines to provide “objective, fair, and authoritative results.”</p><p>Another important aspect of IQ is its role in the context of informed consent, which is a central aspect of medical ethics, along with right to privacy and minimizing harm. It has been pointed out by Shahvisi (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>) that to be informed about a treatment means not only to have knowledge but also to have an understanding of the treatment, and understandability is one of the basic dimensions of IQ (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). This raises the issue of the responsibility of search engines in the context of the existing health information to make informed and autonomous choices. If people make autonomous decisions based on the information obtained on the Internet (something that is often incorrectly called “doctor google” effect), one may argue whether search engines have a responsibility to provide high quality information. This is clearly a new challenge in medical ethics, and has been discussed, for instance, in the context of online information on medical tourism (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). The efforts made by the big internet companies, from search engines to social media, to pay particular attention to the IQ of health information indicates that they are well aware of the responsibility that comes with their role.</p><p>Focusing on data privacy without addressing the aspect of health IQ and its role in informed consent is a deficiency in the smaller search engines that would ultimately impact on the process of informed consent. This study, although limited to the information on vaccines, highlights an unregulated gray area for which search engines and regulators should be ethically responsible and that will need being addressed.</p><p>Moral responsibility follows on the level of harm that misinformation and disinformation on healthcare may cause. It could be argued that there is a greater onus on Google—and other commercial search engine providers—than on alternative search engines to take these considerations into account when designing or interfering with algorithms for the purposes of promoting ethical compliance, given their larger market share. Google currently has over 90% of the worldwide market share<xref ref-type=\"fn\" rid=\"fn0005\"><sup>5</sup></xref> and therefore has the potential to indirectly “cause harm,” through the potential promotion of low IQ webpages on vaccinations, to a great many more people than any of the alternative providers.</p><p>However, studies have shown that those who hold anti-authoritarian views, openness to (potentially) controversial opinions, and an interest in alternative medicine are, in some cases, already more likely to hold vaccine-hesitant beliefs (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). These are also the individuals who are most likely to use alternative search providers given their sensitivity to privacy and tracking concerns. Therefore, while the alternative providers might reach a proportionally smaller audience—they are reaching an audience that is already more receptive to anti-vaccine information (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>) and therefore more vulnerable to its effects. In other words, unless the alternative providers take steps to rank vaccine-related search results according to IQ, they may cause greater harm to those they do reach, meaning that the net negative impact is still greater even though the number of individuals they reach is smaller.</p></sec><sec sec-type=\"conclusions\" id=\"s5\"><title>Conclusion</title><p>Our analysis shows that while it may well be technically possible to design a search engine that manages to balance privacy-preservation with the promotion of high IQ material, this is currently not the case. The current relationship between privacy-preserving design features of search engines and the IQ of the results they return is inverse (although not proportionally). In instances where this can have harm public health, as in the example we have provided of the promotion of anti-vaccine misinformation, not intervening to alter the design of the algorithm—even if this means sacrificing some degree of user privacy—can lead to severe harm for a large population of users and is, therefore, unethical.</p><p>Designing a search engine that is privacy savvy and avoids issues with filter bubbles that can result from user-tracking may be a good thing, and in fact this aspect is, at least in part, regulated from the perspective of data protection—which is primarily interpreted as data security rather than data privacy. Our study suggests that this is necessary but insufficient, and instead mechanisms should be developed to test search engines from the perspective of IQ (particularly for YMYL webpages) before they can be deemed trustworthy providers of health information.</p></sec><sec sec-type=\"data-availability\" id=\"s6\"><title>Data Availability Statement</title><p>All datasets generated for this study are included in the article/<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Material</xref>.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>PG and MG designed the research. PG, PB, GC, AC, MG, MN, AP-B, PS, and TV performed the research and analyzed the data. PG, JM, MT, and LF analyzed the data and wrote the paper. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn id=\"fn0001\"><p><sup>1</sup>Misinformation is incorrect information that stems from human error e.g., a lack of fact checking, whereas disinformation is purposefully and deliberately incorrect. Both matter in this context because whilst the original 1998 study may have been an example of misinformation, it is possible that maleficent actors are now purposefully spreading disinformation for the purpose of undermining public health.</p></fn><fn id=\"fn0002\"><p><sup>2</sup>eHealth literacy is defined by Norman and Skinner (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>) as the ability to seek, find, understand, and appraise online health information and apply the knowledge gained to addressing or solving a health problem. This is why we use the term eHealth literacy even though this article focuses exclusively on the IQ of online health information —not broader sources of eHealth information such as electronic health records or wearable devices.</p></fn><fn id=\"fn0003\"><p><sup>3</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://static.googleusercontent.com/media/guidelines.raterhub.com/en//searchqualityevaluatorguidelines.pdf\">https://static.googleusercontent.com/media/guidelines.raterhub.com/en//searchqualityevaluatorguidelines.pdf</ext-link></p></fn><fn id=\"fn0004\"><p><sup>4</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://medium.com/qwant-blog/web-indexation-where-does-qwants-independence-stand-8eab4f7856f8\">https://medium.com/qwant-blog/web-indexation-where-does-qwants-independence-stand-8eab4f7856f8</ext-link> (Archived at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://web.archive.org/web/20190627090553/https://medium.com/qwant-blog/web-indexation-where-does-qwants-independence-stand-8eab4f7856f8\">https://web.archive.org/web/20190627090553/https://medium.com/qwant-blog/web-indexation-where-does-qwants-independence-stand-8eab4f7856f8</ext-link>).</p></fn><fn id=\"fn0005\"><p><sup>5</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://gs.statcounter.com/search-engine-market-share\">https://gs.statcounter.com/search-engine-market-share</ext-link></p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s9\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fmed.2020.00400/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fmed.2020.00400/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Table_1.XLSX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><collab>World Health Organization</collab></person-group>\n<source>Ten Threats to Global Health in 2019.</source> (<year>2019</year>). 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Cardiovasc Med</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Cardiovasc Med</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Cardiovasc. Med.</journal-id><journal-title-group><journal-title>Frontiers in Cardiovascular Medicine</journal-title></journal-title-group><issn pub-type=\"epub\">2297-055X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850989</article-id><article-id pub-id-type=\"pmc\">PMC7431661</article-id><article-id pub-id-type=\"doi\">10.3389/fcvm.2020.00143</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cardiovascular Medicine</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Renin-Angiotensin System and Coronavirus Disease 2019: A Narrative Review</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Mascolo</surname><given-names>Annamaria</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/667844/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Scavone</surname><given-names>Cristina</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/460124/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Rafaniello</surname><given-names>Concetta</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ferrajolo</surname><given-names>Carmen</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/592376/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Racagni</surname><given-names>Giorgio</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Berrino</surname><given-names>Liberato</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/548949/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Paolisso</surname><given-names>Giuseppe</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Rossi</surname><given-names>Francesco</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/266124/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Capuano</surname><given-names>Annalisa</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/514712/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Section of Pharmacology “L. Donatelli”, Department of Experimental Medicine, University of Campania “Luigi Vanvitelli”</institution>, <addr-line>Naples</addr-line>, <country>Italy</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Campania Regional Centre for Pharmacovigilance and Pharmacoepidemiology</institution>, <addr-line>Naples</addr-line>, <country>Italy</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano</institution>, <addr-line>Milan</addr-line>, <country>Italy</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”</institution>, <addr-line>Naples</addr-line>, <country>Italy</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Shuyang Zhang, Peking Union Medical College Hospital, China</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Zhengyuan Xia, The University of Hong Kong, Hong Kong; Zamaneh Kassiri, University of Alberta, Canada</p></fn><corresp id=\"c001\">*Correspondence: Annamaria Mascolo <email>annamaria.mascolo@unicampania.it</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to General Cardiovascular Medicine, a section of the journal Frontiers in Cardiovascular Medicine</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†These authors have contributed equally to this work and share lead authorship</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>11</day><month>8</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>7</volume><elocation-id>143</elocation-id><history><date date-type=\"received\"><day>05</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>06</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Mascolo, Scavone, Rafaniello, Ferrajolo, Racagni, Berrino, Paolisso, Rossi and Capuano.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Mascolo, Scavone, Rafaniello, Ferrajolo, Racagni, Berrino, Paolisso, Rossi and Capuano</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Although clinical manifestations of the 2019 novel coronavirus disease pandemic (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), are mainly respiratory symptoms, patients can also develop severe cardiovascular damage. Therefore, understanding the damage caused by SARS-COV-2 to the cardiovascular system and the underlying mechanisms is fundamental. The cardiovascular damage may be related to the imbalance of the renin-angiotensin-system (RAS) as this virus binds the Angiotensin-Converting-Enzyme 2 (ACE2), expressed on the lung alveolar epithelial cells, to enter into cells. Virus internalization may cause a downregulation of ACE2 on host cell surface that could lead to a local increased level of angiotensin II (AII) and a reduced level of angiotensin 1-7 (A1-7). An imbalance between these angiotensins may be responsible for the lung and heart damage. Pharmacological strategies that interfere with the viral attachment to ACE2 (umifenovir and hydroxychloroquine/chloroquine) or that modulate the RAS (analogous of A1-7 and ACE2, losartan) are in clinical development for COVID-19. The use of RAS inhibitors has also become a matter of public concern as these drugs may increase the mRNA expression and levels of ACE2 and impact the virulence and transmission of SARS-COV-2. Data on the effect of RAS inhibitors on ACE2 mRNA expression are scarce. Scientific societies expressed their opinion on continuing the therapy with RAS inhibitors in patients with COVID-19 and underlying cardiovascular diseases. In conclusion, RAS may play a role in SARS-COV-2-induced cardiac and pulmonary damage. Further studies are needed to better understand the role of RAS in COVID-19 and to guide decision on the use of RAS inhibitors.</p></abstract><kwd-group><kwd>COVID-19</kwd><kwd>renin-angiotensin system</kwd><kwd>SARS-COV-2</kwd><kwd>heart damage</kwd><kwd>pulmonary damage</kwd><kwd>RAS inhibitors</kwd></kwd-group><counts><fig-count count=\"1\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"89\"/><page-count count=\"13\"/><word-count count=\"9419\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>The renin–angiotensin system (RAS) is a complex hormonal system composed by different mediators that can affect the cardiovascular, renal, immune, and nervous functions (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B2\" ref-type=\"bibr\">2</xref>). Many components of the RAS have been isolated from different tissues (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>), including the lung (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). This system is composed by two pathways: the classic RAS and the non-classic RAS, which have opposite activities, especially for renal, and cardiovascular functions (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B5\" ref-type=\"bibr\">5</xref>). A component of the non-classic RAS, the Angiotensin-Converting-Enzyme 2 (ACE2) present on the lung surface, has been discovered to be a functional receptor for coronaviruses, essential for triggering their infection (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Severe acute respiratory syndrome coronavirus 1 (SARS-COV-1) and SARS-COV-2, which are responsible for the SARS and the more recent coronavirus disease 2019 (COVID-19), respectively, are both able to bind the ACE2 in the lung (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Patients affected with COVID-19 show respiratory and flu-like symptoms, which can be complicated by lymphopenia and interstitial pneumonia with high levels of pro-inflammatory cytokines that can lead to acute respiratory distress syndrome (ARDS) and organ failure (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). Although the clinical manifestations of COVID-19 are mainly represented by respiratory symptoms, some patients also developed severe cardiovascular damage (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). In addition, an increased risk of death was found in patients with cardiovascular diseases (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>).</p><p>Understanding the mechanisms by which the RAS interacts with SARS-COV-2 is fundamental for the treatment of patients with cardiac diseases as showed in the context of metabolic diseases (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). Moreover, considering the interaction between these viruses and the ACE2, concerns were also raised about the use of RAS inhibitors in patients with COVID-19 as they may alter ACE2 mRNA expression and levels and, in this way, impact the virulence and transmission of SARS-COV-2 (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Therefore, in this review, we aim to summarize the physiological role of the RAS, its implication in the SARS-COV-2 infection, the actual evidence and recommendation on the use of RAS inhibitors, and the ongoing researches of drugs with a potential for the treatment of COVID-19 and acting either by influencing the RAS or disrupting the viral attachment to ACE2.</p></sec><sec id=\"s2\"><title>Clinical Characteristics of COVID-19</title><p>First evidence regarding the clinical characteristics of patients with COVID-19 showed the presence of bilateral lung ground glass opacity on computed tomography (CT) imaging (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). CT abnormalities were observed in both asymptomatic or symptomatic patients with SARS-CoV-2 infection, making it a useful diagnostic tool. Asymptomatic individuals with CT abnormalities rarely developed severe pneumonia (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Initial symptoms were fever, cough, dyspnea, myalgia or fatigue, sputum production, headache, hemoptysis, and diarrhea. In most severe cases, there was a progression to ARDS, to acute cardiac injury, to acute kidney injury (AKI), or to shock. Other symptoms that were identified pertained to the gastrointestinal system (nausea and diarrhea) (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). However, other studies showed a lower development of gastrointestinal symptoms (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>, <xref rid=\"B15\" ref-type=\"bibr\">15</xref>). Moreover, an increase in serum lactate dehydrogenase as marker of lung tissue damage was observed in COVID-19 patients (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>), and it was associated with higher odds of severe disease (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>). Additionally, older age and lymphopenia were identified as potential risk factors for severe COVID-19 (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>).</p></sec><sec id=\"s3\"><title>Classic and Non-classic RAS</title><p>The classic RAS involves as main effector peptide the angiotensin II (AII), whose synthesis starts with the cleavage of angiotensinogen into angiotensin I (AI) by the renin and then its conversion into AII by the ACE (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Despite this represent the main pathway for the AII production, also other enzymes can be involved (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). The main effects of AII are explained by its interaction with three receptors (AT1, AT2, and nonAT1nonAT2). AT1 and AT2 are classified as G protein-coupled receptors (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>), while nonAT1nonAT2 seems more prone to be an angiotensin clearance receptor or an angiotensinase (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). The stimulation of the AT1 receptor can induce vasoconstriction, increase the release of catecholamines and the synthesis of aldosterone (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Moreover, AT1 receptors can stimulate fibrosis, inflammatory processes, reduction of collagenase activity, and expression of mitogen-activated protein kinase (MAPK) (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B5\" ref-type=\"bibr\">5</xref>). As pro-inflammatory action, these receptors seem to be involved in several pathways: down-regulation of the NADPH oxidase expression in smooth muscle cells; enhancement of the production of reactive oxygen species (ROS) and the activity of pro-inflammatory transcription nuclear factors like nuclear factor-kappaB (NF-kB) and E26 transformation-specific sequence (Ets) (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>); release of different types of cytokines such as TNF-α, IL-6, and MCP-1 (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>); shifting of the macrophage phenotype toward the pro-inflammatory M1 polarization state (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). The stimulation of AT2 receptors, instead, has a protective role in the RAS activation inducing anti-inflammatory, anti-oxidative, and anti-fibrotic effects (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Classic and non-classic renin-angiotensin system (RAS) and its interaction with SARS-COV-2.</p></caption><graphic xlink:href=\"fcvm-07-00143-g0001\"/></fig><p>The non-classic RAS involves, instead, other peptide mediators and enzymes. Specifically, the main mediator is the angiotensin 1-7 (A1-7), whose synthesis can involve two different pathways. One starts with the cleavage of AII into A1-7 by the carboxypeptidase ACE2, while another through the cleavage of AI into angiotensin 1–9 (A1–9) by ACE2 and its subsequent conversion into A1–7 by ACE (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Today, two forms of ACE2 are recognized, one soluble and another transmembrane, both contributing to the generation of A1-7. The A1–7 stimulates the G protein-coupled receptor MAS1, promoting the nitric oxide release (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>), Akt phosphorylation (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>), and anti-inflammatory effects (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). Moreover, the activation of MAS1 receptors, expressed on the macrophage surface, inhibits the inflammatory macrophage phenotype and the release of pro-inflammatory cytokines (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Therefore, A1-7 is a component of a beneficial axis of the RAS that exerts opposite cardiovascular and renal effects compared to the ACE/AII/AT1 axis (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>).</p><p>Interestingly, it has been found that human monocytes can express ACE and ACE2 and metabolize AI to multiple angiotensin peptides. In particular, classical monocytes (CD14<sup>++</sup>CD16<sup>−</sup>) produce both AII and A1–9/A1–7, whereas the non-classical subtype (CD14<sup>+</sup>CD16<sup>++</sup>) produces mainly A1–7 (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). This indicates that ACE and ACE2 participate to the inflammation also as components of a local RAS at sites infiltrated by monocytes/macrophages.</p></sec><sec id=\"s4\"><title>SARS-COV-2 and ACE2 in the Lung</title><p>SARS-COV-2 is a betacoronavirus with a single-stranded positive-sense RNA genome encapsulated within a membrane envelope (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). The genome encodes for several structural proteins, including the glycosylated spike (S) protein that is a major inducer of host immune response. The S protein is also important because mediates host cell invasion by SARS-COV-2 via binding to the receptor protein ACE2 present on the surface of lung alveolar epithelial cells (host cells) (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B27\" ref-type=\"bibr\">27</xref>). The affinity of S protein binding region to the extracellular domain of ACE2 has been estimated of 15 nM (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B28\" ref-type=\"bibr\">28</xref>). The invasion process requires the activation of the S protein, which is facilitated by the human androgen-sensitive transmembrane serine protease type 2 (TMPRSS211) (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B26\" ref-type=\"bibr\">26</xref>). Specifically, TMPRSS211 cleaves the S protein and generates the S1 and S2 subunits. This is a critical step as both subunits are essential for viral entry in the host cells (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). S1 is the subunit recognized by ACE2 and the one that facilitates viral attachment, whereas S2 is the subunit that drives membrane fusion and viral internalization in the pulmonary epithelium (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). The greater virulence of SARS-COV-2 compared to SARS-COV-1 was supposed to be related to the higher affinity of S1 subunit for ACE2 (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>, <xref rid=\"B28\" ref-type=\"bibr\">28</xref>). In fact, a Cryo-EM structure analysis revealed that the affinity of the S protein of SARS-COV-2 to ACE2 is about 10–20 times greater than that observed with the S protein of SARS-COV-1 (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>).</p><p>Another important consideration is that the ACE2 internalization mediated by SARS-COV-2 could potentially result in a reduced presence of ACE2 on cell surface, leading to the absence of a key factor for AII degradation and A1-7 synthesis. An imbalance between AII and A1-7 levels may further exacerbate the damage of lung provoked by SARS-COV-2. Therefore, a decrease in ACE2 may contribute to the reduction of pulmonary function and the increase of tissue fibrosis and inflammation due to COVID-19 (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). This hypothesis was already investigated with SARS-COV-1 infection, which was associated with a reduced presence of ACE2 on cell membranes and an increased severity of lung injury (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). Because SARS-COV-1 and SARS-COV-2 share the same cellular invasion process, they may also share similar pathogenesis and pathological manifestations of lung injury (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>).</p></sec><sec id=\"s5\"><title>SARS-COV-2 and ACE2 in the Heart</title><p>Potentially, once the SARS-COV-2 enters the circulation, it can infect any tissue expressing the ACE2, including the heart or other cardiovascular tissues (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). Evidence showed that patients with COVID-19 had a high occurrence of cardiovascular symptoms, in addition to respiratory ones, and that these symptoms were also reported in patients without underlying cardiovascular diseases (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). The National Health Commission of China (NHC) reported that cardiovascular symptoms (such as heart palpitations and chest tightness) occurred at the beginning of the SARS-COV-2 infection in some of confirmed cases. Moreover, the 11.8% of patients who died for COVID-19 but without underlying cardiovascular diseases had substantial heart damage (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). These data suggest the necessity of involving cardiologists in the management of patients with COVID-19 (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). However, the real contribute of SARS-COV-2 in the development of myocardial injury is not clear (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). It is known that the infection itself may directly impact cardiovascular diseases and the development of cardiovascular complications (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>, <xref rid=\"B33\" ref-type=\"bibr\">33</xref>). Another factor that should be considered is also the expression in the tissue of TMPRSS211 or other proteases able to trigger the viral entry (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). Another hypothesis for the induction of heart damage considers the reduction of ACE2 caused by SARS-COV-2, which might exacerbate symptoms in patients with underlying cardiovascular diseases (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>, <xref rid=\"B34\" ref-type=\"bibr\">34</xref>). This could be due to the imbalance between the classic and non-classic RAS in favor of AII that may further compromise cardiac function apart from the viral infection (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). In fact, a preclinical study shows that ACE2 knockout animal models had a worse left ventricular remodeling in response to the AII-induced acute injury, suggesting a protective role of non-classic RAS in myocardial recovery (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). This finding may also explain the heart damage found in patients with COVID-19 but without cardiovascular diseases (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). To corroborate this hypothesis, a study demonstrated that the AII level in the plasma sample of SARS-COV-2 infected patients was markedly high and linearly associated with the viral load and lung injury (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). Moreover, another study found in the 35% of heart samples from patients with SARS the presence of viral RNA associated with a reduced ACE2 protein expression (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). Another proposed mechanism of myocardial injury includes the cytokine storm (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>) as the systemic inflammatory response and immune system disorders during disease progression may be responsible for the myocardial damage (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Also, in this case, other than the viral infection itself, a minor role in potentiating the inflammation might be played by the classic RAS cascade. Moreover, needs to be considered that also some drugs that are being investigated for COVID-19 are potential risk factors for the cardiovascular toxicity (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>).</p><p>Finally, evidence showed that COVID-19 may produce a form of disseminated intravascular coagulation (DIC) as the presence of microthrombi have been reported from the autopsy of patients with COVID-19 (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>). To date, the exact causes of DIC are many and unclear. Potential suggested mechanisms are as follows: inflammation (e.g., IL-6) stimulates the synthesis of fibrinogen (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>); or the virus may directly bind to endothelial cells; or a mutual relationship between DIC and cytokine storm (wherein each exacerbates the other) exists.</p></sec><sec id=\"s6\"><title>Concerns, Evidence and Recommendation on the Use of RAS Inhibitors in Patients With COVID-19</title><p>Concerns were raised on the use of RAS inhibitors in patients with COVID-19 as the use of these drugs may determine an increase of ACE2 and then of SARS-COV-2 virulence (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Among drugs able to inhibit the RAS, there are renin inhibitors, ACE inhibitors, and the Angiotensin Receptor Blockers (ARBs). ACE inhibitors and ARBs are among drugs most commonly used worldwide for the treatment of cardiovascular diseases. Therefore, concerns on their use in patients with COVID-19 are even more important. Initial evidence showed that patients with COVID-19 and coexisting cardiovascular conditions had a more severe illness, a more frequent admission to the intensive care unit, were more prone to receive mechanical ventilation, or to die (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). The first hypothesis was that the medical management of these conditions, including the use of RAS inhibitors, may have contributed to the adverse health outcomes. So far, there is no rigorous report accounting for key factors as potential confounders in risk prediction; moreover, available evidence on the effect of RAS inhibitors on ACE2 mRNA expression and levels are conflicting and scarce, highlighting also the absence of data on lung-specific mRNA expression of ACE2 (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Researches have also suggested that this effect of RAS inhibitors may not be uniform among molecules (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B39\" ref-type=\"bibr\">39</xref>). Moreover, even if there was a relationship between the RAS inhibition and the up-regulation of ACE2, there is no evidence demonstrating a causal relationship between the ACE2 activity and the SARS-COV-2 associated mortality (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>). Furthermore, the presence of ACE2 on cell surface may not be the only factor participating in the infection process. In fact, additional co-factors might participate in the cell invasion process as SARS-COV-1 infection was not observed in some cells expressing ACE2 on the surface, whereas it was found in cells apparently without ACE2 (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>). Moreover, the lethal outcome observed in patients with COVID-19 may also be driven by the severity of the lung damage. In this regard, a preclinical study suggested a beneficial role of RAS blockers in limiting the SARS-COV-1-induced lung injury (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>), so that, a protective role is played by RAS inhibitors. This finding could rise a new hypothesis in which the activation of the classic RAS, rather than its inhibition, may predispose patients toward a more deleterious outcome.</p><p>Finally, another aspect that should be considered is the potential harm associated with the withdrawal of a RAS inhibitor in a patient with a stable cardiovascular condition. In fact, RAS inhibitors are known to determine clinical benefits and to protect both myocardium and kidney. Therefore, their sudden withdrawal may expose patients to an unjustified risk related to decompensation and symptoms exacerbation, especially in high cardiovascular risk patients. In this regard, clinical trials have demonstrated a rapid relapse of the dilated cardiomyopathy or a decline of the clinical condition after the discontinuation of the pharmacological treatment with a RAS inhibitor (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>).</p><p>Moreover, there are solid evidence on the effect of RAS inhibitors in reducing mortality in patients with cardiovascular diseases. These drugs are indeed the cornerstone therapy for a favorable prognosis in patients with heart failure, with the highest level of evidence in reducing mortality (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>). Finally, Scientific Societies have expressed their opinion on the use of RAS inhibitors, highlighting the absence of evidence suggesting an eventual discontinuation of ACE-inhibitors, or ARBs in patients with COVID-19. Therefore, they recommend to continue the treatment with the usual anti-hypertensive agent in patients with COVID-19 (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>–<xref rid=\"B49\" ref-type=\"bibr\">49</xref>). This recommendation has been supported by different observational studies published in the last few months. In this regards, a population-based case–control study carried out in the Lombardy region of Italy did not show any association between the use of ARBs or ACE-inhibitors with COVID-19 among all patients (adjusted odds ratio, 0.95 [95% confidence interval (CI), 0.86 to 1.05] for ARBs and 0.96 [95% CI, 0.87 to 1.07] for ACE inhibitors) or among patients with a severe or fatal course of the disease (adjusted odds ratio, 0.83 [95% CI, 0.63 to 1.10] for ARBs and 0.91 [95% CI, 0.69 to 1.21] for ACE inhibitors) (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>).</p><p>Accordingly, another Italian nested case-control study showed no increased risk of being infected by SARS-COV-2 in patients treated with RAS inhibitors (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>). Moreover, a case-population study showed that RAS inhibitors had an adjusted odds ratio for COVID-19 requiring admission to hospital of 0.94 (95% CI, 0.77 to 1.15) compared with users of other antihypertensive drugs (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>). In relation to the mortality outcome, instead, a retrospective observational study showed similar mortality rates between the RAS inhibitor and non-RAS inhibitor cohorts (2.2 vs. 3.6%, adjusted hazard ratio [HR] 0.85; 95% CI, 0.28 to 2.58) (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>). Similarly, a Korean nationwide population-based cohort study showed no difference for mortality between RAS inhibitors users and non-users (adjusted odds ratio, 0.88; 95% CI, 0.53 to 1.44) (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>). Finally, a retrospective, multi-center study demonstrated a lower risk of COVID-19 mortality in inhospital patients with hypertension and hospitalized due to COVID-19 who received ACE inhibitor/ARB compared to those who did not receive an ACE inhibitor/ARB (adjusted HR, 0.37; 95% CI, 0.15 to 0.89) (<xref rid=\"B55\" ref-type=\"bibr\">55</xref>). Different other published studies supported the aforementioned findings (<xref rid=\"B56\" ref-type=\"bibr\">56</xref>–<xref rid=\"B58\" ref-type=\"bibr\">58</xref>). Moreover, it is ongoing an observational study that will enroll about 2,000 participants to assess if the chronic intake of RAS inhibitors modifies the prevalence and severity of clinical manifestations of COVID-19 (<ext-link ext-link-type=\"uri\" xlink:href=\"https://ClinicalTrials.gov\">ClinicalTrials.gov</ext-link> identifier, NCT04331574).</p><p>Clinical trials are also ongoing to assess instead clinical benefits of continuing or not the treatment with ARBs or ACE inhibitors in patients with COVID-19 (NCT04330300, NCT04351581, NCT04353596, and NCT04329195). In particular, the NCT04330300 is a randomized, open label, parallel assignment clinical trial that will randomize patients with primary essential hypertension who are already taking ACE inhibitor/ARB to either switch to an alternative antihypertensive agent or continue with the ACE inhibitor/ARB treatment. The NCT04351581 is a randomized, single mask (outcome assessor), parallel assignment clinical trial that will randomize hospitalized patients with COVID-19 to continue or discontinue their treatment with the ACE inhibitor or ARB. The NCT04353596 is also a randomized, single mask (outcome assessor), parallel assignment clinical trial that will randomize symptomatic SARS-CoV2-infected patients to stop/replace the chronic treatment with the ACE inhibitor/ARB or to continue this chronic treatment. The NCT04329195 is instead a randomized, open label, parallel assignment clinical trial that will randomize patients with a history of cardiovascular disease treated with RAS blockers, and infected by SARS-CoV-2 to stop or continue the treatment with the RAS blocker. Moreover, the substudy of the Austrian Coronavirus Adaptive Clinical Trial (ACOVACT), which is a randomized, controlled, multicenter, open-label basket trial that aims to compare various antiviral treatments for COVID-19, will also compare the sub-arm with RAS blockade vs. no RAS blockade for patients with blood pressure >120/80 mmHg (NCT04351724). Characteristics of the ongoing clinical trials are showed in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Characteristics of ongoing clinical trials on drugs acting either by influencing the RAS or disrupting the viral attachment to ACE2 in patients with COVID-19.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Clinical trial number</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Clinical phase; multicenter</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Arms</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Estimated enrollment</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Primary outcome</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Estimated study completion date</bold></th></tr></thead><tbody><tr style=\"border-bottom: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT04330300</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4; No</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Experimental arm: switching to an alternative anti-hypertensive medication (specifically a calcium channel blocker or thiazide/thiazide-like diuretic at an equipotent blood pressure lowering dose). The choice of the alternative anti-hypertensive will be at the discretion of the patient's treating physician.<break/> • Comparator arm: continuing the treatment with ACE inhibitor/ARB</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2,414</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. Number of COVID-19 positive participants who die, require intubation in intensive care unit, or require hospitalization for non-invasive ventilation at 12 months. Time from randomization to the first occurrence of any of the clinical events above.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">March 1, 2021</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT04351581</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Not reported; No</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Experimental arm: continuing the treatment with ACE inhibitor/ARB. The clinicians will be encouraged to continue the medication throughout the hospital admission but it will be permissible for the clinician to stop treatment if necessary (e.g., due to hypotension).<break/> • Experimental arm: discontinuing the treatment with ACE inhibitor/ARB. If hypertensive treatment is necessary during hospital admission, the clinicians will first be encouraged to start non-ACE inhibitor/non-ARB treatment.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">215</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. Days alive and out of hospital within 14 days after recruitment</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">December 2020</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT04353596</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4; Yes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Experimental arm: chronic treatment with ACE inhibitor or ARB will be stopped or replaced.<break/> • Comparator arm: no intervention, which means to continue the treatment with ACE inhibitor or ARB.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">208</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. Combination of maximum Sequential Organ Failure Assessment (SOFA) Score and death at 30 days. 2. Composite of admission to an intensive care unit, the use of mechanical ventilation, or all-cause death at 30 days.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">May 15, 2022</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT04329195</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3; No</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Experimental arm: discontinuation of RAS blocker therapy<break/> • Comparator arm: continuation of RAS blocker therapy</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">554</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. Time to clinical improvement from day 0 to day 28 (improvement of two points on a seven-category ordinal scale, or live discharge from the hospital, whichever comes first)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">August 9, 2020</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT04351724substudy</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2/3; Yes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Experimental arm: candesartan at 4 mg once daily and titrated to normotension<break/> • Comparator arm: non-RAS antihypertensive agents titrated to normotension. Those with normal blood pressure may be controlled without further treatment.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">500</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. Sustained improvement (>48 h) of one point on the WHO Scale within 29 days (daily evaluation).</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">December 31, 2020</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT04260594</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4; Not reported</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Experimental arm: umifenovir tablets (2 tablets/time, 3 times/day for 14–20 days) + basic treatment<break/> • Comparator arm: basic treatment<break/> • The basic treatment is based on the condition of the patient.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">380</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. Virus negative conversion rate in the first week</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">December 30, 2020</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT04252885</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4; No</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Experimental arm: standard treatment + lopinavir/ritonavir. Specifically, 50 participants are given ordinary treatment plus a regimen of lopinavir (200 mg) and ritonavir (50 mg) (oral, q12h, every time 2 tablets of each, taking for 7–14 days).</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">125</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. The rate of virus inhibition at Day 0, 2, 4, 7, 10, 14, and 21. Novel corona viral nucleic acid is measured in nose/throat swab at each time point.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">July 31, 2020</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Comparator arm: standard treatment + umifenovir. Specifically, 50 participants are given ordinary treatment plus a regimen of umifenovir (100 mg) (oral, tid, 200 mg each time, taking for 7–14 days).<break/> • No intervention arm: standard treatment. Specifically, 25 cases are only given ordinary treatment.</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr style=\"border-bottom: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT04255017</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4; No</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Experimental arm: addition of umifenovir (0.2 g once, 3 times a day for 2 weeks)<break/> • Experimental arm: addition of oseltamivir (75 mg once, twice a day for 2 weeks)<break/> • Experimental arm: addition of lopinavir/ritonavir (500 mg once, twice a day for 2 weeks)<break/> • No intervention arm: symptomatic supportive treatment</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">400</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. Rate of disease remission at 2 weeks. Defined for mild patients as fever, cough and other symptoms relieved with improved lung CT, and for severe patients as fever, cough and other symptoms relieved with improved lung CT, SPO2> 93% or PaO2/FiO2 > 300 mmHg (1 mmHg = 0.133 Kpa); 2. Time for lung recovery at 2 weeks. Defined as the comparison of the average time of lung imaging recovery after 2 weeks of treatment in each group.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">July 1, 2020</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT04350684</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4; No</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Experimental arm: umifenovir + interferon-β 1a + lopinavir/ritonavir + single dose of hydroxychloroquine + standards of care<break/> • Comparator arm: interferon-β 1a + lopinavir/ritonavir + single dose of hydroxychloroquine + standards of care</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">40</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. Time to clinical improvement from the date of randomization until 14 days later. Improvement of two points on a seven-category ordinal scale (recommended by the World Health Organization: COVID-2019) R&D. Geneva: World Health Organization) or discharge from the hospital, whichever came first.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">April 24, 2020</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT04312009</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2; Yes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Experimental arm: losartan (50 mg daily, oral)<break/> • Control arm: placebo (microcrystalline methylcellulose, gelatin capsule, oral)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">200</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. Difference in Estimated Positive End-expiratory Pressure (PEEP adjusted) P/F Ratio at 7 days. Outcome calculated from the partial pressure of oxygen or peripheral saturation of oxygen by pulse oximetry divided by the fraction of inspired oxygen (PaO2 or SaO2: FiO2 ratio). PaO2 is preferentially used if available. A correction is applied for endotracheal intubation and/or positive end-expiratory pressure. Patients discharged prior to day 7 will have a home pulse oximeter send home for measurement of the day 7 value, and will be adjusted for home O2 use, if applicable. Patients who died will be applied a penalty with a P/F ratio of 0.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">April 1, 2021</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT04311177</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2; Yes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Experimental arm: losartan (25 mg daily, oral)<break/> • Comparator arm: placebo (microcrystalline methylcellulose, gelatin capsule, oral)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">580</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. Hospital Admission within 15 days. Outcome reported as the number of participants per arm admitted to inpatient hospital care due to COVID-19-related disease within 15 days of randomization.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">April 1, 2021</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT04328012</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2/3; Yes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Experimental arm: lopinavir/ritonavir (400 mg/200 mg, oral, BID X 5–14 days depending on availability)<break/> • Experimental arm: hydroxychloroquine (400 mg BID on Day 0, and 200 mg BID Days 1–4, days 1–13 if available)<break/> • Experimental arm: losartan (25 mg, oral, daily X 5–14 days depending on availability)<break/> • Comparator arm: placebo (BID X 14 days)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4,000</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. National Institute of Allergy and Infectious Diseases COVID-19 Ordinal Severity Scale (NCOSS) at 60 days. Difference in NCOSS scores between the different treatment groups</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">April 1, 2021</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT04335786</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4; Yes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Experimental arm: valsartan for 14 days at a dosage and frequency titrated to blood pressure with 80 mg or 160 mg tablets up to a maximum dose of 160 mg b.i.d.<break/> • Comparator arm: placebo for 14 days (matching 80 or 160 mg placebo tablets at a dosage and frequency titrated to systolic blood pressure)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">651</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. First occurrence of intensive care unit admission, mechanical ventilation or death within 14 days. Death is defined as all-cause mortality</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">December 2021</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT04360551</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2; No</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Experimental arm: telmisartan (40 mg, oral, daily X 21 days)<break/> • Comparator arm: placebo (once daily X 21 days)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">40</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. Maximum clinical severity of disease over the 21 day period of study. Based on a modified World Health Organization (WHO) COVID-19 7-point ordinal scale</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">June 30, 2021</td></tr></tbody></table></table-wrap></sec><sec id=\"s7\"><title>New Pharmacological Approaches for Preventing Viral Entry of SARS-COV-2 With a Focus on the Disruption of S Protein/ACE2 Interaction</title><p>To prevent viral infection, molecules like camostat mesylate, nafamostat mesylate, gabexate, umifenovir, and hydroxychloroquine/chloroquine are being considered (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). Nafamostat and camostat are inhibitors of the protease TMPRSS211 (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). Gabexate has instead multiple mechanisms of action. It has anticoagulant and anti-platelet activities on one hand, and it is a serine protease inhibitor with antiviral and anti-inflammatory properties on the other (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>, <xref rid=\"B60\" ref-type=\"bibr\">60</xref>).</p><p>While these drugs act on the protease inhibition, umifenovir and hydroxychloroquine/chloroquine directly influence the S protein/ACE2 interaction (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>) (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). Hydroxychloroquine and chloroquine, in addition to their use for malaria and autoimmune diseases, may be effective also for the treatment of COVID-19. These drugs are able to elevate endosomal pH and interfere with ACE2 glycosylation (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>, <xref rid=\"B70\" ref-type=\"bibr\">70</xref>). The efficacy of chloroquine was already demonstrated with SARS-COV-1 infection, in which the treatment was effective either if administrated prior or after the infection, suggesting that chloroquine may have both a prophylactic and therapeutic use (<xref rid=\"B70\" ref-type=\"bibr\">70</xref>). Moreover, preliminary <italic>in vitro</italic> results demonstrated that remdesivir and chloroquine are highly effective in the inhibition of SARS-COV-2 infection (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>). Clinical findings also confirmed the efficacy of chloroquine in terms of reduction of exacerbation of pneumonia and duration of symptoms in a cohort of 100 subjects (<xref rid=\"B72\" ref-type=\"bibr\">72</xref>, <xref rid=\"B73\" ref-type=\"bibr\">73</xref>). This finding led the China Authority to include these medicines in the recommendations for the prevention and treatment of COVID-19 pneumonia (<xref rid=\"B73\" ref-type=\"bibr\">73</xref>). Many other clinical studies are ongoing to evaluate the efficacy and safety of hydroxychloroquine for the pre-exposure prophylaxis, post-exposure prophylaxis, and treatment of COVID-19 (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.clinicaltrials.gov\">www.clinicaltrials.gov</ext-link>) (<xref rid=\"B74\" ref-type=\"bibr\">74</xref>). However, it should be noted that current evidence on the effects of chloroquine is conflicting. Authors of a recent systematic review underlined that, even though a rationale to justify clinical research on chloroquine in patients with COVID-19 exists, high-quality clinical trials are urgently needed (<xref rid=\"B75\" ref-type=\"bibr\">75</xref>). In addition, a further literature review (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>) reported that there is limited <italic>in vitro</italic> evidence on the efficacy of this drug against SARS-COV-2 and that clinical data based on studies with small sample size and affected by methodological limitations (<xref rid=\"B77\" ref-type=\"bibr\">77</xref>, <xref rid=\"B78\" ref-type=\"bibr\">78</xref>). Therefore, high quality randomized clinical trials are strongly needed. Umifenovir interferes instead with the attachment of viral envelope protein to host cells (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). Umifenovir is an antiviral agent actually authorized in Russia, but not in Europe, for the treatment of Influenza A and B. This drug is considered safe and it is patented for the SARS treatment (<xref rid=\"B79\" ref-type=\"bibr\">79</xref>). The opinion of the Italian Medicine Agency on this drug is that evidence on its efficacy are not sufficient to support its use in patients with COVID-19 (<xref rid=\"B80\" ref-type=\"bibr\">80</xref>). Currently, a randomized, open label, parallel assignment clinical study is evaluating the efficacy and safety of umifenovir for the treatment of pneumonia in patients infected with SARS-COV-2 (NCT04260594). In this study, patients will be randomized to receive umifenovir plus basic treatment or just the basic treatment (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Moreover, two clinical trials are ongoing to assess the efficacy and safety of umifenovir and lopinavir/ritonavir (NCT04252885) or umifenovir, oseltamivir, and lopinavir/ritonavir (NCT04255017). Specifically, the NCT04252885 is a randomized, open label, parallel assignment clinical trial that will randomize patients with SARS-COV-2 infection in three groups (2:2:1). One group will receive the standard treatment plus lopinavir/ritonavir; the second group will receive standard treatment plus umifenovir; finally, the third group will just receive the standard treatment. The NCT04255017 is instead a randomized, single mask (participants), parallel assignment clinical trial that will randomize COVID-19 patients in four arms. One arm will receive the treatment with umifenovir; the second arm will receive the treatment with oseltamivir; the third arm will receive the treatment with lopinavir/ritonavir; the last arm will just receive the symptomatic supportive treatment (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Another small, randomized, triple mask (Participant, Care Provider, Investigator), parallel assignment clinical trial will be conducted on patients who have a positive test confirming COVID-19 to evaluate the combined treatment with umifenovir, interferon-β 1a, lopinavir/ritonavir, single dose of hydroxychloroquine, and the standards of care compared to the same combined treatment without umifenovir (NCT04350684).</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Mechanism of action, main adverse events and potential drug-drug interactions of inhibitors of viral invasion interfering with the S protein/ACE2 interaction, RAS inhibitors, and analogous ACE2 and A1-7 under clinical evaluation for the treatment of COVID-19.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Therapeutic class</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Drugs</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Main mechanism of action</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Main adverse events</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Drug-drug interactions</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>References</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibitors of S protein/ACE2 interaction</td><td valign=\"top\" align=\"left\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\"><italic>Chloroquine/Hydroxychloroquine</italic></td><td valign=\"top\" align=\"left\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\">Increase of endosomal pH and interference with ACE2 glycosylation</td><td valign=\"top\" align=\"left\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\">Cardiovascular disorders, including prolongation of QT</td><td valign=\"top\" align=\"left\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\">Digoxin, class IA and III antiarrhythmic, tricyclic antidepressants, antipsychotics</td><td valign=\"top\" align=\"center\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B61\" ref-type=\"bibr\">61</xref>, <xref rid=\"B62\" ref-type=\"bibr\">62</xref>)</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Umifenovir</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interference with the attachment of the viral protein to host cells</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gastrointestinal symptoms and increased transaminase</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">As UDP-glucuronosyltransferase 1A9 and 2B7 inhibitor, umifenovir can increase levels of its substrates (paracetamol, buprenorphine, etc.) Cytochrome 3A4 inducers can reduce umifenovir levels</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B63\" ref-type=\"bibr\">63</xref>, <xref rid=\"B64\" ref-type=\"bibr\">64</xref>)</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ARBs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Losartan</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Blocks the AII-induced lung injury</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dizziness, anemia, renal failure, asthenia, hyperkaliemia</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fluconazole and Rifampicine can increase losartan levels, Potassium-sparing diuretics can increase the risk of hyperkaelemia</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B65\" ref-type=\"bibr\">65</xref>, <xref rid=\"B66\" ref-type=\"bibr\">66</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Analogous of ACE2 and A1-7</td><td valign=\"top\" align=\"left\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\"><italic>A1-7</italic></td><td valign=\"top\" align=\"left\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\">Restores the beneficial effect of the non-classic RAS</td><td valign=\"top\" align=\"left\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\">Headache, fatigue, injection site reaction</td><td valign=\"top\" align=\"left\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\">Not Available</td><td valign=\"top\" align=\"center\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B29\" ref-type=\"bibr\">29</xref>, <xref rid=\"B67\" ref-type=\"bibr\">67</xref>, <xref rid=\"B68\" ref-type=\"bibr\">68</xref>)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>ACE2</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Restores the beneficial effect of the non-classic RAS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hypernatremia, rash, dysphagia, and pneumonia</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Not Available</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B69\" ref-type=\"bibr\">69</xref>)</td></tr></tbody></table></table-wrap><p>In addition, speculations were done on the possible use for COVID-19 of new compounds, never approved before, which have shown the ability of interfering with S protein/ACE2 interaction (<xref rid=\"B74\" ref-type=\"bibr\">74</xref>). The compound SSAA09E2 showed the ability of blocking the early interaction of SARS-S protein with ACE2 in ACE2-expressing 293T cells (<xref rid=\"B81\" ref-type=\"bibr\">81</xref>). Moreover, the agent VE607 also showed a significant inhibition of SARS-pseudovirus entry in the same cellular model (<xref rid=\"B82\" ref-type=\"bibr\">82</xref>).</p></sec><sec id=\"s8\"><title>New Pharmacological Perspective for COVID-19 Acting on the RAS</title><p>Based on the beneficial role of the non-classic RAS, which seems lacking in patients with COVID-19, hypotheses have been made on the potential therapeutic approach of restoring the ACE2/A1-7 pathway. This hypothesis is based on preclinical evidence showing an improvement of oxygenation, reduction of inflammation, and reduction of tissue fibrosis after infusion of A1-7 in two models of ARDS (<xref rid=\"B65\" ref-type=\"bibr\">65</xref>, <xref rid=\"B83\" ref-type=\"bibr\">83</xref>). Evidence also showed that the administration of the soluble human recombinant ACE2 was able to reverse the lung-injury process in preclinical models of other viral infections (<xref rid=\"B84\" ref-type=\"bibr\">84</xref>, <xref rid=\"B85\" ref-type=\"bibr\">85</xref>). The rationale to administer soluble ACE2 is to stimulate the RAS protective pathway without increasing the ACE2 transmembrane form that could instead potentiate the viral entry into the cells. Clinical evidence on this aspect is scarce (<xref rid=\"B86\" ref-type=\"bibr\">86</xref>). A phase 2 trial conducted in patients with ARDS showed that ACE2 infusion safely reduced the AII level, but this trial was not powered enough to show efficacy in terms of pulmonary function (<xref rid=\"B69\" ref-type=\"bibr\">69</xref>). Restoring the ACE2 activity may also be beneficial for the myocardial protection in patients with COVID-19 (<xref rid=\"B87\" ref-type=\"bibr\">87</xref>). To date, clinical researches are ongoing to assess the clinical impact of a restoration of the non-classic RAS (ACE2 and A1-7) in patients with COVID-19. Is underway a controlled trial aimed to assess the efficacy, safety and clinical impact of A1-7 infusion in a cohort of COVID-19 patients requiring mechanical ventilation (NCT04332666). It was, instead, suspended a further clinical trial that aimed to assess preliminary biologic, physiologic, and clinical data with the use of ACE2 recombinant compared to the standard care in patients with COVID-19 (NCT04287686).</p><p>In addition, based on the organ protective effects of RAS inhibitors, many studies are being conducted to investigate their efficacy in COVID-19 patients. The beneficial effects of ACE inhibitors and ARB may be related to the prevalence of ACE2/A1-7 effects as demonstrated in experimental studies (<xref rid=\"B88\" ref-type=\"bibr\">88</xref>, <xref rid=\"B89\" ref-type=\"bibr\">89</xref>). Moreover, experimental evidence strongly suggests that AII could promote acute lung injury induced by different coronaviruses, including SARS-COV-1 and SARS-COV-2 (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>, <xref rid=\"B65\" ref-type=\"bibr\">65</xref>). Therefore, the use of RAS inhibitors may block the deleterious effect associated with AII. Two trials are ongoing to investigate the role of losartan for the treatment of COVID-19 in patients who have not previously received a RAS inhibitor and are either hospitalized (NCT04312009) or not hospitalized (NCT04311177). In particular, both trails (NCT04312009 and NCT04311177) are randomized, quadruple mask (participant, care provider, investigator, outcomes assessor), parallel assignment clinical trials that will compare the treatment with losartan vs. placebo in COVID-19 patients, including those with ARDS. Moreover, a pragmatic adaptive, randomized, quadruple mask (participant, care provider, investigator, outcomes assessor), parallel assignment trial is comparing the treatment with lopinavir/ritonavir, or hydroxychloroquine, or losartan vs. placebo in patients with COVID-19 (NCT04328012). Another randomized, quadruple mask (participant, care provider, investigator, outcomes assessor), parallel assignment clinical trial will evaluate the treatment with valsartan compared to placebo for the prevention of ARDS in hospitalized patients with COVID-19 (NCT04335786). Finally, a pilot, randomized, triple mask (participant, care provider, investigator), parallel assignment clinical trial is ongoing to assess the safety and efficacy of telmisartan compared to placebo for the mitigation of pulmonary and cardiac complications in COVID-19 patients (NCT04360551). Characteristics of the mentioned clinical trials are showed in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>. The mechanism of action, main adverse events and potential drug-drug interactions of RAS inhibitors and analogous of A1-7 and ACE2 under clinical evaluation for COVID-19 are summarized in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>.</p><p>Finally, other compounds that may be useful for the treatment of COVID-19, but not currently evaluated, are molecules that may adjust the imbalance between AT1 and AT2 receptors such as compound 21 (C-21), CGP-42112A, and L-163491 (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). C-21 and CGP-42112A are two agonists of AT2 receptors, whereas L-163491 has a dual action as a partial agonist of AT2 receptors and a partial antagonist of AT1 receptors (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>).</p></sec><sec sec-type=\"conclusions\" id=\"s9\"><title>Conclusion</title><p>The RAS may play a complex role in SARS-COV-2 infection. SARS-COV-2 internalization may cause a reduction of ACE2 on cell surface. A reduction in ACE2 can further contribute to the pulmonary function deterioration and the myocardial damage. However, there is a paucity of clinical evidence on the efficacy of restoring the ACE2 functionality for the treatment of viral-induced lung injury. A clinical trial is ongoing to evaluate the effect of A1-7 in COVID-19 patients. To date, there is no effective drug for the treatment of COVID-19 and few clinical data are available. Some clinical trials are ongoing to evaluate the efficacy of drugs that could interfere with the S protein/ACE2 interaction such as umifenovir and hydroxychloroquine/chloroquine.</p><p>Data instead on the increased mRNA expression and levels of ACE2 after treatment with RAS inhibitors are scarce and to date not associated with an increased mortality in patients with COVID-19. Currently, clinical trials are ongoing to investigate the use of a RAS inhibitor for the reduction of the lung damage in patients with COVID-19. Substantial evidence is needed to guide decision-making on the use of ACE inhibitors and ARBs in such patients, until then we need to base on the available data that place RAS inhibitors among the safe choices for cardiovascular diseases.</p></sec><sec id=\"s10\"><title>Author Contributions</title><p>AM, CS, CR, CF, GR, LB, GP, FR, and AC: drafting the work, revising it for important intellectual content, final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately discussed. FR and AC developed the concept and designed the study. AM wrote the paper. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s11\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>We are grateful for the help and support of the Italian Society of Pharmacology (SIF) and its Section of Clinical Pharmacology Giampaolo Velo. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Physiol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Physiol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Physiol.</journal-id><journal-title-group><journal-title>Frontiers in Physiology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-042X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32848876</article-id><article-id pub-id-type=\"pmc\">PMC7431662</article-id><article-id pub-id-type=\"doi\">10.3389/fphys.2020.00949</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Physiology</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Role of Histone Deacetylases in Skeletal Muscle Physiology and Systemic Energy Homeostasis: Implications for Metabolic Diseases and Therapy</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Tian</surname><given-names>Haili</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/677629/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Liu</surname><given-names>Sujuan</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ren</surname><given-names>Jun</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/52428/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Lee</surname><given-names>Jason Kai Wei</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/210309/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Ru</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Peijie</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/641077/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>School of Kinesiology, Shanghai University of Sport</institution>, <addr-line>Shanghai</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Anatomy and Histology, School of Basic Medical Sciences, Tianjin Medical University</institution>, <addr-line>Tianjin</addr-line>, <country>China</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Cardiology, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital Fudan University</institution>, <addr-line>Shanghai</addr-line>, <country>China</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore</institution>, <addr-line>Singapore</addr-line>, <country>Singapore</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Global Asia Institute, National University of Singapore</institution>, <addr-line>Singapore</addr-line>, <country>Singapore</country></aff><aff id=\"aff6\"><sup>6</sup><institution>N.1 Institute for Health, National University of Singapore</institution>, <addr-line>Singapore</addr-line>, <country>Singapore</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Brian James Morris, The University of Sydney, Australia</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Sean L. McGee, Deakin University, Australia; Viviana Moresi, Sapienza University of Rome, Italy</p></fn><corresp id=\"c001\">*Correspondence: Ru Wang, <email>wangru@ty.sus.edu.cn</email></corresp><corresp id=\"c002\">Peijie Chen, <email>chenpeijie@sus.edu.cn</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>These authors have contributed equally to this work</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>949</elocation-id><history><date date-type=\"received\"><day>27</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>14</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Tian, Liu, Ren, Lee, Wang and Chen.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Tian, Liu, Ren, Lee, Wang and Chen</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Skeletal muscle is the largest metabolic organ in the human body and is able to rapidly adapt to drastic changes during exercise. Histone acetyltransferases (HATs) and histone deacetylases (HDACs), which target histone and non-histone proteins, are two major enzyme families that control the biological process of histone acetylation and deacetylation. Balance between these two enzymes serves as an essential element for gene expression and metabolic and physiological function. Genetic KO/TG murine models reveal that HDACs possess pivotal roles in maintaining skeletal muscles’ metabolic homeostasis, regulating skeletal muscles motor adaptation and exercise capacity. HDACs may be involved in mitochondrial remodeling, insulin sensitivity regulation, turn on/off of metabolic fuel switching and orchestrating physiological homeostasis of skeletal muscles from the process of myogenesis. Moreover, many myogenic factors and metabolic factors are modulated by HDACs. HDACs are considered as therapeutic targets in clinical research for treatment of cancer, inflammation, and neurological and metabolic-related diseases. This review will focus on physiological function of HDACs in skeletal muscles and provide new ideas for the treatment of metabolic diseases.</p></abstract><kwd-group><kwd>histone deacetylases</kwd><kwd>exercise capacity</kwd><kwd>skeletal muscle</kwd><kwd>metabolism</kwd><kwd>muscle physiology</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">China Postdoctoral Science Foundation<named-content content-type=\"fundref-id\">10.13039/501100002858</named-content></funding-source></award-group><award-group><funding-source id=\"cn002\">National Natural Science Foundation of China<named-content content-type=\"fundref-id\">10.13039/501100001809</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"4\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"99\"/><page-count count=\"11\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Skeletal muscle is the largest metabolic organ in the human body, consuming about 18% of the entire body daily expenditure of energy (<xref rid=\"B15\" ref-type=\"bibr\">Durnin, 1981</xref>). It produces various secrete factors and participates in the interplay among multiple tissues and organs (<xref rid=\"B65\" ref-type=\"bibr\">Pedersen and Febbraio, 2012</xref>; <xref rid=\"B58\" ref-type=\"bibr\">Mizgier et al., 2019</xref>). Muscular contraction is one of the main physiological functions of skeletal muscles and plays a role in maintaining organ and systemic metabolic homeostasis (<xref rid=\"B24\" ref-type=\"bibr\">Guo et al., 2020</xref>). Proper exercises help build up body defense to combat various diseases including obesity, type 2 diabetes, Alzheimer disease, osteoarthritis, and so on (<xref rid=\"B47\" ref-type=\"bibr\">Luan et al., 2019</xref>). These important functions require delicate regulations in muscle from the level of genome to signal transduction, establishing muscle plasticity, and responses to environmental stress. Acetyl-CoA as a central metabolite of amino acid/fatty acid/glucose not only serves as fuel for energy expenditure but also bridges the gap between environmental stress and organismal response (<xref rid=\"B43\" ref-type=\"bibr\">Lempradl et al., 2015</xref>; <xref rid=\"B45\" ref-type=\"bibr\">Liu et al., 2020</xref>). This control occurs through post-translational modification on nucleosomes histone lysine residues and other signal transducing proteins, which in turn controls accessibility of DNA to regulatory factors and protein activity. Acetylation, one of the most prevalent modifications of protein, is believed to function as a key modulator of chromatin structure and signal transduction, and provides an avenue to couple extracellular stimuli with genome during muscle metabolism process by regulating acetylation and deacetylation (<xref rid=\"B26\" ref-type=\"bibr\">Haberland et al., 2009</xref>).</p><p>histone deacetylases (HDACs) as a family of protein deacetylases have been demonstrated to moderate physiological homeostasis and development by deacetylation (<xref rid=\"B26\" ref-type=\"bibr\">Haberland et al., 2009</xref>) (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). In humans, there are 18 types of HDACs, which are classified into four categories based on homologous proteins in yeast, namely: Class I Rpd3-like protein (HDAC1, -2, -3, -8); Class II Hda1-like proteins are classified as Class IIa (HDAC4, -5, -7, -9) and Class IIb (HDAC6, -10); Class III Sir2p-like, nicotinamide adenine dinucleotide (NAD<sup>+</sup>)-dependent (SIRT1, -2, -3, -4, -5, -6, -7); and Class IV HDAC11, containing homologous domain with both Rpd3 and Hda1 (<xref rid=\"B76\" ref-type=\"bibr\">Seto and Yoshida, 2014</xref>). HDACs modulate gene expression and protein activity through deacetylating proteins. When histone lysine ε-amino acid in the nucleosome is acetylated, it can neutralize positive charge, before loosening chromatin structure, to promote binding of transcription factors to DNA and expression of downstream target genes. By contrast, histone deacetylation compresses chromatin structure, thereby inhibiting transcriptional gene expression (<xref rid=\"B77\" ref-type=\"bibr\">Shahbazian and Grunstein, 2007</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>The targets of HADCs in skeletal muscles and their physiological functions.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Class</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Members</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Down targets</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Genetic KO/TG phenotype</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">References</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HDAC1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MyoD↓, <italic>PTEN</italic>↓, FoxO?</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Functionally redundant HDAC1/2 DKO mice causes mitochondrial abnormalities and sarcomere degeneration</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B69\" ref-type=\"bibr\">Puri et al., 2001</xref>; <xref rid=\"B63\" ref-type=\"bibr\">Ohkawa et al., 2006</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Beharry et al., 2014</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Cho et al., 2015</xref>; <xref rid=\"B99\" ref-type=\"bibr\">Zhu et al., 2016</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Bin et al., 2019</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HDAC2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MyoD↓, MEF2D↓, <italic>NF-</italic>κ<italic>Bp65</italic>↓</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HDAC3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MEF2D, PCAF↓, <italic>Ampd3</italic>↓ RCAN1? MEF2A-<italic>Cpt1b</italic>↓</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">mKO mice: Enhanced amino acid\\lipid metabolism and endurance exercise Causes IR under basal condition</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B23\" ref-type=\"bibr\">Gregoire et al., 2007</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Han et al., 2014</xref>; <xref rid=\"B96\" ref-type=\"bibr\">Yuan et al., 2014</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Hong et al., 2017</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HDAC8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IIA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HDAC4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MEF2↓, <italic>Glut4</italic>↓, Myosin Heavy Chain, PGC-1α, and Hsc70-, <italic>Pax7</italic>↓</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Functionally redundant HDAC4/5 DKO, HDAC5/9 DKO, HDAC4/5/9 TKO increases type I fiber percentage and oxidation metabolism</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B55\" ref-type=\"bibr\">McKinsey et al., 2000a</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Choi et al., 2014</xref>; <xref rid=\"B62\" ref-type=\"bibr\">Niu et al., 2017</xref>; <xref rid=\"B48\" ref-type=\"bibr\">Luo et al., 2019</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HDAC5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MEF2↓, <italic>Glut4</italic>↓, <italic>Baf60c</italic>↓</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B96\" ref-type=\"bibr\">Yuan et al., 2014</xref>; <xref rid=\"B57\" ref-type=\"bibr\">Meng et al., 2017</xref>; <xref rid=\"B62\" ref-type=\"bibr\">Niu et al., 2017</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HDAC9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Atg7, Beclin1, LC3</italic>↓ Dach2-<italic>Myog, Gdf5</italic>↓</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B49\" ref-type=\"bibr\">Macpherson et al., 2015</xref>; <xref rid=\"B98\" ref-type=\"bibr\">Zhang et al., 2019</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HDAC7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MEF2↓</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B14\" ref-type=\"bibr\">Dressel et al., 2001</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IIB</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HDAC6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Pax7</italic>, MFN1↓ Fam65b, dysferlin and MAFbx-</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">KO mice: Causes mitochondrial oxidative damage, mitofusion defect Protect against muscle wasting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B3\" ref-type=\"bibr\">Balasubramanian et al., 2014</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Lee et al., 2014</xref>; <xref rid=\"B70\" ref-type=\"bibr\">Ratti et al., 2015</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HDAC10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">III</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SIRT1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PGC-1α? STAT3↓</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">mKO mice: Mediates CR induced insulin sensitivity, has no effect on glucose homeostasis or exercise capacity TG mice: no effect on glucose homeostasis or exercise capacity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B72\" ref-type=\"bibr\">Rodgers et al., 2005</xref>; <xref rid=\"B75\" ref-type=\"bibr\">Schenk et al., 2011</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SIRT2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">KO mice: Exacerbates obesity and IR in HFD</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SIRT3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MnSOD?, Hexokinase II? PDH subunit E1α?</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">KO mice: Exacerbates obesity and IR in HFD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B8\" ref-type=\"bibr\">Boyle et al., 2013</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Jing et al., 2013</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Lantier et al., 2015</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SIRT4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Malonyl CoA decarboxylase↓</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">KO mice: Increased exercise tolerance and protect against HFD-induced obesity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B40\" ref-type=\"bibr\">Laurent et al., 2013</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SIRT5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SIRT6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NF-κB-<italic>Mstn</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">KO mice: Abnormal hypoglycemia TG mice: Protect against HFD mKO mice: Impaired insulin sensitivity, loss of muscle mass</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B34\" ref-type=\"bibr\">Kanfi et al., 2010</xref>; <xref rid=\"B93\" ref-type=\"bibr\">Xiao et al., 2010</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Cui et al., 2017</xref>; <xref rid=\"B74\" ref-type=\"bibr\">Samant et al., 2017</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SIRT7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IV</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HDAC11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr></tbody></table><table-wrap-foot><attrib><italic>Activation?, inhibition↓, unknown. KO, whole-body deletion; TG, whole-body overexpression; DKO, double knockout; TKO, triple knockout; mKO, muscle-specific knockout; CR, caloric restriction; HFD, high fat diet; IR, insulin resistance.</italic></attrib></table-wrap-foot></table-wrap><p>Biochemical properties of HDACs have been comprehensively reviewed (<xref rid=\"B77\" ref-type=\"bibr\">Shahbazian and Grunstein, 2007</xref>). However, the physiological functions of HDACs have yet to be well examined in skeletal muscles. A series of researches shed light on the potential of drugs targeting HDACs to improve muscle fitness and cardiac muscle disease, which needs further clarification of HDACs physiological function to understand the mechanisms (<xref rid=\"B2\" ref-type=\"bibr\">Bai et al., 2011</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Galmozzi et al., 2013</xref>; <xref rid=\"B94\" ref-type=\"bibr\">Xie et al., 2014</xref>; <xref rid=\"B97\" ref-type=\"bibr\">Zhang and Ren, 2014</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Gaur et al., 2016</xref>). Ample work has revealed the role of HDAC in muscle physiology such as skeletal muscles me olism and thus exercise capacity (<xref rid=\"B52\" ref-type=\"bibr\">McGee and Hargreaves, 2010</xref>) (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Class I HDACs can interact with myocyte enhancer factor 2 (MEF2), MyoD, regulating myogenesis, and exercise capacity (<xref rid=\"B50\" ref-type=\"bibr\">Mal et al., 2001</xref>; <xref rid=\"B69\" ref-type=\"bibr\">Puri et al., 2001</xref>; <xref rid=\"B63\" ref-type=\"bibr\">Ohkawa et al., 2006</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Gregoire et al., 2007</xref>). HDAC3 participates in myocytes differentiation and is linked to skeletal muscles metabolic fuel switching (<xref rid=\"B23\" ref-type=\"bibr\">Gregoire et al., 2007</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Hong et al., 2017</xref>). Class II HDACs can be phosphorylated by HDAC kinases and are transduced from nucleus to cytoplasm in response to cellular stress, mediating muscle fiber switch and affecting the pathway of insulin sensitivity such as <italic>Glut4</italic> and AKT (<xref rid=\"B26\" ref-type=\"bibr\">Haberland et al., 2009</xref>; <xref rid=\"B52\" ref-type=\"bibr\">McGee and Hargreaves, 2010</xref>). Class III HDACs target a crucial mitochondrial biogenesis factor peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α) in skeletal muscles, liver and fat tissue (<xref rid=\"B91\" ref-type=\"bibr\">White and Schenk, 2012</xref>). Therefore, we will further discuss how these HDACs influence respective downstream targets, exercise capacity, and therapeutic effects in human diseases.</p></sec><sec id=\"S2\"><title>Class I HDAC: HDAC1/2/3</title><sec id=\"S2.SS1\"><title>Regulation of Myogenesis by Class I HDACs</title><p>In previous studies, HDAC1/2/3 was shown to be associated with the process of myogenesis or myocyte differentiation (<xref rid=\"B50\" ref-type=\"bibr\">Mal et al., 2001</xref>; <xref rid=\"B69\" ref-type=\"bibr\">Puri et al., 2001</xref>; <xref rid=\"B63\" ref-type=\"bibr\">Ohkawa et al., 2006</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Gregoire et al., 2007</xref>). HDAC1 tightly binds to MyoD and deacetylates specific sites to inhibit the expression of muscle-specific genes such as <italic>MHC</italic> and <italic>MCK</italic> in myoblasts. Mimicking muscle differentiation condition by serum deprivation, HDAC1 protein gradually decreases during myocyte differentiation and transfers to bind to the tumor suppressor pRb, accompanied by isolation of HDAC1-MyoD and transcriptional activation of muscle-specific genes (<xref rid=\"B69\" ref-type=\"bibr\">Puri et al., 2001</xref>). A HDAC1 (H141A) mutant incapable of binding with MyoD loses its inhibitory property on muscle-specific genes in myoblast state (<xref rid=\"B50\" ref-type=\"bibr\">Mal et al., 2001</xref>). In the late stage of myocyte differentiation, activating factor gradually switches from MyoD to myogenin, occurring with a decrease in the MEF2D inhibitory regulator HDAC2. Simultaneous overexpression of myogenin and MEF2D can enhance the expression of the muscle-specific gene <italic>MHC</italic> in the absence of MyoD (<xref rid=\"B63\" ref-type=\"bibr\">Ohkawa et al., 2006</xref>). MEF2D, a key factor that controls myocyte differentiation, can only be effectively deacetylated by HDAC3, rather than HDAC1/2/8 (<xref rid=\"B23\" ref-type=\"bibr\">Gregoire et al., 2007</xref>). In addition, HDAC3 can inhibit autoacetylation of acetyltransferases p300 and p300/CBP-associated factor (PCAF). Thus, HDAC3 impedes MyoD-MEF2-PCAF to form a multicomplex, disturbing MEF2-dependent myogenic transcription (<xref rid=\"B23\" ref-type=\"bibr\">Gregoire et al., 2007</xref>; <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Controls of myogenesis by Class I HDACs. The inhibitory effects of HDACs on myogenic factors MEF2 and MyoD maintain a primary myoblast state. During myocyte differentiation and myogenic process, the inhibition is lifted by other factors, facilitating MEF2-MyoD complex formation to promote myogenesis. –Ac, deacetylation.</p></caption><graphic xlink:href=\"fphys-11-00949-g001\"/></fig></sec><sec id=\"S2.SS2\"><title>Redundant Roles of HDAC1/2 in Maintaining Sarcomere Homeostasis in Muscle</title><p>Loss of function of HDAC1/2 inhibits autophagy flux in skeletal muscles tissue, accompanied by decreased LC3 II/I ratio and p62 accumulation under fasting condition (<xref rid=\"B60\" ref-type=\"bibr\">Moresi et al., 2012</xref>). Toxic autophagy intermediates accumulate in muscle fibers in which class I HDACs deficiency led to impaired exercise capacity (<xref rid=\"B60\" ref-type=\"bibr\">Moresi et al., 2012</xref>). This offers convincing evidence on the role of HDAC1/2 in stabilizing basic structure and facilitating development in muscles. Genetic models suggest that Class I HDACs control the physiological homeostasis of skeletal muscles. A germline deletion shows that whole-body knockout of either HDAC1 or HDAC2 leads to mortality in mice prior to the perinatal period (<xref rid=\"B59\" ref-type=\"bibr\">Montgomery et al., 2007</xref>). However, a tissue-specific single deletion of HDAC1 or HDAC2 in myocardium does not cause any phenotype (<xref rid=\"B59\" ref-type=\"bibr\">Montgomery et al., 2007</xref>). Double knockout HDAC1/2 in the heart leads to severe cardiomyopathy in mice, indicating the obligatory role for HDAC1/2 in specific tissues (<xref rid=\"B59\" ref-type=\"bibr\">Montgomery et al., 2007</xref>). In skeletal muscle, double knockout of HDAC1/2 by myogenin-Cre causes mitochondrial abnormalities and sarcomere degeneration, disrupting fundamental structural units of myofibers (<xref rid=\"B60\" ref-type=\"bibr\">Moresi et al., 2012</xref>). Therefore, these findings demonstrate that HDAC1/2 redundantly maintains sarcomere homeostasis in skeletal muscle.</p></sec><sec id=\"S2.SS3\"><title>HDAC3 Functions as a Fuel Switch in Muscle Energy Metabolism</title><p>Histone deacetylases 3 is the enzymatic core of nuclear receptor corepressor (N-CoR) and silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) co-repressor; correspondingly, N-CoR and SMRT co-repressor activate HDAC3 through its SANT domain enzyme activity (<xref rid=\"B35\" ref-type=\"bibr\">Karagianni and Wong, 2007</xref>). Mice suffer from insulin resistance after specifically knocking out HDAC3 in skeletal muscles-mKO, while their endurance exercise abilities are enhanced (<xref rid=\"B30\" ref-type=\"bibr\">Hong et al., 2017</xref>). It seems a self-contradictory phenomenon because former studies pointed out that increased endurance exercise capacity enhances insulin sensitivity. The study shows that insulin signaling cascades including pAKT/AKT, pIRS1-S1101, and pGSK/GSK have no change in HDAC3 mKO muscle, while glucose uptake and insulin sensitivity are impaired in glucose tolerance test (GTT) and insulin tolerance test (ITT) (<xref rid=\"B30\" ref-type=\"bibr\">Hong et al., 2017</xref>), which is called the “dissociation effect” by researchers. Lipid tends to be used as energy fuel in HDAC mKO mice, which inhibits glucose absorption but does not influence insulin signaling sensitivity (<xref rid=\"B30\" ref-type=\"bibr\">Hong et al., 2017</xref>). Further work found that the HDAC3 knockout upregulates the expression of AMP deaminase 3 (AMPD3), the first rate-limiting enzyme in purine metabolism, in skeletal muscles (<xref rid=\"B17\" ref-type=\"bibr\">Fortuin et al., 1996</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Hong et al., 2017</xref>). AMPD3 can deaminate AMP to form IMP, facilitating aspartic acid to transmit into fumarate and malate, the intermediate metabolites of tricarboxylic acid cycle (TCA cycle) (<xref rid=\"B30\" ref-type=\"bibr\">Hong et al., 2017</xref>). Research studies found that exercise induced glucose labeled 13C6 expressed a lower glycolysis flux rate in muscles of HDAC3 mKO but a higher expression in the TCA cycle intermediates (<xref rid=\"B30\" ref-type=\"bibr\">Hong et al., 2017</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Gong et al., 2018</xref>). Thus, a general increase in TCA cycle metabolites activates the oxidation in mitochondria. <italic>In vitro</italic> radioactive aspartic acid isotope tracer test showed that inhibiting HDAC3 or overexpressing AMPD3 can increase amino acid metabolism rate and enhance fatty acid metabolism, thereby downregulating glucose metabolism (<xref rid=\"B30\" ref-type=\"bibr\">Hong et al., 2017</xref><xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). In liver, knockout HDAC3 causes severe liver steatosis and can be rescued by wild-type or catalytically inactive mutants of HDAC3, indicating an enzymatic activity independent function (<xref rid=\"B80\" ref-type=\"bibr\">Sun et al., 2013</xref>). However, the muscle fuel switching in HDAC3 mKO mice cannot be rescued by an enzymatic inactivity mutant HDAC3 (<xref rid=\"B78\" ref-type=\"bibr\">Song et al., 2019</xref>). This finding demonstrates that HDAC3 controls the fuel utilization in skeletal muscles and is dependent on its enzymatic activity. The relationship between exercise and glucose uptake is far more complex than one could expect.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Class I HDACs regulates sarcomere structure and exercise capacity in mature muscle. A schematic diagram shows Class I HDACs regulate exercise capacity on different levels. HDAC1/2 have redundant roles in modulating autophagy flux in muscle, eliminating toxic factors in muscle. HDAC3 serves as a fuel switch and promotes muscle to use fatty acid to adapt endurance exercise.</p></caption><graphic xlink:href=\"fphys-11-00949-g002\"/></fig></sec></sec><sec id=\"S3\"><title>Class II HDACs: HDAC4/5/7/9/6</title><sec id=\"S3.SS1\"><title>Phosphorylation and Nucleus-Cytoplasm Shuttle of Class II HDACs</title><p>The MEF2 family is a class of transcription factors that are closely associated with myogenic basic helix-loop-helix domains, including MyoD (<xref rid=\"B84\" ref-type=\"bibr\">Taylor and Hughes, 2017</xref>). MEF2 may interact with Class IIa histone deacetylase HDAC4/5 to inhibit the transcription of MEF2-dependent genes, thereby impeding the differentiation from myoblast to myotube (<xref rid=\"B55\" ref-type=\"bibr\">McKinsey et al., 2000a</xref>). During exercise, Ca<sup>2+</sup> flows out from sarcoplasmic reticulum and activate calcium/calmodulin-dependent protein kinase (CaMK), which phosphorylates HDAC4/5 and mediates HDAC4/5 shuttle from the nucleus to the cytoplasm, thus releasing the repressive effect of HDAC4/5 on MEF2-dependent genes (<xref rid=\"B56\" ref-type=\"bibr\">McKinsey et al., 2000b</xref>; <xref rid=\"B96\" ref-type=\"bibr\">Yuan et al., 2014</xref>). Further research demonstrates that the nucleoplasmic shuttle of HDAC4 relies on 14-3-3 binding, while the shuttle of HDAC5 depends on CaMK phosphorylation at serine -259 and -498 sites first, and then exporting from the nucleus by binding with 14-3-3 (<xref rid=\"B56\" ref-type=\"bibr\">McKinsey et al., 2000b</xref>). As expected, HDAC7 in Class IIa also possesses the ability to be exported from nucleus to cytoplasm (<xref rid=\"B19\" ref-type=\"bibr\">Gao et al., 2010</xref>), suggesting that there may be redundant functions of Class IIa HDACs. Except for Class IIa, knocking out Class IIb HDAC6 in embryonic stem cells (ESCs) can promote ESCs to differentiate into myoblast cells, and transplantation of ESCs with HDAC6 knockdown can also help the regeneration of wound skeletal muscles (<xref rid=\"B42\" ref-type=\"bibr\">Lee et al., 2015</xref>).</p></sec><sec id=\"S3.SS2\"><title>Regulation of Class II HDACs on Fiber-Type Switch, Mitochondria Remodeling, and Energy Stress in Skeletal Muscle</title><p>Evidence shows that the proportion of type I fiber in skeletal muscles has no change in animal knocked out alone any member of Class IIa (<xref rid=\"B68\" ref-type=\"bibr\">Potthoff et al., 2007</xref>). However, the proportion of type I fiber is significantly increased in HDAC4/5 DKO, HDAC5/9 DKO, HDAC4/5/9 TKO mice as well as exercise capacity of skeletal muscles (<xref rid=\"B68\" ref-type=\"bibr\">Potthoff et al., 2007</xref>). Altogether, the finding indicated a functional redundant mechanism in Class II HDACs family. Previous study found that Class IIa HDACs are closely associated with the MEF2 family and can repress their downstream target genes (<xref rid=\"B55\" ref-type=\"bibr\">McKinsey et al., 2000a</xref>). Therefore, similar to Class IIa HDACs DKO, the proportion of type I fiber is decreased in the MEF2C and MEF2D knockout mouse models, overexpressing MEF2C results in increasing type I fiber and exercise capacity. HDAC4/5 not only regulates exercise capacity but also governs motor adaptation. Several studies had shown that exercise promotes phosphorylation of HDAC4/5 mediated by CaMK and adenosine monophosphate-activated protein kinase (AMPK), then increasing a MEF2-dependent transcription of <italic>Glut4</italic> and muscle-specific genes, which participates in the adaptation and plasticity of skeletal muscles (<xref rid=\"B54\" ref-type=\"bibr\">McGee et al., 2008</xref>, <xref rid=\"B51\" ref-type=\"bibr\">2009</xref>; <xref rid=\"B53\" ref-type=\"bibr\">McGee and Hargreaves, 2011</xref>). Besides exercise, glucose can also activate K<sub>ATP</sub> channel-dependent calcium signaling and CaMK, which induce phosphorylation-dependent nuclear-cytoplasm transportation of HDAC5 (<xref rid=\"B57\" ref-type=\"bibr\">Meng et al., 2017</xref>). The leaving HDAC5 releases its repressive effect on <italic>Baf60c</italic> and enhancing insulin-independent AKT activation (<xref rid=\"B57\" ref-type=\"bibr\">Meng et al., 2017</xref>). Using specific inhibitor of class IIa HDAC activity, myosin heavy chain, PGC-1α, and heat-shock cognate (HSC70) are confirmed to be one of the substrates of HDAC4 in denervation-induced atrophy muscle in comparison with acetylated protein enrichment with untreated group (<xref rid=\"B48\" ref-type=\"bibr\">Luo et al., 2019</xref>). Taken together, Class IIa HDACs play critical roles in exercise capacity, remodeling, and nutrient sensing of skeletal muscles (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Control of muscle exercise adaption and mitochondrial function by Class II HDACs. <bold>(A)</bold> Schematic of the Class IIa HDACs nucleoplasm shuttle. Under stress condition, CaMK and AMPK could be activated and phosphorylate Class IIa HDACs. Phosphorylated HDACs then bind with 14-3-3 and shuttle to cytoplasm, relieving inhibitory effects on MEF2 and <italic>Baf60c</italic>. <bold>(B)</bold> Class IIb HDAC6 can deacetylate MFN1 and facilitates its mito-fusion function. +P, phosphorylation.</p></caption><graphic xlink:href=\"fphys-11-00949-g003\"/></fig><p>For Class IIb, it was revealed that glucose deprivation-induced mitochondrial fusion mediated by mitofusion1 (MFN1) is significantly disrupted in HDAC6 KO mice, causing mitochondria degeneration, which is reversed following application of MFN1 acetylation-resistant mutant (<xref rid=\"B41\" ref-type=\"bibr\">Lee et al., 2014</xref>). This study suggested that MFN1 deacetylation by HDAC6 plays an important role in mitochondrial adaptive energy production and remodeling of skeletal muscles.</p></sec></sec><sec id=\"S4\"><title>Class III HDACs/Sir2-Homolog: SIRT1/6/3/4</title><sec id=\"S4.SS1\"><title>Nuclear-Located SIRT1/6</title><p>Numerous studies have found that caloric restriction (CR) extends lifespan of mammals, <italic>Caenorhabditis elegans</italic>, and fruit flies, while such effect is lost in <italic>SIR2</italic> or <italic>NPT1</italic> mutant <italic>C. elegans</italic> strains (<xref rid=\"B32\" ref-type=\"bibr\">Imai et al., 2000</xref>; <xref rid=\"B44\" ref-type=\"bibr\">Lin et al., 2000</xref>). <italic>SIR2</italic> encodes the silencing protein, Sir2p, and NPT1 participates in the synthesis of NAD<sup>+</sup>. NAD<sup>+</sup> is an essential cofactor for Sir2p-Class III HDACs to exert enzyme activity rather than Class I, II, and IV relaying on the zinc (<xref rid=\"B32\" ref-type=\"bibr\">Imai et al., 2000</xref>; <xref rid=\"B44\" ref-type=\"bibr\">Lin et al., 2000</xref>). In mammals, Sir2 has 7 homologous proteins and these proteins are essential to mitochondrial energy homeostasis, antioxidant defense, cell proliferation, and DNA repair (<xref rid=\"B86\" ref-type=\"bibr\">Vargas-Ortiz et al., 2019</xref>).</p><sec id=\"S4.SS1.SSS1\"><title>SIRT1 Mediates Energy Stress Adaptation in Skeletal Muscles</title><p><xref rid=\"B72\" ref-type=\"bibr\">Rodgers et al. (2005)</xref> reported that SIRT1 promotes the transcriptional activity of PGC-1α by deacetylating the PGC-1α at K13 site and mediates CR-induced gluconeogenesis-related genes <italic>G6P</italic>, <italic>PEPCK</italic> expression in liver. Further research showed that the NAD<sup>+</sup>/NADH ratio and the enzymatic activity of Sir2, a sensor of redox state, were decreased in differentiating myocytes, which relieved the inhibitory effect on MyoD and then promoted cell differentiation. Resveratrol (RSV), a compound that may target SIRT1-PGC-1α, can improve mitochondrial function and metabolic homeostasis, owning a potential to prolong lifespan. RSV loses its activating effect on PGC-1α in SIRT1 (<sup>–/–</sup>) MEFs (<xref rid=\"B38\" ref-type=\"bibr\">Lagouge et al., 2006</xref>). Moreover, RSV-treated mice at a dose of 4 g/kg increased their exercise capacity, oxygen consumption, and improved insulin sensitivity against high-fat induced obesity (<xref rid=\"B38\" ref-type=\"bibr\">Lagouge et al., 2006</xref>). In addition, RSV could enhance the deacetylation of PGC-1α in brown fat tissue (BAT) and skeletal muscles, which promotes mitochondrial production, oxygen consumption, and thus improves metabolic syndrome (<xref rid=\"B38\" ref-type=\"bibr\">Lagouge et al., 2006</xref>). The function of SIRT1 in regulating skeletal muscles metabolic homeostasis and exercise capacity has attracted great attention from researchers. Researchers further generated skeletal muscle-specific SIRT1-mKO mice and found that mKO mice lost the effect of CR-induced increasing insulin sensitivity and were unable to deacetylate and inactivate STAT3, resulting in upregulating the phosphatidylinositol-3-kinase (PI3K) inhibitory regulator p55α/p50α expression (<xref rid=\"B75\" ref-type=\"bibr\">Schenk et al., 2011</xref>). Although knockout or overexpression of SIRT1 in skeletal muscles has no effect on endurance capacity or glucose homeostasis in mice (<xref rid=\"B25\" ref-type=\"bibr\">Gurd et al., 2009</xref>; <xref rid=\"B66\" ref-type=\"bibr\">Philp et al., 2011</xref>; <xref rid=\"B90\" ref-type=\"bibr\">White et al., 2013</xref>; <xref rid=\"B82\" ref-type=\"bibr\">Svensson et al., 2020</xref>), some studies have shown that AMPK regulates energy metabolism of skeletal muscles partially mediated by SIRT1, and SIRT1 is significantly increased after endurance exercise (<xref rid=\"B81\" ref-type=\"bibr\">Suwa et al., 2008</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Canto et al., 2009</xref>). Overexpression of Sirt1 in skeletal muscles by adeno-associated virus 1 (AAV1) promotes the expression of oxidation-related genes including <italic>Ppargc1a</italic>, <italic>Tfam</italic>, <italic>Cpt1b</italic>, and <italic>Pdk4</italic>, while it has no effect on insulin sensitivity of body (<xref rid=\"B88\" ref-type=\"bibr\">Vila et al., 2016</xref>). But overexpressing Sirt1 in liver by AAV8 can protect from fatty liver induced by high-carbohydrate food (HCD) (<xref rid=\"B87\" ref-type=\"bibr\">Vila et al., 2014</xref>). Taken together, the aforementioned studies suggest that SIRT1 possesses limited regulation capacity of skeletal muscles motility, and SIRT1 may modulate mitochondrial homeostasis and mediate skeletal muscles adaptation under certain physiological conditions, such as CR, aging, and regeneration (<xref rid=\"B21\" ref-type=\"bibr\">Gomes et al., 2013</xref>; <xref rid=\"B73\" ref-type=\"bibr\">Ryall et al., 2015</xref>; <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). In addition, there may be more beneficial effects of SIRT1 on metabolism in the liver or fat tissue (<xref rid=\"B38\" ref-type=\"bibr\">Lagouge et al., 2006</xref>; <xref rid=\"B87\" ref-type=\"bibr\">Vila et al., 2014</xref>, <xref rid=\"B88\" ref-type=\"bibr\">2016</xref>; <xref rid=\"B79\" ref-type=\"bibr\">Stefanowicz et al., 2018</xref>).</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Class III HDACs promote a stress-induced adaptation in muscle and regulate mitochondrial function. Class III HDACs are NAD<sup>+</sup>-dependent deacetylases and may function as a redox sensor in muscle cell. SIRT1 could activate PGC-1alpha by directly deacetylasing K13 site and regulate insulin sensitivity by targeting STAT3. Mitochondria-located Class III HDACs control mitochondrial function by deacetylating certain mitochondrial proteins, modulating muscle fuel utilization and exercise capacity.</p></caption><graphic xlink:href=\"fphys-11-00949-g004\"/></fig></sec></sec><sec id=\"S4.SS2\"><title>SIRT6 Improves Muscle Fitness and Exercise Capacity</title><p>SIRT6 is another Sir2-like deacetylase located in the nucleus. Whole body deletion of SIRT6 causes 60% mice death around 4 weeks owning to hypoglycemia (<xref rid=\"B93\" ref-type=\"bibr\">Xiao et al., 2010</xref>). Nevertheless, the skeletal muscle-specific knockout of SIRT6 causes insulin resistance and impairs glucose homeostasis of mKO mice (<xref rid=\"B12\" ref-type=\"bibr\">Cui et al., 2017</xref>). Because the activity of AMPK is reduced in SIRT6-mKO mice, the glycolipids absorption and utilization of skeletal muscles are impaired, and the exercise capacity of the mice was also attenuated (<xref rid=\"B12\" ref-type=\"bibr\">Cui et al., 2017</xref>). Furthermore, researchers constructed the overexpressing SIRT6 (Sirt6BAC) mice and found that their body weight and fat content were normal, but the Sirt6BAC mice could protect from HCD-induced hyperglycemia via increasing p-AKT/AKT induced by insulin stimulation and glucose absorption (<xref rid=\"B1\" ref-type=\"bibr\">Anderson et al., 2015</xref>). Vitro experiments further demonstrated that the ability of glucose uptake was enhanced mainly in skeletal muscles, not in brain, iWAT, eWAT tissues of Sirt6BAC mice (<xref rid=\"B1\" ref-type=\"bibr\">Anderson et al., 2015</xref>). These results shows the potential effects of SIRT6 targeted in skeletal muscles on improving exercise performance and treating metabolic diseases (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>).</p></sec><sec id=\"S4.SS3\"><title>Mitochondria-Located SIRT3/4</title><sec id=\"S4.SS3.SSS1\"><title>SIRT3/4 Maintains Mitochondria Function in Skeletal Muscles</title><p>SIRT3/4 are mitochondria-localized deacetylases and responsible for regulating the deacetylation of proteins in mitochondria and maintaining mitochondrial homeostasis. After feeding high-fat-diet (HFD), accelerated obesity, attenuated insulin sensitivity, worsened fatty liver and increased acetylation of proteins in mitochondria were observed in SIRT3 KO mice (<xref rid=\"B29\" ref-type=\"bibr\">Hirschey et al., 2011</xref>). Multi-tissue proteomics and physiological examination reveals that SIRT3 is responsible for mitochondrial acetylated proteome regulation and metabolic fuel switching in brain, heart, kidney, liver, and skeletal muscles (<xref rid=\"B13\" ref-type=\"bibr\">Dittenhafer-Reed et al., 2015</xref>). Protein enrichment analysis discovers that its regulatory proteins were mainly concentrated in lipid metabolism (<xref rid=\"B13\" ref-type=\"bibr\">Dittenhafer-Reed et al., 2015</xref>). Knocking out SIRT3 leads to reduction in the levels of deacetylation of manganese superoxide dismutase (MnSOD) mitochondrial complex II and pyruvate dehydrogenase (PDH) subunit E1α, a downregulation of hexokinase II (HK II) binding with mitochondria, resulting in impaired glucose and lipid metabolism of skeletal muscles (<xref rid=\"B39\" ref-type=\"bibr\">Lantier et al., 2015</xref>). Interestingly, SIRT3 liver-specific knockout (hep<sup>–/–</sup>) and skeletal muscle-specific knockout (skm<sup>–/–</sup>) mice did not affect glucose homeostasis under chow or HFD conditions (<xref rid=\"B16\" ref-type=\"bibr\">Fernandez-Marcos et al., 2012</xref>). The above results indicate that SIRT3 has an important role in metabolic regulation, but in specific physiological processes such as redox state, exercise, and aging, and its regulatory mechanism is yet defined in skeletal muscles (<xref rid=\"B36\" ref-type=\"bibr\">Kong et al., 2010</xref>; <xref rid=\"B71\" ref-type=\"bibr\">Robin et al., 2020</xref>; <xref rid=\"B92\" ref-type=\"bibr\">Williams et al., 2020</xref>). A recent study found that SIRT4 knockout can resist HFD-induced obesity and increase endurance exercise in mice by repressing malonyl CoA decarboxylase, which was a key enzyme controlling fatty acid beta-oxidation and was reported to regulate muscle fuel switching between carbohydrates and fatty acids (<xref rid=\"B37\" ref-type=\"bibr\">Koves et al., 2008</xref>; <xref rid=\"B40\" ref-type=\"bibr\">Laurent et al., 2013</xref>). Moreover, knockdown of SIRT4 by adenoviral shRNA can increase the mRNA and protein content of SIRT1, thereby enhancing the expression of fatty acid oxidation genes and mitochondrial oxidation capacity in hepatocytes and myotubes (<xref rid=\"B61\" ref-type=\"bibr\">Nasrin et al., 2010</xref>) (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p></sec></sec></sec><sec id=\"S5\"><title>Therapeutic Targets of Hdacs and Their Potential for Metabolic Diseases</title><p>A comparative analysis used HDAC pan-inhibitor SAHA, a class I HDAC selective inhibitor (MS275) and a class II HDAC selective inhibitor (MC1568) to treat C2C12 separately. It was found that only MS275 could significantly stimulate mitochondria biogenesis and oxygen consumption (<xref rid=\"B18\" ref-type=\"bibr\">Galmozzi et al., 2013</xref>). In obese diabetic mice, it was found that specifically inhibiting Class I rather than Class II HDACs improved GTT and insulin sensitivity, increased oxidative metabolism of skeletal muscles and adipose tissue, and reduced body weight (<xref rid=\"B18\" ref-type=\"bibr\">Galmozzi et al., 2013</xref>). Similar to the effect of MS275, knockdown HDAC3 in C2C12 could also increase <italic>Pgc-1</italic>α, <italic>Glut4</italic>, <italic>Tfam</italic>, <italic>Idh3</italic>α transcription, suggesting that HDAC3 and its target genes played an important role in the above events (<xref rid=\"B18\" ref-type=\"bibr\">Galmozzi et al., 2013</xref>).</p><p>For Class II HDACs, studies have found that scriptaid, a Class IIa HDAC inhibitor, has similar effects to exercise. Six weeks of scriptaid administration significantly improved endurance performance, and significantly increased the whole-body energy expenditure and the expression of lipid oxidation related genes (<italic>Pdk4</italic>, <italic>Cpt1b</italic>, <italic>Pgc-1</italic>α, <italic>Ppar</italic>δ, etc.) of C57BL/6 mice (<xref rid=\"B20\" ref-type=\"bibr\">Gaur et al., 2016</xref>). One possible explanation of above observation is the potential target of scriptaid inhibition of titin (a structure protein of sarcomere) deacetylation and downstream genes of Class IIa HDACs (<xref rid=\"B20\" ref-type=\"bibr\">Gaur et al., 2016</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Huang et al., 2019</xref>).</p><p>In mice treated with SPT1720, another activator of SIRT1, overtly enhanced endurance exercise ability was noted. Moreover, these mice were protected from HFD-induced obesity and insulin resistance, owing to an upregulation of the oxidative metabolism in skeletal muscles, liver, and BAT tissues (<xref rid=\"B64\" ref-type=\"bibr\">Pacholec et al., 2010</xref>). In order to further explore the principle of SIRT1 activation, the researchers purified SIRT1 <italic>in vitro</italic> and added SRT1720 and its analogs SRT2183, SRT1460, and RSV, and they found that the enzymatic activity of SIRT1 was not enhanced, suggesting that these drugs may indirectly activate SIRT1 (<xref rid=\"B64\" ref-type=\"bibr\">Pacholec et al., 2010</xref>). The enzymatic activity of SIRT1 largely depends on the content of cofactor NAD<sup>+</sup>. This implied that the enhancement of SIRT1 activity may be achieved through indirect upregulation of NAD<sup>+</sup>. Study has discovered that knocking out poly (ADP-ribose) polymerase-1 (PARP-1), which is a NAD<sup>+</sup> consuming enzyme, could increase NAD<sup>+</sup> content and enhance the activity of SIRT1 in BAT and skeletal muscles (<xref rid=\"B2\" ref-type=\"bibr\">Bai et al., 2011</xref>). PARP-1 inhibitors can upregulate the proteins of mitochondrial respiratory chain complexes in mice, enhance the aerobic oxidation capacity of mitochondria, and improve mitochondrial defects in the primary myotubes of obese humans (<xref rid=\"B2\" ref-type=\"bibr\">Bai et al., 2011</xref>; <xref rid=\"B67\" ref-type=\"bibr\">Pirinen et al., 2014</xref>).</p><p>Resveratrol was initially reported as an SIRT1 activator that improves mitochondrial function and exercise capacity in mice, and resists HFD-induced obesity (<xref rid=\"B38\" ref-type=\"bibr\">Lagouge et al., 2006</xref>). However, subsequent studies have discovered that administering the same dose of RSV to rats or mice does not increase mitochondrial protein content (<xref rid=\"B28\" ref-type=\"bibr\">Higashida et al., 2013</xref>). By contrast, overexpression of SIRT1 in the triceps muscle of rats decreases the mitochondrial protein content (<xref rid=\"B28\" ref-type=\"bibr\">Higashida et al., 2013</xref>). In a double-blind human trial, 11 healthy and obese men were supplied for 30-day RSV, in which the data showed that RSV can improve systolic blood pressure and homeostasis model assessment (HOMA) index-indicating glucose metabolism ability, simulating the effect of CR (<xref rid=\"B85\" ref-type=\"bibr\">Timmers et al., 2011</xref>). However, some researchers found that the overexpression of SIRT1 alone cannot mimic the CR effect in transgenic mice, and the transcriptomic changes in various tissues were quite different or even opposite (<xref rid=\"B7\" ref-type=\"bibr\">Boutant et al., 2016</xref>). Such an opposite situation may explain that the genetic model and compound stimulation are not completely consistent. RSV as a potential metabolic syndrome treatment drug still needs large-scale population sample verification.</p></sec><sec id=\"S6\"><title>Conclusion</title><p>The very first mammalian histone deacetylase HDAC1 was cloned and isolated by <xref rid=\"B83\" ref-type=\"bibr\">Taunton et al. (1996)</xref>, and 15,000 articles about HDACs have been published in the last 20 years. Currently, we know at least 18 HDAC proteins. They are responsible for eradicating epigenetic modifications, establishing an epigenetic off chromatin state, and regulating heritable gene expression (<xref rid=\"B95\" ref-type=\"bibr\">Yang and Seto, 2008</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Haberland et al., 2009</xref>). In these processes, each HDACs may play a different role (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). What kind of gene/protein is the specific downstream target of these HDACs? Is its enzyme activity related to intracellular localization and the formation of multi-protein complex? It is still one of the key and difficult issues in this field. Based on previous research experience, some techniques may be used to further experiments as follows: (1) HDACs interacting proteins/complex co-IP; (2) Protein-acetylation western; (3) Histone acetylation target gene ChIP; (4) High through put proteomics/acetylome with specific HDACs inhibitor, etc.</p><p>Histone deacetylases are potential therapeutic targets, and clinical drugs such as SAHA (Vorinostat) and FK228 (romidepsin) have been used for antitumor treatment (<xref rid=\"B6\" ref-type=\"bibr\">Bolden et al., 2006</xref>). However, they have two disadvantages: (1) great toxic and side effects and (2) difficulty in specific inhibition of HDACs activity. With the development of computer simulation technology and structural biology, it is believed that more specific HDACs inhibitors/activators can be constructed. And researches should attach importance to HDACs regulatory factors like PARP-1 when direct targeting on HDACs fails to show its effects. Further development of their inhibitors with more specificity, and trials for the treatment of metabolic diseases, may have great potential as well. Additionally, with the further development of biotechnology, some RNA therapies presented by AAV can specifically inhibit/overexpression of certain protein in skeletal muscles tissue, which may be able to target HDACs and treat related diseases (<xref rid=\"B89\" ref-type=\"bibr\">Wang et al., 2005</xref>; <xref rid=\"B46\" ref-type=\"bibr\">Long et al., 2016</xref>). However, its safety and effectiveness still need further clinical trials. Future research can discover known or unknown HDACs targeted drugs for the treatment of metabolic diseases.</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>HT, SL, and JR designed the literature search and wrote the review. JL, RW, and PC critically analyzed and revised the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by grants from the China Postdoctoral Science Foundation (2019M661042 to SL and 2019M651553 to HT) and National Natural Science Foundation of China (81501071 to SL).</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Anderson</surname><given-names>J. G.</given-names></name><name><surname>Ramadori</surname><given-names>G.</given-names></name><name><surname>Ioris</surname><given-names>R. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"systematic-review\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Genet</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Genet</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Genet.</journal-id><journal-title-group><journal-title>Frontiers in Genetics</journal-title></journal-title-group><issn pub-type=\"epub\">1664-8021</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849795</article-id><article-id pub-id-type=\"pmc\">PMC7431663</article-id><article-id pub-id-type=\"doi\">10.3389/fgene.2020.00789</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Genetics</subject><subj-group><subject>Systematic Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Serum-Derived microRNAs as Prognostic Biomarkers in Osteosarcoma: A Meta-Analysis</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Luo</surname><given-names>Huan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Peng</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ye</surname><given-names>Hua</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Shi</surname><given-names>Jianxiang</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/920623/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Dai</surname><given-names>Liping</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Xiao</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Song</surname><given-names>Chunhua</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Jianying</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Jitian</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/922284/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>College of Public Health, Zhengzhou University</institution>, <addr-line>Zhengzhou</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Henan Key Laboratory of Tumor Epidemiology, Zhengzhou University</institution>, <addr-line>Zhengzhou</addr-line>, <country>China</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Zhengzhou University, Henan Academy of Medical and Pharmaceutical Sciences</institution>, <addr-line>Zhengzhou</addr-line>, <country>China</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Laboratory of Molecular Biology, Henan Luoyang Orthopedic Hospital (Henan Provincial Orthopedic Hospital)</institution>, <addr-line>Zhengzhou</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Dongqing Wei, Shanghai Jiao Tong University, China</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Leda Torres, National Institute of Pediatrics, Mexico; Debashis Sahoo, University of California, San Diego, United States</p></fn><corresp id=\"c001\">*Correspondence: Jianying Zhang <email>jianyingzhang@hotmail.com</email></corresp><corresp id=\"c002\">Jitian Li <email>jitianlee@hotmail.com</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Systems Biology, a section of the journal Frontiers in Genetics</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>789</elocation-id><history><date date-type=\"received\"><day>31</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>02</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Luo, Wang, Ye, Shi, Dai, Wang, Song, Zhang and Li.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Luo, Wang, Ye, Shi, Dai, Wang, Song, Zhang and Li</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Recent reports suggest that microRNAs (miRNAs) may serve as prognostic biomarkers in osteosarcoma. Due to osteosarcoma's early metastasis and poor prognosis, it is very important to find novel prognostic biomarkers for improving osteosarcoma's prognosis. Herein we propose a meta-analysis for serum miRNA's prognostic value in osteosarcoma. In this study, the literature available from PubMed, Web of Science, Embase, and Cochrane Library databases was reviewed. The pooled hazard ratios (HRs) with their 95% confidence intervals (CIs) were calculated to evaluate miRNAs prognostic values. A total of 20 studies investigating serum miRNAs were included in this meta-analysis; the initial terminal point of these reports included overall survival (OS), progression-free survival (PFS), disease-free survival (DFS), and recurrence-free survival (RFS). For prognostic meta-analyses, the pooled HR for terminal events of higher expression of miRNAs and lower expression of miRNAs were 5.68 (95% CI 4.73–6.82, <italic>P</italic> < 0.05) and 3.78 (95% CI 3.27–4.37, <italic>P</italic> < 0.05), respectively. Additionally, subgroup analyses were conducted based on the analysis methods applied and clinicopathological features reported. In the pooled analyses, the miRNA expression levels are associated with poor prognosis according to both univariate and multivariate analyses. Furthermore, serum miRNAs (miRNA-195, miRNA-27a, miRNA-191, miRNA-300, miRNA-326, miRNA-497, miRNA-95-3p, miRNA-223, miRNA-491-5p, miRNA-124, miRNA-101, miRNA-139-5p, miRNA-194) were associated with poor OS and found to be closely correlated with clinical stage and distant metastasis in osteosarcoma. The results illustrate that low or high expression of these specific miRNAs are both potentially useful as prognostic serum biomarkers in osteosarcoma, and miRNAs (miRNA-195, miRNA-27a, miRNA-191, miRNA-300, miRNA-326, miRNA-497, miRNA-95-3p, miRNA-223, miRNA-491-5p, miRNA-124, miRNA-101, miRNA-139-5p, miRNA-194) may indicate clinical stage and metastasis in this form of cancer.</p></abstract><kwd-group><kwd>miRNA</kwd><kwd>osteosarcoma</kwd><kwd>prognosis</kwd><kwd>serum</kwd><kwd>biomarker</kwd><kwd>meta-analysis</kwd></kwd-group><counts><fig-count count=\"5\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"52\"/><page-count count=\"10\"/><word-count count=\"5860\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Patient survival in osteosarcoma has improved in recent decades. Osteosarcoma is the most common malignant bone tumor, with a worldwide incidence of approximately one to three cases annually per million (Kansara et al., <xref rid=\"B21\" ref-type=\"bibr\">2014</xref>). Current therapies include surgical resection and combination neoadjuvant chemotherapy, which is reported to have a curative effect in ~70% of patients (Collins et al., <xref rid=\"B7\" ref-type=\"bibr\">2013</xref>). Metastasis and recurrence are common challenges in refractory osteosarcoma, that worsen patient prognosis (Bielack et al., <xref rid=\"B2\" ref-type=\"bibr\">2002</xref>). The highly malignant nature of osteosarcoma, as well as its high rates of recurrence and lung metastasis represent strong concerns (Jones et al., <xref rid=\"B20\" ref-type=\"bibr\">2012</xref>; Ogawa et al., <xref rid=\"B32\" ref-type=\"bibr\">2013</xref>). Clinically, histological examination of the biopsy specimens is preferred for the diagnosis or prognostic evaluation of osteosarcoma. However, such invasive tests may be burdensome when monitoring the progression of the disease, and the accuracy of diagnosis and prognostic evaluation may vary because of differences in sample collection and personnel. Therefore, it is essential to develop novel approaches for the timely diagnosis of osteosarcoma in order to achieve better prognosis (Gu et al., <xref rid=\"B16\" ref-type=\"bibr\">2014</xref>).</p><p>MiRNAs are small (about 21-nucleotide-long) non-coding RNAs which can regulate gene expression (Filipowicz et al., <xref rid=\"B13\" ref-type=\"bibr\">2008</xref>). Elevated or downregulated miRNAs may act as oncogenes or tumor suppressors in various cancers (Hayashita et al., <xref rid=\"B17\" ref-type=\"bibr\">2005</xref>; He et al., <xref rid=\"B18\" ref-type=\"bibr\">2005</xref>; Kent and Mendell, <xref rid=\"B22\" ref-type=\"bibr\">2006</xref>; Tian et al., <xref rid=\"B39\" ref-type=\"bibr\">2019</xref>). Additionally, miRNAs that are stable in serum or plasma, or in other biological samples, may have potential utility as diagnostic or prognostic biomarkers in different cancers (Calin and Croce, <xref rid=\"B4\" ref-type=\"bibr\">2006</xref>; Esquela-Kerscher and Slack, <xref rid=\"B12\" ref-type=\"bibr\">2006</xref>; Mitchell et al., <xref rid=\"B28\" ref-type=\"bibr\">2008</xref>; Zhou et al., <xref rid=\"B52\" ref-type=\"bibr\">2016</xref>). These findings show that miRNAs warrant attention as potential novel biomarkers for diagnosis or prognosis in osteosarcoma.</p><p>Although numerous recent studies have reported a correlation between prognosis in osteosarcoma and miRNA expression, none have demonstrated sufficient evidence for clinical translation of their findings. For instance, two previous meta-analyses have concluded the prognostic value of miRNA expression in osteosarcoma (Cheng et al., <xref rid=\"B6\" ref-type=\"bibr\">2017</xref>; Kim et al., <xref rid=\"B23\" ref-type=\"bibr\">2017</xref>); however, in these studies, either tissue or both tissue and blood were used as samples. Tissue samples' obtainment are invasive for patients than serum samples. To optimally obtain samples from patients and increase patients' acceptability, it is important for us to find novel serum biomarker for osteosarcoma. To the best of our knowledge, very few studies have provided robust evidence on the potential prognostic utility of serum miRNAs in osteosarcoma. Therefore, in the present work, we conducted a meta-analysis of studies in which serum samples were analyzed, to explore the prognostic value of miRNAs in osteosarcoma. Following which, subgroup analyses included analysis method and clinicopathological features were also explored to better analyze the prognostic value of various groups.</p></sec><sec sec-type=\"methods\" id=\"s2\"><title>Methods</title><p>This study was implemented according to the guidelines of the Meta-analysis of Observational Studies in Epidemiology (MOOSE) (Stroup et al., <xref rid=\"B36\" ref-type=\"bibr\">2000</xref>), and the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines (Moher et al., <xref rid=\"B29\" ref-type=\"bibr\">2009</xref>). We have completed the prognostic value of serum microRNA. In constructing the prognostic value of serum microRNA, we comply with the population, interventions, comparators, out-comes, and study designs (PICOS) principle to complete the research design.</p><sec><title>Selection of Studies</title><p>The literature available in PubMed, Web of Science, Embase, and Cochrane Library databases, up to June 20, 2020, was investigated. The combination of search terms used was (osteosarcoma OR osteogenic sarcoma) AND (microRNA OR miRNA OR miR) AND (prognosis OR survival OR prognostic OR outcome). Only studies of the Chinese population published in English were included, and studies analyzing samples other than serum were excluded.</p></sec><sec><title>Inclusion and Exclusion Criteria</title><p>Inclusion criteria for studies in this review were as follows: (1) studies investigating the utility of miRNAs for evaluating prognosis in osteosarcoma, (2) serum miRNAs' assay method based on quantitative real-time polymerase chain reaction, (3) studies presenting sufficient data to allow calculation of HR and 95% CI, and (4) studies in which a cut-off value was defined. Studies were subject to the following exclusion criteria: (1) studies reporting duplicate data; studies in non-Chinese populations, (2) non-English publications, review articles, or meta-analysis, (3) studies reporting insufficient data for pooled analysis, and (4) studies of tissue, cell lines, or animal experiments.</p></sec><sec><title>Quality Assessment and Data Extraction</title><p>For prognostic meta-analyses, the quality of included studies was assessed using the Newcastle–Ottawa Scale (NOS), based on the following categories: selection, comparability, and outcome; the highest score was 9, with scores ≥6 indicating studies of high quality (Stang, <xref rid=\"B35\" ref-type=\"bibr\">2010</xref>). The extracted data and information included were as follows: the first author, the year of publication, the country of origin, osteosarcoma sample size, sample type, cut off value, miRNAs characteristics, analysis methods, clinical outcomes, and detection methods. Two investigators retrieved and assessed the literature, respectively, and disagreements were resolved by extensive discussion.</p></sec><sec><title>Statistical Methods</title><p>All analyses were performed using STATA 12.0 software. Based on the information provided in the included studies, the pooled HRs with 95% CIs were calculated using this meta-analysis model. Forest plots were used to estimate the effect of miRNA expression on overall survival (OS), progression-free survival (PFS), disease-free survival (DFS), and recurrence free-survival (RFS) (Hong et al., <xref rid=\"B19\" ref-type=\"bibr\">2014</xref>; Zhang et al., <xref rid=\"B47\" ref-type=\"bibr\">2014a</xref>,<xref rid=\"B49\" ref-type=\"bibr\">b</xref>; Cai et al., <xref rid=\"B3\" ref-type=\"bibr\">2015</xref>; Tang et al., <xref rid=\"B38\" ref-type=\"bibr\">2015</xref>; Wang N. G. et al., <xref rid=\"B40\" ref-type=\"bibr\">2015</xref>; Wang T. et al., <xref rid=\"B42\" ref-type=\"bibr\">2015</xref>; Yang et al., <xref rid=\"B45\" ref-type=\"bibr\">2015</xref>; Cao et al., <xref rid=\"B5\" ref-type=\"bibr\">2016</xref>; Dong et al., <xref rid=\"B9\" ref-type=\"bibr\">2016</xref>; Liu et al., <xref rid=\"B27\" ref-type=\"bibr\">2016</xref>; Niu et al., <xref rid=\"B31\" ref-type=\"bibr\">2016</xref>; Pang et al., <xref rid=\"B33\" ref-type=\"bibr\">2016</xref>; Wang S. N. et al., <xref rid=\"B41\" ref-type=\"bibr\">2017</xref>; Wang Z. et al., <xref rid=\"B43\" ref-type=\"bibr\">2017</xref>; Cong et al., <xref rid=\"B8\" ref-type=\"bibr\">2018</xref>; Li et al., <xref rid=\"B26\" ref-type=\"bibr\">2018</xref>; Yao et al., <xref rid=\"B46\" ref-type=\"bibr\">2018</xref>; Zhou et al., <xref rid=\"B51\" ref-type=\"bibr\">2018</xref>; Shi et al., <xref rid=\"B34\" ref-type=\"bibr\">2020</xref>). <italic>I</italic><sup>2</sup> index was used to assess the between-study heterogeneity, with <italic>I</italic><sup>2</sup> > 50% indicating a large degree of heterogeneity; in this case, a random effect model was applied. <italic>I</italic><sup>2</sup> ≤ 50% implied that there was no significant heterogeneity, and the fixed effect model was used. Next, subgroup analyses were conducted to identify potential sources of heterogeneity and assess the prognostic value of different subgroups; the level of significance was set at <italic>P</italic> < 0.05. In addition, Begg's test (Begg and Mazumdar, <xref rid=\"B1\" ref-type=\"bibr\">1994</xref>) and Egger's test (Egger et al., <xref rid=\"B10\" ref-type=\"bibr\">1997</xref>) were performed to assess the publication bias; values of <italic>P</italic> < 0.05 indicated significant publication bias.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>Characteristics of the Included Studies and Quality Assessment</title><p>The screening process for the studies is shown in detail in <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>. A primary search of the PubMed, Web of Science, Embase, and Cochrane Library databases, using the search strategy described, identified 2,596 articles. The innovative contribution of this work is focused on studies which using serum sample, herein studies using plasma or tissue were excluded, as such, all included studies examined serum samples. The data extracted from the included studies, the quality of the reports and heterogeneity are shown in <xref rid=\"T1\" ref-type=\"table\">Tables 1</xref>, <xref rid=\"T2\" ref-type=\"table\">2</xref>. The osteosarcoma sample size ranged from 60 to 185 subjects. The assay method was based on qRT-PCR. Cut off values were defined in the included studies to differentiate between high-expression miRNAs and low-expression miRNAs, and multivariate or univariate analyses were performed. The quality of each study was high according to the NOS (Stang, <xref rid=\"B35\" ref-type=\"bibr\">2010</xref>). A total of 20 studies and 2,242 osteosarcoma patients were included in this prognostic meta-analysis.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Flow-process diagram of the study selection process.</p></caption><graphic xlink:href=\"fgene-11-00789-g0001\"/></fig><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>The extracted data and quality assessment of literature on the prognostic utility of miRNAs in osteosarcoma.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Author/miRNA</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Year</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Country</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Sample size</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Sample type</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Cut off value</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>miRNA expression with poor prognosis</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Assay method</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Analysis method</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Outcome</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>NOS score</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hong miRNA-29a/29b</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2014</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2.85/3.27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">High</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS, DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Zhang miRNA-196a/196b</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2014</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4.86/5.48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">High</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS, DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cai miRNA-195</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2014</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">166</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.44</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS, DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Zhang miRNA-133b/206</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2014</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2.66/2.84</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS, DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yang miRNA-221</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2015</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">108</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2.42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">High</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RFS, OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Wang miRNA-152</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2015</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tang miRNA-27a</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2015</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">166</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3.70</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">High</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS, DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Wang miRNA-191</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2015</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3.56</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">High</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS, DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dong miRNA-223</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2016</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">112</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Liu miRNA-300</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2016</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">114</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">High</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">U/M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cao miRNA-326</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2016</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mean level</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Niu miRNA-95-3p</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2016</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">133</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.75</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pang miRNA-497</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2016</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">185</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4.80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">U/M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Li miRNA-542-3p</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2017</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">76</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.87</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">High</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">U/M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS, PFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Wang miRNA-491</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2017</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">102</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mean level</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">U/M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Wang miRNA-491-5p</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2017</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">72</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS, DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cong miRNA-124</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2017</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">114</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.37-fold</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS, DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yao miRNA-101</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2018</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">152</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Median level</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">U/M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS, RFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Zhou miRNA-139-5p</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2018</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">98</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Median level</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">U/M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Shi miRNA-194</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2020</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">124</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Median level</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">qRT-PCR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS,DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td></tr></tbody></table><table-wrap-foot><p><italic>M, Multivariate analysis; U, Univariate analysis; DFS, Disease-free survival; OS, Overall survival; RFS, Recurrence-free survival; PFS, Progression-free survival; NR, Not reported; high, high expression; low, low expression</italic>.</p></table-wrap-foot></table-wrap><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Prognostic value of the microRNAs expression profile mentioned in the literature.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>microRNA</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>HR (95% CI)</bold></th><th valign=\"top\" align=\"center\" colspan=\"2\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Heterogeneity test</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Sample size</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Expression</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Outcome</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Number of microRNA</bold></th></tr><tr><th rowspan=\"1\" colspan=\"1\"/><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>I</italic><sup><bold>2</bold></sup> (%)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Chi<sup><bold>2</bold></sup> (<italic>P</italic>)</bold></th><th rowspan=\"1\" colspan=\"1\"/><th rowspan=\"1\" colspan=\"1\"/><th rowspan=\"1\" colspan=\"1\"/><th rowspan=\"1\" colspan=\"1\"/></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-133b</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.53 (2.58–11.83)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.939</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-206</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.66 (2.69–11.88)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.914</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-133b/206</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.48 (4.59–19.57)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.953</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-195</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.23 (2.31–7.73)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.568</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">166</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-152</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.13 (0.02–0.70)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-223</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.59 (1.84–11.45)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">112</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-326</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.90 (1.13–12.53)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-95-3p</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.22 (2.31–8.07)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">133</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-497</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.96 (2.39–6.58)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.868</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">185</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-491</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.06 (1.56–6.00)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.928</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">102</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-124</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.73 (2.27–6.12)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.841</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">114</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-491-5p</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.68 (1.66–4.32)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.951</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">72</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DFS/OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-101</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.16 (2.80–6.19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.995</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">152</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/RFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-139-5p</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.03 (2.17–4.23)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.707</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">98</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-29a</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.68 (2.50–12.92)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.903</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">High expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-29b</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.71 (2.58–12.67)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.904</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">High expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-196a</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.59 (3.08–14.11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.896</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">High expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-196b</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.64 (3.09–14.24)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.900</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">High expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-196a/196b</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.99 (4.82–20.69)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.979</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">High expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-221</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.26 (3.29–16.04)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.886</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">108</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">High expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RFS/OS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-27a</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.36 (1.90–5.95)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.851</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">166</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">High expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-191</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.05 (1.75–5.31)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.593</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">High expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-300</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.07 (3.41–7.54)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.958</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">114</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">High expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-542-3p</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.83 (5.41–11.34)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.431</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">76</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">High expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/PFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-194</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.01 (2.53–6.36)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.695</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">124</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OS/DFS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr></tbody></table><table-wrap-foot><p><italic>DFS, Disease-free survival; OS, Overall survival; RFS, Recurrence-free survival; PFS, Progression-free survival; –, Not available</italic>.</p></table-wrap-foot></table-wrap></sec><sec><title>Prognostic Accuracy and Subgroup Analyses</title><p>To analyze the prognostic value of miRNA expression in osteosarcoma, forest plots of data from the 19 studies, in accordance with HRs and their 95% CIs, are shown in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>. The HRs were calculated on the basis of low-expression or high-expression miRNAs, respectively. HR >1 or <1 implied poor or good prognosis for patients with osteosarcoma, respectively. The pooled HRs for low- and high-expression miRNAs were 3.78 (95% CI 3.27–4.37, <italic>P</italic> < 0.05) and 5.68 (95% CI 4.73–6.82, <italic>P</italic> < 0.05), respectively, and both tended to be associated with a poorer outcome. Additionally, low-expression miRNAs were stratified by outcomes, including OS (pooled HR = 3.59, 95% CI 3.02–4.26, <italic>P</italic> < 0.05), DFS (pooled HR = 4.25, 95% CI 3.14–5.76, <italic>P</italic> < 0.05), and RFS (pooled HR = 4.34, 95% CI 2.48–7.60, <italic>P</italic> < 0.05). Furthermore, high-expression miRNAs were classified by outcomes, including OS (pooled HR = 5.98, 95% CI 4.58–7.80, <italic>P</italic> < 0.05), DFS (pooled HR = 4.80, 95% CI 3.53–6.53, <italic>P</italic> < 0.05), RFS (pooled HR = 6.82, 95% CI 2.13–21.88, <italic>P</italic> < 0.05), and PFS (pooled HR = 6.95, 95% CI 4.34–11.12, <italic>P</italic> < 0.05), and these miRNAs were also associated with poor prognosis in osteosarcoma.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Forest plot for miRNA expression and prognosis in osteosarcoma, stratified by outcomes included (OS, DFS, RFS, PFS). <bold>(A)</bold> Low-expression miRNAs correlated with poor prognosis in osteosarcoma, stratified by outcomes (OS, DFS, RFS). <bold>(B)</bold> High-expression miRNAs correlated with poor prognosis in osteosarcoma, stratified by event times (OS, DFS, RFS, PFS).</p></caption><graphic xlink:href=\"fgene-11-00789-g0002\"/></fig><p>Subgroup analyses were performed according to analysis method and clinicopathological features in order to explore the correlation of miRNA expression on prognosis in osteosarcoma, as shown in <xref ref-type=\"fig\" rid=\"F3\">Figures 3</xref>, <xref ref-type=\"fig\" rid=\"F4\">4</xref>, respectively. As shown in <xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>, subgroup analyses reveal that low expression levels of miRNA were significantly correlated with poor prognosis in osteosarcoma according to both multivariate (pooled HR = 3.88, 95% CI 3.29–4.58, <italic>P</italic> < 0.05) and univariate analyses (pooled HR = 3.47, 95% CI 2.57–4.68, <italic>P</italic> < 0.05). Similar results are shown in <xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>: multivariate (pooled HR = 5.28, 95% CI 4.29–6.51, <italic>P</italic> < 0.05) and univariate analyses (pooled HR = 7.23, 95% CI 4.93–10.59, <italic>P</italic> < 0.05) both indicate that high expression of miRNA are correlated with poor prognosis in osteosarcoma. Additionally, as shown in <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>, the level of expression of serum miRNAs is closely correlated with distant metastasis (pooled HR = 3.30, 95% CI 2.77–3.94, <italic>P</italic> < 0.05), and clinical stage (pooled HR = 3.48, 95% CI 2.91–4.15, <italic>P</italic> < 0.05) in osteosarcoma.</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Forest plot for miRNA expression and prognosis of osteosarcoma, stratified by analysis method. <bold>(A)</bold> Low-expression miRNAs correlated with poor prognosis in osteosarcoma, stratified by analysis method (M and U). <bold>(B)</bold> High-expression miRNAs with poor prognosis in osteosarcoma, stratified by analysis method (M and U). The <italic>p</italic>-values for heterogeneity of HR by subgroup, and overall, are shown. M, Multivariate analysis; U, Univariate analysis.</p></caption><graphic xlink:href=\"fgene-11-00789-g0003\"/></fig><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Forest plot of the correlation between metastasis, clinical stage, and miRNA expression in studies with poor overall survival. <bold>(A)</bold> Correlation between miRNA level and tumor metastasis in osteosarcoma with poor overall survival, stratified by miRNAs expression (high-expression miRNA or low-expression miRNA). <bold>(B)</bold> Correlation between miRNA level and clinical stage in osteosarcoma with poor overall survival, stratified by miRNA expression (high-expression miRNA or low-expression miRNA).</p></caption><graphic xlink:href=\"fgene-11-00789-g0004\"/></fig></sec><sec><title>Publication Bias and Sensitivity Analysis</title><p>The <italic>P</italic>-values for Begg's tests of low-expression miRNAs and high-expression miRNAs were 0.028 and 0.602, respectively, and the corresponding <italic>P</italic>-values for Egger's tests were 0.544 and 0.283. Furthermore, the funnel plots of Begg's and Egger's are all symmetrical demonstrating that there is no significant publication bias in this research (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>). Sensitivity analyses revealed that none of the studies were outliers, suggesting that the pooled results of this research are credible.</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>Forest plot of the publication bias. <bold>(A)</bold> Begg's funnel plot of publication bias for the association between miRNA low expression and poor prognosis. <bold>(B)</bold> Egger's test of publication bias for the association between miRNA low expression and poor prognosis. <bold>(C)</bold> Begg's funnel plot of publication bias for the association between miRNA high expression and poor prognosis. <bold>(D)</bold> Egger's test of publication bias for the association between miRNA high expression and poor prognosis.</p></caption><graphic xlink:href=\"fgene-11-00789-g0005\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Although osteosarcoma is the common malignant bone tumor (Kansara et al., <xref rid=\"B21\" ref-type=\"bibr\">2014</xref>) and extensive progress has been made in the development of effective therapies (Bielack et al., <xref rid=\"B2\" ref-type=\"bibr\">2002</xref>; Collins et al., <xref rid=\"B7\" ref-type=\"bibr\">2013</xref>), patient prognosis remains unsatisfactory. Therefore, for improved treatment, management, and patient prognosis in osteosarcoma, the identification of novel prognostic biomarkers is critical. The potential utility of circulating miRNAs as non-invasive biomarkers has been demonstrated in several types of cancers (Zhou et al., <xref rid=\"B52\" ref-type=\"bibr\">2016</xref>; Tan et al., <xref rid=\"B37\" ref-type=\"bibr\">2019</xref>). Furthermore, blood testing is more easily accepted by patients than other invasive tests. Therefore, we conducted a meta-analysis of studies investigating the prognostic capacity of serum miRNAs in osteosarcoma. A more detailed subgroup analysis was also conducted to further examine the association between analysis methods, clinical stage, metastasis, and miRNA expression level in osteosarcoma.</p><p>We investigated 20 studies on 23 different miRNAs in osteosarcoma in this meta-analysis; these included 9 highly expressed miRNAs (miRNA-29a, miRNA-29b, miRNA-196a, miRNA-196b, miRNA-221, miRNA-27a, miRNA-191, miRNA-542-3p, and miRNA-300) (Hong et al., <xref rid=\"B19\" ref-type=\"bibr\">2014</xref>; Zhang et al., <xref rid=\"B47\" ref-type=\"bibr\">2014a</xref>; Tang et al., <xref rid=\"B38\" ref-type=\"bibr\">2015</xref>; Wang T. et al., <xref rid=\"B42\" ref-type=\"bibr\">2015</xref>; Yang et al., <xref rid=\"B45\" ref-type=\"bibr\">2015</xref>; Li et al., <xref rid=\"B26\" ref-type=\"bibr\">2018</xref>), and 14 miRNAs with low expression (miRNA-195, miRNA-223, miRNA-497, miRNA-491, miRNA-124, miRNA-101, miRNA-139-5p, miRNA-326, miRNA-133b, miRNA-206, miRNA-152, miRNA-95-3p, and miRNA-491-5p, miR-194) (Cai et al., <xref rid=\"B3\" ref-type=\"bibr\">2015</xref>; Cao et al., <xref rid=\"B5\" ref-type=\"bibr\">2016</xref>; Dong et al., <xref rid=\"B9\" ref-type=\"bibr\">2016</xref>; Pang et al., <xref rid=\"B33\" ref-type=\"bibr\">2016</xref>; Wang S. N. et al., <xref rid=\"B41\" ref-type=\"bibr\">2017</xref>; Cong et al., <xref rid=\"B8\" ref-type=\"bibr\">2018</xref>; Yao et al., <xref rid=\"B46\" ref-type=\"bibr\">2018</xref>; Zhou et al., <xref rid=\"B51\" ref-type=\"bibr\">2018</xref>; Shi et al., <xref rid=\"B34\" ref-type=\"bibr\">2020</xref>). A previous meta-analysis has reported that aberrant expression of miRNAs, in terms of both elevated and downregulated expression, are associated with poor prognosis in osteosarcoma (Cheng et al., <xref rid=\"B6\" ref-type=\"bibr\">2017</xref>). Similar to the above findings, our data confirm the observation that serum miRNAs with aberrantly elevated or downregulated levels of expression are strongly correlated with poor prognosis for osteosarcoma. Among osteosarcoma patients, high expression of serum miRNAs (miRNA-196a and miRNA-196b) and combined expression of miRNA-196a/miRNA-196b were independent prognostic factors for OS and DFS (Zhang et al., <xref rid=\"B47\" ref-type=\"bibr\">2014a</xref>). Further, Frampton et al. (<xref rid=\"B14\" ref-type=\"bibr\">2014</xref>) reported that miRNA-21, miRNA-23a, and miRNA-27a are highly expressed in pancreatic tumor, and their combination could serve as a prognostic biomarker in this tumor. The above results suggest that the development of an optimal panel of miRNA expression would be useful biomarker to improve prognosis in osteosarcoma.</p><p>The levels of miRNA-27a, miRNA-191, miRNA-195, miRNA-497, miRNA-223, miRNA-124, miRNA-101, miRNA-139-5p, miRNA-326, miRNA-95-3p, miRNA-491-5p, and miRNA-300, miRNA-194 (Cai et al., <xref rid=\"B3\" ref-type=\"bibr\">2015</xref>; Tang et al., <xref rid=\"B38\" ref-type=\"bibr\">2015</xref>; Wang T. et al., <xref rid=\"B42\" ref-type=\"bibr\">2015</xref>; Cao et al., <xref rid=\"B5\" ref-type=\"bibr\">2016</xref>; Dong et al., <xref rid=\"B9\" ref-type=\"bibr\">2016</xref>; Liu et al., <xref rid=\"B27\" ref-type=\"bibr\">2016</xref>; Niu et al., <xref rid=\"B31\" ref-type=\"bibr\">2016</xref>; Pang et al., <xref rid=\"B33\" ref-type=\"bibr\">2016</xref>; Wang Z. et al., <xref rid=\"B43\" ref-type=\"bibr\">2017</xref>; Cong et al., <xref rid=\"B8\" ref-type=\"bibr\">2018</xref>; Yao et al., <xref rid=\"B46\" ref-type=\"bibr\">2018</xref>; Zhou et al., <xref rid=\"B51\" ref-type=\"bibr\">2018</xref>; Shi et al., <xref rid=\"B34\" ref-type=\"bibr\">2020</xref>) are potentially associated with clinical stage as well as the tumor metastasis in osteosarcoma. miRNA-27a, miRNA-191, and miRNA-300 are highly expressed in osteosarcoma, while miRNA-195, miRNA-497, miRNA-223, miRNA-124, miRNA-101, miRNA-139-5p, miRNA-326, miRNA-95-3p, miRNA-194, and miRNA-491-5p are expressed at low levels. In particular, the high expression of miR-191 may affect cancer progression through various pathways, and is associated with therapeutic outcomes or poor prognosis in cancers (Elyakim et al., <xref rid=\"B11\" ref-type=\"bibr\">2010</xref>; Li et al., <xref rid=\"B25\" ref-type=\"bibr\">2017</xref>). As such, miR-27a and miR-191 serve as regulatory factors in osteosarcoma. Low expression of miR-195 may act as a regulator in hepatocellular carcinoma cells, with potential utility in cancer therapy (Xu et al., <xref rid=\"B44\" ref-type=\"bibr\">2009</xref>). In addition, low expression of serum miR-497 may be associated with tumor development (Kong et al., <xref rid=\"B24\" ref-type=\"bibr\">2015</xref>). miR-124 was found to be expressed at low levels and correlated with invasion and metastasis in hepatocellular carcinoma (HCC), which may predict poor prognosis (Zheng et al., <xref rid=\"B50\" ref-type=\"bibr\">2012</xref>). Low expression of miR-101 has been shown to inhibit the progression of bladder transitional cell carcinoma, and may serve as a tumor suppressor gene in this disease (Friedman et al., <xref rid=\"B15\" ref-type=\"bibr\">2009</xref>). miR-139-5p has been shown to be expressed at low levels, and is considered to act as a regulator of cell proliferation, metastasis, apoptosis, and the cell cycle (Zhang et al., <xref rid=\"B48\" ref-type=\"bibr\">2014c</xref>). In glioblastomas, miR-326 shows low expression and acts as a tumor suppressor (Nawaz et al., <xref rid=\"B30\" ref-type=\"bibr\">2016</xref>). These results demonstrate, at a molecular level, that miRNAs have diagnostic or prognostic utility that could be extended to other tumors. miR-195, miR-497, miR-223, miR-124, miR-101, miR-139-5p, and miR-326 act as tumor suppressors in cancers, which is consistent with the results obtained in our study. Therefore, the conclusions that high-expression or low-expression miRNAs are potential novel biomarkers for predicting prognosis in osteosarcoma are reliable.</p><p>However, this study has some limitations. All relevant publications may not have been included in the databases, and specific subgroup analyses showed mild heterogeneity. HRs and RRs were merged into HRs in the included literature, potentially leading to slight logical errors, finally the included studies' population limited to Chinese. Despite these limitations, this study suggests that miRNAs have potential utility as novel prognostic markers in osteosarcoma. Further investigation of the dynamic expressional profile of miRNAs during the entire course of the development and treatment of osteosarcoma is needed for clinical implementation of miRNAs as biomarkers in either diagnosis or prognosis.</p><p>Despite these limitations, the results of this study suggest that serum miRNAs represent excellent biomarkers of prognosis in osteosarcoma. We conclude that miRNAs in this systematic review with aberrantly low or high expression are indicative of poor prognosis in osteosarcoma. Further, the expression of miRNAs(miRNA-195, miRNA-27a, miRNA-191, miRNA-300, miRNA-326, miRNA-497, miRNA-95-3p, miRNA-223, miRNA-491-5p, miRNA-124, miRNA-101, miRNA-139-5p, miRNA-194) is correlated with clinical stage and tumor metastasis in this disease. However, the implementation of miRNAs as biomarkers for monitoring cancer progression, clinical stage and distant metastasis and guiding therapeutic interventions improving poor prognosis. Future studies should aim to address these critical aspects.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p></sec><sec id=\"s6\"><title>Author Contributions</title><p>HL designed the study and collected data. HL and PW drafted the manuscript. PW, HY, JS, LD, XW, and CS contributed to the writing. JZ and JL contributed to the writing and review of the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s7\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>The authors gratefully acknowledge JZ (Henan Academy of Medical and Pharmaceutical Sciences, Zhengzhou University) for providing financial support and language help, and JL (Laboratory of Molecular Biology, Henan Luoyang Orthopedic Hospital) for providing assistance with language and writing of this manuscript.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This study was supported by the National Science and Technology Major Project of China (No. 2018ZX10302205), the Major Project of Science and Technology in Henan Province (No. 161100311400), the Program of Natural Science Foundation of Henan Province (No. 182300410009), and the Major Project of TCM research in Henan Province (No. 2018ZYZD01).</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s8\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fgene.2020.00789/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fgene.2020.00789/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Data_Sheet_1.zip\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Begg</surname><given-names>C. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Chem</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Chem</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Chem.</journal-id><journal-title-group><journal-title>Frontiers in Chemistry</journal-title></journal-title-group><issn pub-type=\"epub\">2296-2646</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850663</article-id><article-id pub-id-type=\"pmc\">PMC7431664</article-id><article-id pub-id-type=\"doi\">10.3389/fchem.2020.00649</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Chemistry</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>A Novel CD133- and EpCAM-Targeted Liposome With Redox-Responsive Properties Capable of Synergistically Eliminating Liver Cancer Stem Cells</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Zihua</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/883585/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Sun</surname><given-names>Mengqi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Wang</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Fan</surname><given-names>Linyang</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhou</surname><given-names>Ying</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/968031/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Hu</surname><given-names>Zhiyuan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Key Laboratory of Brain Aging and Neurodegenerative Diseases of Fujian Provincial Universities and Colleges, School of Basic Medical Sciences, Fujian Medical University</institution>, <addr-line>Fuzhou</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>CAS Key Laboratory of Standardization and Measurement for Nanotechnology, CAS Key Laboratory for BiomedicalEffects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China</institution>, <addr-line>Beijing</addr-line>, <country>China</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Key Laboratory of Colloid Interface and Chemical Thermodynamics, Institute of Chemistry Chinese Academy of Sciences</institution>, <addr-line>Beijing</addr-line>, <country>China</country></aff><aff id=\"aff4\"><sup>4</sup><institution>School of Nanoscience and Technology, Sino-Danish College, University of Chinese Academy of Sciences</institution>, <addr-line>Beijing</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Yi Hou, Beijing University of Technology, China</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Jianfeng Zeng, Soochow University, China; Lihong Jing, Institute of Chemistry (CAS), China</p></fn><corresp id=\"c001\">*Correspondence: Zihua Wang <email>wangzh@iccas.ac.cn</email></corresp><corresp id=\"c002\">Zhiyuan Hu <email>huzy@nanoctr.cn</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Nanoscience, a section of the journal Frontiers in Chemistry</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>649</elocation-id><history><date date-type=\"received\"><day>05</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>22</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Wang, Sun, Li, Fan, Zhou and Hu.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Wang, Sun, Li, Fan, Zhou and Hu</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Cancer stem cells (CSCs) are a small subset of cells that sit atop the hierarchical ladder in many cancer types. Liver CSCs have been associated with high chemoresistance and recurrence rates in hepatocellular carcinoma (HCC). However, as of yet, no satisfactorily effective liver CSC-targeted treatment is available, which drove us to design and investigate the efficacy of a liposome-based delivery system. Here, we introduce a redox-triggered dual-targeted liposome, CEP-LP@S/D, capable of co-delivering doxorubicin (Dox) and salinomycin (Sal) for the synergistic treatment of liver cancer. This system is based on the association of CD133- and EpCAM-targeted peptides to form Y-shaped CEP ligands that were anchored to the surface of the liposome and allowed the selective targeting of CD133<sup>+</sup> EpCAM<sup>+</sup> liver CSCs. After arriving to the CSCs, the CEP-LP@S/D liposome undergoes endocytosis to the cytoplasm, where a high concentration of glutathione (GSH) breaks its disulfide bonds, thereby degrading the liposome. This then induces a rapid release of Dox and Sal to synergistically inhibit tumor growth. Notably, this effect occurs through Dox-induced apoptosis and concurrent lysosomal iron sequestration by Sal. Interestingly, both <italic>in vitro</italic> and <italic>in vivo</italic> studies indicated that our GSH-responsive co-delivery system not only effectively enhanced CSC targeting but also eliminated the non-CSC faction, thereby exhibiting high antitumor efficacy. We believe that the smart liposome nanocarrier-based co-delivery system is a promising strategy to combat liver cancer, which may also lay the groundwork for more enhanced approaches to target other cancer types as well.</p></abstract><kwd-group><kwd>targeting peptide</kwd><kwd>glutathione responsive</kwd><kwd>liver cancer stem cell</kwd><kwd>targeted drug delivery</kwd><kwd>synergistic therapy</kwd></kwd-group><counts><fig-count count=\"6\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"32\"/><page-count count=\"11\"/><word-count count=\"5920\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China with high mortality and incidence rates. Despite advances in diagnostic techniques and treatment approaches, most patients with advanced HCC have a poor prognosis, which may be partly attributed to a high ratio of cancer stem cells (CSCs). CSCs are a rare subset of cells that are involved in tumor maintenance, metastasis, drug resistance, and relapse (Wang et al., <xref rid=\"B26\" ref-type=\"bibr\">2015a</xref>). Unfortunately, conventional chemotherapy and radiotherapy approaches are ineffective against CSCs and are prone to tumor recurrence, treatment failure, and ultimately death (Yarchoan et al., <xref rid=\"B29\" ref-type=\"bibr\">2019</xref>). Salinomycin (Sal) is a potent drug that has been recently shown to selectively inhibit CSCs in various types of cancers, including liver CSCs (Gupta et al., <xref rid=\"B6\" ref-type=\"bibr\">2009</xref>; Mai et al., <xref rid=\"B14\" ref-type=\"bibr\">2017</xref>). However, Sal possesses unfavorable properties, such as hydrophobicity and nerve and muscle toxicity, that greatly hinder its clinical application (Wang et al., <xref rid=\"B24\" ref-type=\"bibr\">2017</xref>). In the past decades, CD133 and EpCAM have been widely studied as stem cell markers in liver cancer (Mikhail and He, <xref rid=\"B16\" ref-type=\"bibr\">2011</xref>). These surface markers serve not only as tools for identifying and isolating liver CSCs but also as therapeutic targets for eradicating these cells (Chan et al., <xref rid=\"B2\" ref-type=\"bibr\">2014</xref>; Jiang et al., <xref rid=\"B8\" ref-type=\"bibr\">2015</xref>; Saygin et al., <xref rid=\"B19\" ref-type=\"bibr\">2019</xref>). Therefore, targeting these CSC-specific markers by optimized drug combinations would be an ideal method for overcoming the stemness of liver CSCs and ameliorating the disease (Clevers, <xref rid=\"B3\" ref-type=\"bibr\">2011</xref>). Considering the importance of CSCs and the shortcomings of conventional anticancer therapies, dual-targeted CSC-specific delivery systems can possibly overcome this dilemma and leave an impact on the clinical setting (Dianat-Moghadam et al., <xref rid=\"B4\" ref-type=\"bibr\">2018</xref>; Guo et al., <xref rid=\"B5\" ref-type=\"bibr\">2019</xref>).</p><p>One way to address this issue is by using multifunctional nanoparticle (NP) systems, such as polymeric NPs, liposomes, and micelles, that can simultaneously deliver multiple therapeutic agents to induce a synergistic effect on CSCs (Zhao et al., <xref rid=\"B32\" ref-type=\"bibr\">2014</xref>; Rao et al., <xref rid=\"B17\" ref-type=\"bibr\">2015</xref>; Shen et al., <xref rid=\"B20\" ref-type=\"bibr\">2015</xref>). Liposomes are an FDA-approved drug delivery system that can be loaded with both hydrophilic and hydrophobic drugs in amphiphilic lipid bilayers (Kim et al., <xref rid=\"B9\" ref-type=\"bibr\">2015</xref>; Dianat-Moghadam et al., <xref rid=\"B4\" ref-type=\"bibr\">2018</xref>). Peptide ligands are widely recognized as the surface modification elements in targeted-delivery therapeutic approaches (Zhang et al., <xref rid=\"B31\" ref-type=\"bibr\">2012</xref>; Mao et al., <xref rid=\"B15\" ref-type=\"bibr\">2015</xref>). Our previous studies have demonstrated that peptide-conjugated liposomes can facilitate drug accumulation at tumor sites and improve the anticancer effect of different drugs (Wang et al., <xref rid=\"B27\" ref-type=\"bibr\">2019</xref>). Recently, the use of pro-drug-loaded stimuli-responsive drug delivery systems to facilitate the delivery of anticancer drugs has become a notable trend (Jia et al., <xref rid=\"B7\" ref-type=\"bibr\">2018</xref>; Yang et al., <xref rid=\"B28\" ref-type=\"bibr\">2018</xref>). Notably, stimuli-responsive NPs can respond to external stimuli (e.g., redox, reactive oxygen species, pH, and enzymes) to release drugs in a controlled manner (Ling et al., <xref rid=\"B11\" ref-type=\"bibr\">2019</xref>; Liu et al., <xref rid=\"B12\" ref-type=\"bibr\">2019</xref>). Glutathione (GSH) is a major antioxidant involved in many physiological processes that is abundant in cancer cells (Yu et al., <xref rid=\"B30\" ref-type=\"bibr\">2019</xref>). Therefore, it is no surprise that numerous GSH-responsive nanocarriers have been developed to deliver drugs and as imaging agents for better diagnostic and therapeutic efficacy (Li et al., <xref rid=\"B10\" ref-type=\"bibr\">2020</xref>). Nonetheless, this technology still has ways to go, as developing more efficient and smarter nanocarriers that can overcome the current challenges associated with CSCs is crucial (Shen et al., <xref rid=\"B21\" ref-type=\"bibr\">2016</xref>; Tan et al., <xref rid=\"B23\" ref-type=\"bibr\">2018</xref>; Reda et al., <xref rid=\"B18\" ref-type=\"bibr\">2019</xref>).</p><p>Herein, we aimed to develop a redox-responsive liposome, hereafter known as CEP-LP@S/D, that is capable of potentially targeting both liver CSCs and bulk cancer cells. The basis of this approach was the incorporation of Sal, a hydrophobic drug, into the lipid layers of the liposome and of doxorubicin (Dox), a hydrophilic drug, into the aqueous cavity of the liposome. Considering that biomarkers are heterogeneously expressed in HCC, a CD133, and EpCAM dual-targeted Y-shaped peptide ligand, CEP, was employed to decorate the surface of this liposome in order to improve both recognition and binding to CSC subpopulations. Moreover, we went further and endowed CEP-LP@S/D with GSH-responsive properties to initiate anticancer drug release in an intracellular manner once the liposomes have made contact with the tumor cells and investigated the effects both <italic>in vitro</italic> and <italic>in vivo</italic> (<xref ref-type=\"fig\" rid=\"F6\">Scheme 1</xref>).</p><fig id=\"F6\" position=\"float\"><label>Scheme 1</label><caption><p>Schematic illustration of glutathione (GSH)-responsive CEP-LP@S/D liposome for controlled drug delivery. <bold>(A)</bold> Schematic illustration of the structure and formation of the CEP-LP@S/D liposome. <bold>(B)</bold> Schematic illustration of the CEP-LP@S/D <italic>in vivo</italic> mechanism of action and transport.</p></caption><graphic xlink:href=\"fchem-08-00649-g0006\"/></fig></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Materials</title><p>We obtained 1-palmitoyl-2-oleoyl-<italic>sn</italic>-glycero-3-phosphocholine (POPC) from A.V.T. Pharmaceutical Co., Ltd. (Shanghai, China), and phosphoethanolamine-<italic>N</italic>-[methoxy(polyethylene glycol)-2000] (DSPE-SS-PEG-2000) from Xi'an ruixi Biological Technology Co., Ltd. (Xi'an, China). Meanwhile, we acquired 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR), LysoTracker Green, and 3-(4,5)-dimethylthiahiazol(-z-y1)-3,5-di-phenytetrazoliumbromide (MTT) from Sigma-Aldrich. We purchased Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) from Gibco (USA) and Sal from Selleck Chemicals (USA). Moreover, we obtained FITC-EpCAM and PE-CD133 antibodies from BioLegend (Shanghai, China).</p></sec><sec><title>Preparation and Characterization of CEP-Liposome NPs</title><p>In our previous work, we screened EP1 (YEVHTYYLD) and CY (CYIVFYDSPLE) as specific peptides toward EpCAM and CD133, respectively, using a high-throughput library (Wang et al., <xref rid=\"B25\" ref-type=\"bibr\">2015b</xref>). These two peptide ligands were connected via a GG linker, leading to the formation of Y-shape peptides (CEP), which was synthesized by the Fmoc solid-phase synthesis technique. The CEP was then covalently conjugated to DSPE-SS-PEG2000-Mal by adding thiol groups and maleimide (Michael addition). Liposomes were fabricated according to the literature (Guo et al., <xref rid=\"B5\" ref-type=\"bibr\">2019</xref>). Briefly, POPC/DSPE-SS-PEG2000-CEP (molar ratio 19:1) was mixed and dissolved in chloroform/methanol (v/v, 2:1). Next, a certain amount of Sal and Dox (mole ratio 1:1.5) was dissolved in 1 ml of methanol at room temperature and mixed with the lipid solution. The mixed solvent was then dried by rotary evaporation at 45°C to form a lipid film. After that, the dried film was hydrated with 5 ml of phosphate-buffered saline (PBS; pH 7.4) for 30 min and sonicated for 10 min. Finally, the prepared drug-loaded liposome, CEP-LP@S/D, was filtered through a 200-nm membrane filter to remove any precipitates. As for our <italic>in vivo</italic> imaging studies, we used DiR as the fluorescent probe; thus, DiR-loaded CEP-LP@S/D liposomes were prepared following the same procedures as above. The morphology and size of CEP-LP@S/D were measured by transmission electron microscopy (TEM), while their hydrodynamic size and polydispersity (PDI) were further measured in aqueous solutions by dynamic light scattering (DLS).</p></sec><sec><title><italic>In vitro</italic> Encapsulation and Release Profiles</title><p>The amount of Sal encapsulated in CEP-LP@S/D was detected at 392 nm by high-performance liquid chromatography (HPLC), and Dox fluorescence (λ<sub>Ex</sub>: 480 nm; λ<sub>Em</sub>: 590 nm) was measured using a fluorescence spectrometer. The drug encapsulation efficiency (EE) and the loading efficiency (LE) were then calculated according to the equations established by Zhang et al. (<xref rid=\"B31\" ref-type=\"bibr\">2012</xref>). Next, <italic>in vitro</italic> drug release from the liposomes was investigated by using a dialysis method. In short, the CEP-LP@S/D liposomes were dispersed in 1 ml of PBS (pH 7.4) and transferred to a dialysis device (MWCO: 10 kDa); they were then immersed in a GSH-containing PBS solution at 37°C and gently stirred. The Dox or Sal content released in the medium was determined by fluorescence spectrometry and HPLC at different time points as described above.</p></sec><sec><title>Cytotoxicity and Mammosphere Formation Assays</title><p>To evaluate the efficacy of the combination therapy <italic>in vitro</italic>, Huh-7 cells, or human HCC cells, were seeded in 96-well plates (5 × 10<sup>3</sup> cells per well) at 37°C for 12 h. After that, the cells were incubated with various concentrations of Dox, Sal, LP@S/D, or CEP-LP@S/D for 48 h, and cell viability was assessed by the MTT assay.</p><p>We further performed the mammosphere formation assay to assess treatment-induced changes in the stemness of these cells, that is, the CSC self-renewal ability. Thus, EpCAM<sup>+</sup> CD133<sup>+</sup> Huh-7 single-cell suspensions were sorted and seeded in ultralow-attachment six-well plates at a density of 5 × 10<sup>3</sup> cells per well. Then, various concentrations of free Sal, LP@S/D, or CEP-LP@S/D were added, and the cells were incubated at 37°C for 24 h. Thereafter, the cells were washed with 1 × BS and cultured in a serum-free DMEM/F12 medium, supplemented with 1 × B27 (Invitrogen), 20-ng/ml recombinant human epidermal growth factor (PeproTech), and 20-ng/ml basic fibroblast growth factor (PeproTech). After that, the cells were cultured in a 5% CO<sub>2</sub> incubator at 37°C for 7 days. The formed mammospheres in each treatment condition were counted and visualized under an optical microscope. Furthermore, to determine whether CEP-LP@S/D could induce a durable mammosphere inhibitory response, the primary mammospheres were trypsinized, prepared into single-cell suspensions, and then cultured in ultralow-adherent six-well plates as previously described. After 5 days, the number and morphology of the secondary mammospheres in each treatment condition were monitored and imaged under a microscope. The saline-treated group was considered as the control.</p></sec><sec><title>Cellular Uptake and Localization</title><p>The cellular internalization of LP@S/D and CEP-LP@S/D was studied by confocal laser scanning microscopy (CLSM). Huh-7 cells were cultured into glass-bottom dishes and incubated for 24 h, after which the cells were incubated with either CEP-LP@S/D or LP@S/D (20 μg/ml) as described above. After 4–8 h of incubation, the cells were washed and stained with 4′,6-diamidino-2-phenylindole (DAPI), a cell-permeant fluorescent nuclear dye, for 15 min and then rinsed with PBS. Finally, the cells were examined using a Zeiss 710 confocal microscope. In addition, we investigated the intracellular distribution of CEP-LP@S/D in the dissociated mammosphere cells via CLSM. Briefly, CD133<sup>+</sup> EpCAM<sup>+</sup> Huh-7 mammosphere cells were seeded into 24-well plates in a DMEM/F12 medium. After 24 h of incubation, they were treated with CEP-LP@S/D or LP@S/D at a concentration of 50 μg/ml for 8 h. Finally, the cells were collected and stained, as described above, and visualized using CLSM.</p></sec><sec><title>The Expression of Stemness-Associated Genes</title><p>To assess the stemness of CD133<sup>+</sup> EpCAM<sup>+</sup> Huh-7 tumorspheres, we extracted total RNA from the tumorspheres using TRizol (Invitrogen Inc., USA). Total RNA was converted into cDNA by using the PrimeScript™ RT Reagent Kit (Takara, China). Next, SYBR Green PCR Master Mix was added to the obtained cDNA, which was then quantified using the ABI PRISM 7,700 real-time polymerase chain reaction (PCR) platform. The mRNA expression levels of <italic>Sox-2, Oct-4, ABCG2</italic>, and <italic>CD133</italic> were normalized against that of <italic>GAPDH</italic> and those of PBS-treated cells.</p></sec><sec><title><italic>In vivo</italic> Imaging to Evaluate the Biodistribution of CEP-LP@S/D</title><p>Tumor-bearing mice were intravenously injected with (i) PBS, (ii) LP@S/D, or (iii) CEP-LP@S/D. After 2 h, the mice were imaged using an <italic>in vivo</italic> imaging system (IVIS). DiR-containing CEP-LP@S/D were detected by the appearance of a fluorescent signal at 748/780 nm (λ<sub>Ex</sub>/λ<sub>Em</sub>), and images were acquired using the IVIS. At the end point of the experiment, the mice were euthanized, and their major organs were harvested and analyzed to assess the biodistribution of CEP-LP@S/D in each organ.</p></sec><sec><title><italic>In vivo</italic> Antitumor Efficacy</title><p>For <italic>in vivo</italic> studies, BALB/C mice (6 weeks old) were subcutaneously (s.c.) injected with 1 × 10<sup>6</sup> sorted CD133<sup>+</sup> EpCAM<sup>+</sup> Huh-7 liver CSCs in their left flanks. When the tumor volume reached about 60–80 mm<sup>3</sup>, the mice were randomly distributed into four groups (<italic>n</italic> = 3), and each group received one of the following treatments: PBS control, Sal, LP@S/D, or CEP-LP@S/D; all the formulations were administered via tail vein injection (6 mg/kg) every other day. The body weight of the mice was monitored every 3 days, and tumor volumes were calculated using the following formula: (width<sup>2</sup> × length)/2; measurements were obtained using a vernier caliper every other day. The mice were sacrificed to harvest their tumors and main organs for further histological examination, namely, hematoxylin–eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis according to the manufacturer's instructions. In addition, we analyzed the CSC fraction in the tumor mass tissues to assess the effectiveness of the treatments in targeting CSCs and associated stemness features; in short, the tumor tissues were cut into small pieces and the extracted RNA was analyzed by real-time quantitative PCR (qPCR) as described above.</p></sec></sec><sec id=\"s3\"><title>Results and Discussion</title><sec><title>Characterization of Drug-Loaded CEP-LP@S/D Liposomes</title><p>Herein, we introduce a Y-shaped surface modification-based material, CEP-DSPE-SS-PEG, that bears two targeting peptides EpCAM and CD133, one in each head (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure 1</xref>). CEP-DSPE-SS-PEG was synthesized through covalent conjugation between the thiolated peptide CY-EP1 and DSPE-SS-PEG2000-Mal as confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure 2</xref>; Belhadj et al., <xref rid=\"B1\" ref-type=\"bibr\">2017</xref>). The anticancer drug Dox and the newly categorized anticancer agent Sal were loaded into CEP-LP@S/D and LP@S/D via the solvent evaporation method. The measurements showed that CEP-LP@S/D and LP@S/D were within a similar size range diameter-wise (<xref ref-type=\"fig\" rid=\"F1\">Figures 1a,b</xref>). The average particle size for all liposome systems was around 115 nm with a small PDI value of <0.28 and good dispersion (<xref ref-type=\"fig\" rid=\"F1\">Figure 1c</xref>). Notably, the particle size was not significantly affected by the CEP peptide modification. Moreover, the TEM images (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure 3</xref>) illustrated that the nanostructure of CEP-LP@S/D was disrupted in a GSH-rich surrounding after 8 h. To further confirm the capability of CEP-LP@S/D to simultaneously deliver two drugs, we tested the encapsulation efficiencies of Sal and Dox in CEP-LP@S/D using HPLC (for Sal) and a fluorescence spectrometer (for Dox). The liposomal EE for both Dox and Sal was higher than 86%. Furthermore, the drug-loading capacities of CEP-LP@S/D and LP@S/D were determined to be 2.4 and 2.2%, respectively. Additionally, the drug-release behaviors of CEP-LP@S/D were investigated with or without GSH (10 mM) in PBS (pH = 7.4) at 37°C. As shown in <xref ref-type=\"fig\" rid=\"F1\">Figures 1d,e</xref>, CEP-LP@S/D exhibited the ability of controlled release, as only about 37 and 29% of the Sal and Dox, respectively, were released from the drug-loaded CEP-LP@S/D after 12 h without the presence of GSH. However, a markedly enhanced drug release rate was detected in the presence of GSH (10 mM). Evidently, the GSH could effectively break the disulfide bonds of the CEP-LP@S/D lipid layers, thereby inducing the disassociation of the liposomal nanostructure; notably, more than 77.6 ± 1.4% of the loaded Dox and 79.2 ± 1.7% of the loaded Sal were released within 48 h in this case. We believe that the accelerated rate of Dox and Sal release by CEP-LP@S/D in the presence of GSH may be attributed to intracellular reducing environment-induced drug release.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Characterization of CEP-LP@S/D. <bold>(a)</bold> Transmission electron microscopy (TEM) image of LP@S/D. <bold>(b)</bold> TEM image of CEP-LP@S/D. <bold>(c)</bold> Hydrodynamic diameters of CEP-LP@S/D and LP@S/D measured by dynamic light scattering (DLS). <bold>(d)</bold> The <italic>in vitro</italic> release profiles of doxorubicin (Dox) in phosphate-buffered saline (PBS; pH 7.4) in the presence or absence of 10 mM glutathione (GSH). <bold>(e)</bold> The <italic>in vitro</italic> release profiles of salinomycin (Sal) in PBS (pH 7.4) in the presence or absence of 10 mM GSH.</p></caption><graphic xlink:href=\"fchem-08-00649-g0001\"/></fig></sec><sec><title>Cellular Uptake and Localization</title><p>The intracellular accumulation and distribution of CEP-LP@S/D in Huh7 cells were studied using confocal microscopy. As shown in <xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>, after 4 h incubation, CEP-LP@S/D-treated cells displayed more fluorescent signals in their cytoplasms than did their LP@S/D-treated counterparts (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure 4</xref>). As time went by, red fluorescent signals indicated that Dox could diffuse into cell nuclei, indicating that the EpCAM and CD133 peptides allowed for receptor-mediated endocytosis, thereby increasing the CEP-LP@S/D uptake and internalization by CSCs. In contrast, CEP-LP@S/D-treated 293T cells showed weak red fluorescence (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure 5</xref>). On another note, these results strongly demonstrate that CEP-LP@S/D could respond to the intracellular redox environment, leading to the disruption of the disulfide bonds in the lipid membranes, which consequently enabled the highly hydrophobic Dox to readily penetrate into the cells and, thus, exhibit greater cellular accumulation. Additionally, to evaluate the CSC-targeted effect, we established CD133<sup>+</sup>EpCAM<sup>+</sup> tumorspheres as CSC models; of course, we confirmed the stemness of these <italic>in vitro</italic> spheroids beforehand by analyzing the widely known stem cell markers <italic>Oct-4, Sox-2</italic>, and <italic>CD133</italic>, which were all overexpressed in these spheres (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure 6</xref>). As shown in <xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>, fluorescently labeled CEP-LP@S/D (red) were obviously notable in the CSC tumorspheres and had a high intensity. Furthermore, after 8 h incubation, the red fluorescence was significantly higher and could be detected in the nuclei. These results indicate that the Y-shaped peptide modification not only led to effective targeting of CSCs but could also facilitate the permeability of liposomal NPs to the inner part of the tumorspheres. Notably, CEP-LP@S/D was efficiently accumulated in the tumors and could effectively recognize and eradicate the CSC faction.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>CEP-LP@S/D uptake by confocal laser scanning microscopy (CLSM) imaging. <bold>(A)</bold> CLSM images of Huh-7 cells incubated with CEP-LP@S/D for 4 and 8 h. Three fluorescent dyes were used to evaluate CEP-LP@S/D uptake; the red signal represents doxorubicin (Dox), the green signal indicates lysosomes (LysoTracker Green), and the blue signal (4′,6-diamidino-2-phenylindole, DAPI) reveals the nuclei. <bold>(B)</bold> CLSM images of tumorsphere cells incubated with CEP-LP@S/D for 4 and 8 h.</p></caption><graphic xlink:href=\"fchem-08-00649-g0002\"/></fig></sec><sec><title>Cytotoxicity and CSC Elimination <italic>in vitro</italic></title><p>The cytotoxicity of the CEP-LP@S/D liposomes was evaluated by the MTT assay. Huh-7 cells were treated with Dox, Sal, LP@S/D, or CEP-LP@S/D at concentrations ranging from 0.01 to 0.5 mg/ml for 48 h. As shown in <xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>, CEP-LP@S/D exhibited higher cytotoxicity than did LP@S/D and the free drugs; notably, both Dox and Sal exhibited low cytotoxicity alone. This likely suggests that the dual-targeted peptide-mediated cellular uptake and GSH-triggered intracellular reductive cleavage improved the release of the loaded drugs. As expected, the tumorsphere formation assay revealed that CEP-LP@S/D induced a dramatic decrease in the number of formed tumorspheres than did the LP@S/D- and Sal-treated groups (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>). Moreover, as shown in <xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>, PBS (the vehicle) did not affect the tumorsphere-forming ability of Huh-7 cells. Interestingly, although the percentage of formed tumorspheres was marginally reduced by Sal and LP@S/D treatment, the size of the spheroids remained largely unaltered. However, the size of the tumorspheres in the CEP-LP@S/D treatment group was notably the smallest among all the groups, which suggests synergistic effects between the two drugs. To determine whether CEP-LP@S/D could induce a durable tumorsphere inhibitory response, CEP-LP@S/D-treated primary tumorspheres were dissociated into single-cell suspensions, and their propensity to form secondary tumorspheres was assessed (<xref ref-type=\"fig\" rid=\"F3\">Figure 3D</xref>). It was shown that the CEP-LP@S/D group had a maximum of 89.2% inhibition rate. Once again, single-cell suspensions of Sal- and LP@S/D-treated primary tumorspheres produced more secondary tumorspheres than did those of the free Sal-treated group; however, their CEP-LP@S/D-treated counterparts exhibited non-clonogenic properties (Suntharalingam et al., <xref rid=\"B22\" ref-type=\"bibr\">2014</xref>). It is worth mentioning that tumorsphere cells are much more resistant to free Sal, a problem that can be effectively overcome by using the liposomal construct prepared in this study. Taken together, these data show that CEP-LP@S/D inhibits the self-renewal of Huh-7 tumorspheres by eliminating the CSC population (CD133- and EpCAM-positive) and that this effect is maintained upon serial passages. Therefore, these results support the use of CEP-LP@S/D for the efficient internalization of drugs into CSC tumorspheres and preferentially inhibiting the proliferation of CSCs.</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>The inhibitory effect of various groups on the tumorsphere-forming ability of Huh-7 cells. <bold>(A)</bold> Viability of Huh-7 cells treated with free doxorubicin (Dox), Sal, LP@S/D, or CEP-LP@S/D for 48 h. <bold>(B)</bold> Quantification of tumorsphere formation efficiency in Huh-7 cells treated with Sal, LP@S/D, or CEP-LP@S/D. <bold>(C)</bold> Images of tumorsphere formation of untreated Huh-7 cells (phosphate-buffered saline, PBS) and those treated with salinomycin (Sal), LP@S/D, or CEP-LP@S/D for 5 days. <bold>(D)</bold> Images of second-generation tumorsphere formation (secondary formation after dissociation of initial spheres) under PBS, Sal, LP@S/D, or CEP-LP@S/D treatment. Scale bar = 300 μm.</p></caption><graphic xlink:href=\"fchem-08-00649-g0003\"/></fig></sec><sec><title>Tumor Accumulation of CEP-LP@S/D <italic>in vivo</italic></title><p>The ability of CEP-LP@S/D to home toward tumors was examined by the IVIS. The CEP-LP@S/D liposomes were loaded with DiR dye prior to being injected into tumor-bearing mice. As shown in <xref ref-type=\"fig\" rid=\"F4\">Figure 4a</xref>, DiR fluorescent signals were obviously detected in the tumor sites 2 h after the injection and gradually increased with time. Notably, CEP-LP@S/D-DiR signals peaked after 20 h and gradually disappeared after the 24-h mark; moreover, they were markedly higher than those of LP@S/D-DiR. These findings clearly demonstrate that the CEP-LP@S/D liposomes remain in the tumors for a satisfactory amount of time (good retention time) and prove that they can efficiently and effectively target the tumor site in both passive and active manners. These characteristics are possible due to the Y-shaped CD133 and EpCAM ligand surface modification, which led to the enhanced permeability and retention (EPR) effect. The <italic>ex vivo</italic> histological analysis (biodistribution study) showed that the fluorescent CEP-LP@S/D-DiR signals were mostly detected in the tumors and spleens of the mice (<xref ref-type=\"fig\" rid=\"F4\">Figure 4b</xref>). However, these signals were weaker in other organs, which exhibited only background or moderate signals, suggesting the preferable accumulation of the liposome in tumor tissues (<xref ref-type=\"fig\" rid=\"F4\">Figure 4c</xref>). This result indicates that the CEP-LP@S/D liposomes favorably accumulate in the tumors, where they have a notable penetration capacity that could significantly increase the possibility of CEP-LP@S/D to eradicate CSCs <italic>in vivo</italic>.</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>The <italic>in vivo</italic> imaging distribution of CEP-LP@S/D. <bold>(a)</bold>\n<italic>In vivo</italic> image of Huh-7 xenograft tumor-bearing nude mice at different time points after injection with DiR-loaded CEP-LP@S/D liposomes. <bold>(b)</bold>\n<italic>Ex vivo</italic> image of tumors and main organs 24 h post injection of the different formulations. <bold>(c)</bold> The quantified biodistribution of CEP-LP@S/D in the major organs 24 h post injection.</p></caption><graphic xlink:href=\"fchem-08-00649-g0004\"/></fig></sec><sec><title><italic>In vivo</italic> Antitumor Efficacy of CEP-LP@S/D</title><p>To further verify whether CEP-LP@S/D could facilitate the accumulation of Sal and Dox in the xenograft-induced tumors, different formulations, with equivalent Dox and Sal doses of 6 mg/kg, were administered via tail vein injection every other day. As shown in <xref ref-type=\"fig\" rid=\"F5\">Figures 5A,B</xref>, CEP-LP@S/D liposomes exhibited the best antitumor effect than did the other two groups. However, free Sal also showed a slight tumor inhibitory effect at the same time points, although this variation was not statistically significant. This suggests that the Y-shaped peptidic construct led to more extensive intracellular delivery via receptor-mediated targeting of EpCAM<sup>+</sup>CD133<sup>+</sup> liver CSCs. In accordance with our <italic>in vitro</italic> results, CEP-LP@S/D disulfide bond breakage in response to elevated GSH levels in the cytosol explains the enhanced release of Dox and Sal, which in turn induce a synergistic cytotoxic effect against bulk tumor cells and CSCs. It is worth noting that none of the mice had any noticeable weight change in any of the treatment groups, suggesting there was no obvious systemic toxicity from CEP-LP@S/D (<xref ref-type=\"fig\" rid=\"F5\">Figure 5C</xref>). A number of studies have indicated that <italic>Sox-2</italic> and <italic>Oct-4</italic> represent strong stemness characteristics that are crucial for the tumor initiation and self-renewal of CSCs (Ma et al., <xref rid=\"B13\" ref-type=\"bibr\">2010</xref>). As shown in <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure 7</xref>, the expression level of <italic>Sox-2</italic> was markedly suppressed by CEP-LP@S/D. Moreover, CEP-LP@S/D treatment also induced significant downregulation of other stemness-associated (<italic>CD133, Oct-4</italic>, and <italic>Sox-2</italic>) and drug efflux (<italic>ABCG2</italic>) genes than did the free Sal-treated group. This indicates that the CEP-LP@S/D liposome was capable of downregulating stemness-associated genes and synergistically enhancing drug cytotoxicity toward CSCs. We went further to assess the antitumor efficacy as well as the potential side effects; histological analysis of tumor slides demonstrated that CEP-LP@S/D was the most effective in inhibiting cell proliferation and inducing cell apoptosis, with only a few side effects (<xref ref-type=\"fig\" rid=\"F5\">Figure 5D</xref>). In contrast, free Sal and LP@S/D did not significantly inhibit cell proliferation and showed a comparable effect to that of PBS, as demonstrated by TUNEL. Apart from these, H&E staining indicated that there is no obvious toxicity in the major organs, including the heart, liver, spleen, lung, and kidney (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure 8</xref>). As shown in <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure 9</xref>, CEP-LP@S/D significantly improved the survival rate of the mice compared to other groups. Taken together, our results indicate that co-delivery of Dox and Sal via CEP-LP@S/D induced a significant synergistic anticancer effect as it combined the ability of Dox to eliminate bulk cancer cells with that of Sal to suppress the CSC population and went further to enhance their absorption and targeting abilities.</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p><italic>In vivo</italic> antitumor activity of the different formulations on Huh-7-bearing nude mice. <bold>(A)</bold> The photographs of tumors in the differently treated Huh-7-bearing nude mice 18 days after tumor excision and experiment end point. <bold>(B)</bold> A curve representing the tumor growth of the differently treated mice. <bold>(C)</bold> The weight of the mice during the treatment. <bold>(D)</bold> Hematoxylin–eosin (HandE) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analyses of tumor tissues after treatment with various formulations. Scale bar = 0.1 cm.</p></caption><graphic xlink:href=\"fchem-08-00649-g0005\"/></fig></sec></sec><sec sec-type=\"conclusions\" id=\"s4\"><title>Conclusions</title><p>To reiterate, in this study, we successfully developed a new redox-responsive liposomal platform for targeted anticancer drug delivery that allows the synergistic amelioration of liver cancer. Our results indicate that the CEP-LP@S/D co-delivery system could enhance the accumulation of drugs in the tumor tissues and target CSCs via the specific peptide recognition receptors CD133 and EpCAM, which are over-expressed on these cells. After cellular uptake, the high concentration of GSH in the cytoplasm is sufficient to break the disulfide bonds in the structure of these liposomes, thereby inducing the fast release of Dox and Sal and leading to the synergistic inhibition of tumor growth and reduction in CSC stemness. We believe that this co-delivery nano-platform could be used as an effective tool for delivering combinatorial therapeutics to synergistically inhibit liver CSCs and tumor cells. This study provides a new perspective for designing specifically responsive drug delivery systems to target CSCs and may lay the groundwork for similar approaches customized for other cancer types.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The original contributions presented in the study are included in the article/<xref ref-type=\"sec\" rid=\"s9\">Supplementary Material</xref>, further inquiries can be directed to the corresponding authors.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by Peking University Animal ethics Committee.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>ZW performed the experiments and wrote the manuscript. WL, MS, and YZ helped to perform <italic>in vitro</italic> experiments. LF helped to characterize materials and incubate cells. ZH revised the manuscript and supervised all the works. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer LJ declared a shared affiliation with authors MS, WL, LF, and ZH to the handling editor at the time of review.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported financially by the National Natural Science Foundation of China (Grant Nos. 81801766, 31870992, and 21775031), Science and Technology Service Network Initiative of the Chinese Academy of Sciences (Grant No. KFJ-STS-ZDTP-079), CAS-JSPS (Grant No. GJHZ2094), Fujian Medical University Foundation for the Introduction of Talents (Grant No. XRCZX2019018).</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s9\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fchem.2020.00649/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fchem.2020.00649/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Belhadj</surname><given-names>Z.</given-names></name><name><surname>Ying</surname><given-names>M.</given-names></name><name><surname>Cao</surname><given-names>X.</given-names></name><name><surname>Hu</surname><given-names>X.</given-names></name><name><surname>Zhan</surname><given-names>C.</given-names></name><name><surname>Wei</surname><given-names>X.</given-names></name><etal/></person-group>. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Mol Biosci</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Mol Biosci</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Mol. Biosci.</journal-id><journal-title-group><journal-title>Frontiers in Molecular Biosciences</journal-title></journal-title-group><issn pub-type=\"epub\">2296-889X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850977</article-id><article-id pub-id-type=\"pmc\">PMC7431665</article-id><article-id pub-id-type=\"doi\">10.3389/fmolb.2020.00196</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Molecular Biosciences</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Molecular Pathogenesis, Immunopathogenesis and Novel Therapeutic Strategy Against COVID-19</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Chatterjee</surname><given-names>Swapan K.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/35536/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Saha</surname><given-names>Snigdha</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/976962/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Munoz</surname><given-names>Maria Nilda M.</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1032982/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Molecular Pharma Pvt., Ltd.</institution>, <addr-line>Kolkata</addr-line>, <country>India</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Cagayan State University</institution>, <addr-line>Tuguegarao City</addr-line>, <country>Philippines</country></aff><aff id=\"aff3\"><sup>3</sup><institution>De La Salle University</institution>, <addr-line>Manila</addr-line>, <country>Philippines</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Pier Paolo Piccaluga, University of Bologna, Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Veronika Butin-Israeli, Northwestern University, United States; Amit Prasad, Indian Institute of Technology Mandi, India</p></fn><corresp id=\"c001\">*Correspondence: Swapan K. Chatterjee, <email>swapan1chatterjee@gmail.com</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Molecular Diagnostics and Therapeutics, a section of the journal Frontiers in Molecular Biosciences</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>11</day><month>8</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>7</volume><elocation-id>196</elocation-id><history><date date-type=\"received\"><day>16</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>22</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Chatterjee, Saha and Munoz.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Chatterjee, Saha and Munoz</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>The coronavirus disease 2019 (COVID-19), is a highly contagious transmittable disease caused by a recently discovered coronavirus, pathogenic SARS-CoV-2. Followed by the emergence of highly pathogenic coronaviruses in 2003 SARS-CoV, in 2012 MERS-CoV, now in 2019 pathogenic SARS-CoV-2, is associated with a global “pandemic” situation. In humans, the effects of these viruses are correlated with viral pneumonia, severe respiratory tract infections. It is believed that interaction between angiotensin converting enzyme 2 (ACE2) cell receptor and viral Spike protein mediates the coronavirus entry into human respiratory epithelial cells and establishes the host tropism. ACE2 receptor is highly expressed in airway epithelial cells. Along with viral-receptor interaction, proteolytic cleavability of S protein has been considered as the determinant of disease severity. Several studies highlight the occurrence of impaired host immune response and expression of excessive inflammatory response especially cytokines against viral infection. The mechanisms of SARS-CoV-2 induced acute lung injury are still undefined; however, the term <italic>cytokine storm</italic> has now been recognized to be closely associated with COVID-19. The levels of inflammatory mediators from <italic>cytokine storm</italic> cause damage to the host cells. In particular, the proinflammatory cytokine IL-6 appears to be the key mediator in early phase of virus-receptor interaction; however, secreted IL-6 might not be representative of lung inflammation. Understanding the cellular, and molecular factors involved in immune dysregulation and the high virulence capacity of COVID-19 will help in potential targeted therapy against it. “Drug repurposing” and “molecular docking analysis” is considered as an attractive alternative approach in analyzing suitable drug candidates to combat SARS-CoV-2 infection. Globally, extensive research is in progress to discover a new vaccine for novel COVID-19. Moreover, our review mainly focuses on the most state-of-the-art therapeutic approach mediated by “Mannose-binding lectin (MBL).” One of the most significant molecules of innate immunity is MBL. It plays a major role in the activation of the complement system as an ante-antibody prior to the response of any particular antibody. Recombinant human MBL can be used as immunomodulators against SARS-CoV-2.</p></abstract><kwd-group><kwd>SARS-CoV-2</kwd><kwd>COVID-19</kwd><kwd>TMPRSS2</kwd><kwd>cytokine storm</kwd><kwd>Mannose-binding lectin</kwd><kwd>innate immunity</kwd><kwd>immunomodulators</kwd></kwd-group><counts><fig-count count=\"2\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"64\"/><page-count count=\"12\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Various viral members belong to the Coronaviridae family (COVs) under the order Nidovirales are continually circulating among the human population and responsible for causing mild to acute respiratory disease (<xref rid=\"B5\" ref-type=\"bibr\">Corman et al., 2020</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Hoffmann et al., 2020</xref>). COVs member coronaviruses belong to the subfamily Coronavirinae and can be classified into alpha-, beta-, gamma-, and delta-CoVs. CoVs family members contain a positive-sense, single-strand RNA genome, 26 to 32 kilobases in length. The extensive distribution and infectivity capacity of CoVs establish them as an important pathogen. Besides numerous avian hosts, various member of CoVs have been recognized in a range of mammals, like bats, mice, masked palm civets, dogs, camels, and cats (<xref rid=\"B12\" ref-type=\"bibr\">Guan et al., 2003</xref>) and responsible for disease related to hepatic, respiratory, gastrointestinal systems, and nervous system in humans (<xref rid=\"B33\" ref-type=\"bibr\">Lu et al., 2020</xref>).</p><p>A new virus called the 2019 novel coronavirus (an enveloped beta-coronavirus) is identified in December 2019 and associated with a <italic>de novo</italic> contagious respiratory disease which primarily transpired in Hubei province, China (<xref rid=\"B19\" ref-type=\"bibr\">Huang et al., 2020</xref>; <xref rid=\"B51\" ref-type=\"bibr\">Wang C. et al., 2020</xref>). Initially, the infection was emerged from the Huanan seafood market and is initiated due to animal contact. Consequently, the disease spread through human-human contact within China and later rapidly spread causing new public health crises worldwide (<xref rid=\"B4\" ref-type=\"bibr\">Chan et al., 2020</xref>; <xref rid=\"B63\" ref-type=\"bibr\">Zhu et al., 2020</xref>). In early March 2020, this disease was documented as “pandemic” by the World Health Organization (<xref rid=\"B7\" ref-type=\"bibr\">Cucinotta and Vanelli, 2020</xref>).</p><p>Virologists are still uncertain about exactly how COVID-19 spreads. Medical doctors and scientists tend to agree that COVID-19 is transmittable through the inhalation of droplets from a person who has viral infection. However, the droplets discharged from cough and sneeze is heavy enough that might not travel more than 1 m (<xref rid=\"B6\" ref-type=\"bibr\">Corman et al., 2019</xref>). Unlike airborne particles that are much smaller than droplets and can stay in the atmosphere for a longer time. According to WHO, COVID-19 is not airborne; however, several experts appear to disagree. Common disinfectants like sodium hypochlorite; hydrogen peroxide etc., can be used to destroy the virus (<xref rid=\"B25\" ref-type=\"bibr\">Kampf et al., 2020</xref>). As per current information, subsequent transmission of this infection is possible through stool and/or contamination of the water supply (<xref rid=\"B19\" ref-type=\"bibr\">Huang et al., 2020</xref>). Although in most people the disease severity is mild, but the clinical manifestation of COVID-19 mainly spans from an asymptomatic state to pneumonia, acute respiratory distress syndrome, followed by multi organ dysfunction. Fever, headache, cough, fatigue, sore throat, myalgia, breathlessness, and conjunctivitis (in some cases) are the common clinical characteristics of this disease (<xref rid=\"B19\" ref-type=\"bibr\">Huang et al., 2020</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Singhal, 2020</xref>).</p><p>Several phylogenetic analysis suggest that bat is likely the animal origin of the SARS-CoV-2 as it is 89% genomically identical with bat SARS-like-CoVZXC21, 50% with MERS-CoV and about 82% identical with human SARS-CoV (<xref rid=\"B61\" ref-type=\"bibr\">Zhou P. et al., 2020</xref>). Genome sequencing data also suggest that pangolins also have approximately 85.5–92.4% conserved sequence to SARS-CoV-2 (<xref rid=\"B59\" ref-type=\"bibr\">Yang et al., 2015</xref>). Other studies also suggest that SARS-CoV-2 could be a recombinant virus of bat coronavirus and snake coronavirus. The truth behind the origin of SARS-CoV-2 is yet to be discovered (<xref rid=\"B57\" ref-type=\"bibr\">Xie and Chen, 2020</xref>). The spike (S) glycoprotein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein are four structural proteins of novel SARS-CoV-2. The most significant surface protein is spike glycoprotein which interferes in establishing the association between the human respiratory epithelial cells to the virus via cell surface membrane receptor angiotensin-converting enzyme 2 (ACE2) and finally establishes the host tropism (<xref rid=\"B32\" ref-type=\"bibr\">Li et al., 2003</xref>). In this review, we emphasize the pandemic potential as well as molecular and immunological events involved in the pathogenesis of novel SARS-CoV-2. As the spike protein of SARS-CoV-2 plays a major role in the disease spreading, we comprehensively summarize the structure of spike protein and their potential role in the mutation of the virus. We also concisely discuss the potential effective treatments as well as new therapeutic approaches to control the transmission of this COVID-19 pandemic.</p></sec><sec id=\"S2\"><title>Molecular Aspect of COVID-19 Pathogenesis</title><sec id=\"S2.SS1\"><title>Structural Configuration of Spike Protein</title><p>SARS-CoV-2 are single-stranded RNA viruses characterized by two groups of proteins, namely – (A) Structural Proteins - such as Spike (S) proteins marking all coronaviruses and bind to receptors on the host cell; Nucleocapsid (N) that protects the genetic information of virus, Matrix (M), and Envelope (E); (B) Replicase Complex Encoding Non-structural Proteins, such as proteases (nsp3 and nsp5) and RdRp (nsp12; <xref rid=\"B61\" ref-type=\"bibr\">Zhou P. et al., 2020</xref>). This enzyme is an ideal target as it helps the virus to replicate, meaning the virus depends on protease. Recent studies have shown that SARS-CoV-2 share a similar type of genomic organization with other beta coronaviruses. Like others, it produces a ∼800 kDa polypeptide upon transcription of genome. The proteolytic processing is mediated by papain-like protease (PLpro) and 3-chymotrypsin-like protease (3CLpro) and produces various non-structural proteins required for replication of viral particles. It is then predicted that these proteolytic enzymes could serve as the probable target site for anti-coronaviruses inhibitors (<xref rid=\"B59\" ref-type=\"bibr\">Yang et al., 2015</xref>). Recently, SARS-CoV showed an open reading frame 3a protein; coded by one of the group specific genes, and showed no sequence similarity to other known structural coronavirus proteins. It interacts with the E and S proteins while the M protein is glycosylated in all coronaviruses. Thus, the close homology of the M protein glycosylation and its likeness to 3a protein could result to investigate the glycosylation of these two membrane proteins (<xref rid=\"B43\" ref-type=\"bibr\">Song et al., 2018</xref>).</p><p>Viral transmembrane spike (S) glycoprotein is a trimeric class I fusion protein promotes the SARS-CoV-2 entry into the cell and the main target of neutralizing antibodies upon infection. Previous studies have shown that the function of spike protein depends upon its cleavage by host proteolysis enzyme into the S1 and S2 subunit. The attachment of the virus was facilitated by the S1 subunit whereas the S2 subunit assists in the fusion of viral and human cell membrane. Additionally, both the N-terminal domain (NTD) and the C-terminal domain (CTD) of the S1 subunit are capable in function as a receptor-binding entity (<xref rid=\"B36\" ref-type=\"bibr\">Millet et al., 2012</xref>). Although the S1 CTD region is utilized by both SARS-CoV and MERS-CoV to identify the receptor [receptor binding domain (RBD)], the region responsible for SARS-CoV-2 S-protein-hACE2 interaction remains unknown. Complete genomic analysis of each monomer of S-protein has shown that it has 22 predicted N-linked glycosylation sites and 4 predicted O-linked glycosylation sites. Studies conducted with Cryo-electron microscopy (Cryo-EM) have shown that 14-16 N-glycans are present on 22 potential sites in S-protein which are responsible for proper protein folding and priming of the protein by host proteases. The glycosylation pattern of the S-protein is one of the major features as it acts as a possible site for mutation and also facilitates the coronavirus to evade both innate as well as adaptive immune responses (<xref rid=\"B43\" ref-type=\"bibr\">Song et al., 2018</xref>). A recent study suggests that prediction of SARS-CoV-2 spike glycoprotein structure, glycan shield pattern and pattern of glycosylation has great inference on understanding the viral camouflage as well as the outline of cell entry, and also facilitate the development of new small-molecule drugs, vaccines, antibodies, and screening of the human host targets (<xref rid=\"B43\" ref-type=\"bibr\">Song et al., 2018</xref>).</p><p>Sequence alignment data done by the Clustal-W has shown that the S2 domain region (aa570–aa1278) of SARS-CoV-2 share a 91% identity with SARS-CoV spike glycoproteins. Though in the S1 domain (aa01–aa550), it shows a larger sequence difference (∼55% identity), which is essential for host cell adhesion, target interaction, and tropism of virulence but it has a conserved domain for ternary folding residue. This finding suggests that SARS-CoV-2 also capable to interact with SARS-CoV host targets like ACE2, CD26, Ezrin, and cyclophilins (<xref rid=\"B48\" ref-type=\"bibr\">Vankadari and Wilce, 2020</xref>).</p></sec><sec id=\"S2.SS2\"><title>ACE2 Receptor – Major Entry Site for SARS-CoV-2</title><p>Receptor recognition is one of the major steps in viral infection of host cells and viral infectivity and pathogenesis. SARS-CoV depends upon ACE2 (angiotensin-converting enzyme 2) receptor which is highly expressed in human epithelial, endothelial, renal, cardiovascular tissue, and lung parenchyma (<xref rid=\"B47\" ref-type=\"bibr\">Tikellis and Thomas, 2012</xref>). Human ACE2 is a type I integral metallocarboxypeptidase act as a key player in the Renin-Angiotensin system (RAS). ACE2 exhibits protective function in the cardiovascular system, and other organs and act as a major target site for the treatment of hypertension. ACE2 protein is more profusely expressed on the apical surface of polarized epithelial cells as well as well-differentiated cells and certain progenitor cells in the bronchi (<xref rid=\"B32\" ref-type=\"bibr\">Li et al., 2003</xref>). Expression of ACE2 receptor in progenitor cells of respiratory tract cells with hair-like projections called cilia serves for the coronavirus entry site in the human body. Well-differentiated epithelial cells expressing ACE2 are readily infected by coronavirus. The viral infection thus correlates with the cell differentiation condition, ACE2 receptors expression, and localization of membrane binding. However, to date, there are still unanswered inquiries remain regarding ACE2 expression in human epithelial cells and its modulatory role in coronavirus. Questions include the type of epithelial cells involved in disease, the polarity of ACE2 expression on epithelial cells, and whether the coronavirus infection is ACE2 dependent. Interestingly, appearances of ACE2 receptor density on the progenitor cell surface increased with age and are generally present higher in men than in women (<xref rid=\"B58\" ref-type=\"bibr\">Xie et al., 2006</xref>). A study by <xref rid=\"B54\" ref-type=\"bibr\">Wu et al. (2020)</xref>, using computer modeling has shown the presence of identical 3-D structures in the receptor-binding domain of the spike proteins of both SARS-CoV-2 as well as SARS-CoV. Biochemical interaction studies and crystal structure analysis by Wan et al. have proved that SARS-CoV-2 receptor-binding domain (RBD) contain residue 394 (glutamine), which has sequence similarity with SARS-CoV residue 479, and both can be accepted by the human ACE2 receptor on the critical lysine 31 (<xref rid=\"B50\" ref-type=\"bibr\">Wan et al., 2020</xref>).</p><p>Angiotensin converting enzyme 2 is an entry receptor for SARS-CoV-2 and shows 76% amino acid sequence homology with the SARS-CoV-S. Structural configuration study shows that, ACE2 contains 17 amino acid N-terminal signal sequences and a 22 amino acid hydrophobic transmembrane sequence near the C-terminus. ACE2 also contains 43 amino acid cytoplasmic domain, a potential phosphorylation sites, eight cysteine residues, and seven potential Af-linked glycosylation sites (<xref rid=\"B23\" ref-type=\"bibr\">Jia, 2016</xref>). The viral spike (S) protein of SARS-CoV-2 binds to cellular receptor ACE2 in a similar way to SARS-CoV-1 but with a 10- to 20-fold higher binding affinity. These findings suggest that increased ACE2 expression might confer easier transmissibility and also increase the susceptibility of SARS-CoV-2 into the host cell (<xref rid=\"B2\" ref-type=\"bibr\">Bourgonje et al., 2020</xref>). Studies using angiotensin-II receptor blockers (ARB) and ACE-inhibitors (ACE-i) suggest that the upregulation of cellular ACE2 expression facilitates the binding of SARS-CoV-2 and associated with severe disease manifestation. This receptor recognition by viral cell leads to host cell entry of the virus in combination with S-protein priming by the host cell protease TMPRSS2. Downregulation of ACE2 expression by SARS-CoV-2 could decrease the angiotensin-II clearance and lead to aggravation of tissue damage. Identification of interaction site and the downstream signaling cascade of SARS-CoV-2 and ACE2 receptor in the human cells will help to design the antibody-based therapeutic strategy (<xref rid=\"B13\" ref-type=\"bibr\">Gue et al., 2020</xref>).</p><p>A study by <xref rid=\"B61\" ref-type=\"bibr\">Zhou P. et al. (2020)</xref> on a mouse model of SARS-CoV infection, demonstrated that overexpression of human ACE2 receptor are associated with the severity of the disease. He also suggested that in pulmonary tissue alveolar epithelial type II (AECII) cells express 83% of ACE2 receptor and provide a suitable site for viral invasion. Additionally, gene ontology enrichment analysis of AECII has demonstrated that increased expression of ACE2 is associated with high levels of various viral process-related genes, including viral life cycle, genome replication, assembly, and regulatory genes for viral processes, etc. (<xref rid=\"B61\" ref-type=\"bibr\">Zhou P. et al., 2020</xref>). These findings imply that the ACE2-expressing AECII is able to assist coronavirus replication in the lung. Like pulmonary tissue some extra-pulmonary tissues such as heart, kidney, endothelium, and intestine express the ACE2 receptor (<xref rid=\"B30\" ref-type=\"bibr\">Li et al., 2020d</xref>). Another observation also suggests that a state of insulin resistance and elevated plasma glucose levels are associated with increased expression of ACE2 in lung epithelial cells and act as the risk factor for morbidity and mortality in SARS-CoV-2 infected patients (<xref rid=\"B10\" ref-type=\"bibr\">Finucane and Davenport, 2020</xref>). According to a study by <xref rid=\"B19\" ref-type=\"bibr\">Huang et al. (2020)</xref> suggest that in addition to lung epithelial tissue SARS-CoV-2 might affect other tissues including male tissues like testis and seminal vesicles. He also reported that SARS-CoV-2 infection might be related to cardiac injury (<xref rid=\"B19\" ref-type=\"bibr\">Huang et al., 2020</xref>). A recent study by, <xref rid=\"B18\" ref-type=\"bibr\">Holshue et al. (2020)</xref> unveiled that stool from a SARS-CoV-2 infected patient was positive for SARS-CoV-2, which suggests that this virus might infect the gastrointestinal tract. Significantly, high expression of ACE2 on the luminal surface of intestinal epithelial cells acts as a co-receptor for amino acid absorption from food. Studies also suggest that intestine might act as a major entry site for SARS-CoV-2 infection and infected epithelium of gut might have significant inference on fecal-oral transmission and viral spread confinement (<xref rid=\"B56\" ref-type=\"bibr\">Xiao et al., 2020</xref>).</p></sec><sec id=\"S2.SS3\"><title>Priming of Spike Protein and Onset of Disease</title><p>Entry of corona-virus into the cell depends upon the priming of S-proteins by host cell proteases which comprise the cleavage at the arginine-rich site (multi-basic) S1/S2 and the S20 sites. The efficient cleavage of S-protein along with cleavage site sequence establishes the zoonotic potential of coronavirus. Various studies have proved that SARS-CoV-2 infection initiation and spread of disease into the host cells mainly depends upon S protein priming by TMPRSS2 (Transmembrane protease serine type 2), the serine protease. In humans, epithelial tissues, especially those lining the upper airways, bronchi, and lower airways show extensive TMPRSS2 expression (<xref rid=\"B17\" ref-type=\"bibr\">Hoffmann et al., 2020</xref>). The protein sequence analysis demonstrates that TMPRSS2 is conserved, with 78% sequence identity in human whereas, in mouse embryos and adult tissues, <italic>In situ</italic> hybridization analyses divulge the presence of TMPRSS2 in the epithelial cell lining the urogenital, gastrointestinal, and bronchi and bronchioles of respiratory tracts. However, the exact physiological function of TMPRSS2 within lung epithelial cells is not clear but various data suggest that it helps in proteolytic cleavage of the epithelial sodium channel and regulate sodium currents (<xref rid=\"B22\" ref-type=\"bibr\">Iwata-Yoshikawa et al., 2019</xref>).</p><p>According to the study, TMPRSS2 belongs to the type II transmembrane serine protease family, and play a major role in the cleavage of hemagglutinin (HA) molecule in the influenza virus upon entry into human airway epithelial cells in human (<xref rid=\"B16\" ref-type=\"bibr\">Hatesuer et al., 2013</xref>). However, it can cleave glycoproteins (Spike protein) and stimulates it to induce the fusion of virus-host cell membrane at the cell surface which in turn assists virus entry into the host cell. Various studies demonstrate that certain TMPRSS2 variants expression increases the threat of disease severity in influenza A (H1N1) infection (<xref rid=\"B16\" ref-type=\"bibr\">Hatesuer et al., 2013</xref>). A study by <xref rid=\"B17\" ref-type=\"bibr\">Hoffmann et al. (2020)</xref> had demonstrated that in infected cells, precleavage at the S1/S2 site by Furin might encourage consequent TMPRSS2-dependent entry and spread of infection. A type I transmembrane protein, Furin is an activating protease that plays a critical role in fusion of viral membrane and viral entry within the host cell. Thus the role of anti-TMPRSS2 as active-site inhibitors or inhibition of furin dependent entry might help us to consider them as probable therapeutic targets for influenza viruses and also for coronaviruses (<xref rid=\"B17\" ref-type=\"bibr\">Hoffmann et al., 2020</xref>).</p></sec><sec id=\"S2.SS4\"><title>Pathogenesis and Immunopathology of COVID-19</title><p>The S protein present in the membrane of SARS-CoV-2 has been considered as the most potent virus entry determinant into the host cell through the receptor ACE2. A study by <xref rid=\"B1\" ref-type=\"bibr\">Belouzard et al. (2009)</xref> revealed that a significant cleavage event takes place by proteolytic enzymes at position S20 of SARS-CoV-2 S protein by TMPRSS2, results in the fusion of membranes as well as viral infectivity mediated by the release of viral RNA. Other studies also revealed that entry of viral RNA into the host cell depends upon not only membrane fusion, but also on the clathrin-dependent, and/or clathrin-independent endocytosis (<xref rid=\"B52\" ref-type=\"bibr\">Wang et al., 2008</xref>). After released into the cytoplasm viral RNA genome is translated into two structural proteins and poly-proteins, which help in viral replication. Upon infection with SARS-CoV-2 genome host cell activates well-coordinated and rapid immune response, i.e., innate and adaptive immune response, which represents the first line of defense against the viral infection. In endosome, membrane specific pattern recognition receptors (PRR), like Toll-like receptor (TLR3, TLR8, TLR7, and TLR9) or the cytosolic RNA sensor, RIG-I/MDA5 or the secretory type PRR like Mannose-binding lectin (MBL) and C-reactive protein (CRP) can recognize viral RNA as pathogen-associated molecular patterns (PAMPs) (<xref rid=\"B38\" ref-type=\"bibr\">Perlman and Netland, 2009</xref>). Interferon (IFN) type I activate a potent innate immune response against viral infection and also induce effective adaptive immune response. This recognition initiate a complex signaling cascade by recruiting adaptor proteins like mitochondrial antiviral-signaling protein (MAVS), IFN-β (TRIF), and stimulator of interferon genes protein (STING) and activate downstream cascades molecules, like adaptor molecule MyD88. This interaction activates transcription factors like nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) and helps in nuclear translocation. In the nuclei, these transcription factors induce the production of type I Interferons (IFN-α/β) and a plethora of pro-inflammatory cytokines especially IL-6 (<xref rid=\"B27\" ref-type=\"bibr\">Li et al., 2020a</xref>). Thus, interactions between the host cell and virus fabricate an assorted set of first line defense against the virus at the entry site. Type I IFN mediated activation of JAK-STAT pathway; initiate the transcription of IFN-stimulated genes (ISGs) under the control of the IFN-stimulated response element (ISRE). Accumulation of type I IFN can suppress viral replication and act as an immune modulator to promote phagocytosis of antigens by macrophage, as well as NK cells mediated restriction of infected cells. Thus, blocking the production of IFNs or disorder of the JAK-STAT signaling pathway or altered expression of macrophages has a direct effect on the survival of the virus within the host cell (<xref rid=\"B62\" ref-type=\"bibr\">Zhou Z. et al., 2020</xref>).</p><p>Generally, Th1 mediated immune response plays a predominant role in adaptive immunity against viral infections. T cell responses majorly depend upon the presence of APC (antigen presenting cells) mediated cytokine microenvironment. CD8<sup>+</sup> cytotoxic T cells (CTLs) which are capable of secreting a cluster of molecules such as, granzymes, perforin, and IFN-γ are essential in the eradication of virus infected cells. CD4<sup>+</sup> Helper T cells facilitate the overall adaptive response by assisting cytotoxic T cells. On the other hand, B-cell mediated humoral immune response, plays a protective role by producing the neutralizing antibody, and also impedes re-infection. According to some recent findings, in COVID-19 patients, an elevated level of chemokines and plasma cytokines like interleukins (IL-1, IL-2, IL-4, IL-7, IL-10, IL-12, IL-13, and IL-17), IP-10, macrophage colony-stimulating factor (MCSF), MCP-1, GCSF, hepatocyte growth factor (HGF), IFN-γ, MIP-1α, and TNF-α, etc., are associated with disease severity (<xref rid=\"B31\" ref-type=\"bibr\">Li et al., 2020e</xref>). Like in SARS and MERS the presence of “lymphopenia” and “<italic>cytokine storm</italic>” may have a significant role in the pathogenesis of COVID-19 (<xref rid=\"B37\" ref-type=\"bibr\">Moore and June, 2020</xref>). Moreover, like in cancer and other chronic infections, persistence of <italic>cytokine storm</italic> might stimulate necrosis or apoptosis of T cells, and leads to their exhaustion (<xref rid=\"B3\" ref-type=\"bibr\">Catanzaro et al., 2020</xref>). This “<italic>cytokine storm</italic>” is responsible for commencing of viral sepsis followed by lung injury induced by inflammation- which is related to other complications like acute respiratory distress syndrome (ARDS), pneumonitis, respiratory failure, sepsis shock, organ failure, and potentially death. Severity of COVID-19 in patients is associated with the marked decrease in the number of circulating B cells, CD8 + cells, CD4 + cells, natural killers (NK) cells, as well as a decrease in eosinophils, monocytes, and basophils (<xref rid=\"B62\" ref-type=\"bibr\">Zhou Z. et al., 2020</xref>; <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Schematic representation of immunopathogenesis of SARS-CoV-2 infection.</p></caption><graphic xlink:href=\"fmolb-07-00196-g001\"/></fig></sec></sec><sec id=\"S3\"><title>Molecular Docking, Prediction of New Drugs and Drug Repurposing</title><p>Novel drug development is a time consuming process that depends upon costly laboratory as well as clinical trials, possibilities of mistakes, and other conditions. So, now a day’s various bioinformatics tools like simulation, molecular docking, chemical stability studies, and target point determination playing an imperative and inventive approach in the designing of new drugs (<xref rid=\"B8\" ref-type=\"bibr\">de Ruyck et al., 2016</xref>). Among them “molecular docking” of any specific place provide a clearer idea about designing new drugs, examining and comparing their efficacy in any particular disease. In the case of “pandemic” caused by SARS-CoV-2 where there is no specific medicine or vaccine available, “drug repositioning” or “drug repurposing” plays an attractive role (<xref rid=\"B41\" ref-type=\"bibr\">Sheahan et al., 2020</xref>). However, such drugs require a clinical trial to show their effectiveness against the disease. In a recent study, Neda Shaghaghi used the crystal structure of SARS-CoV-2 proteinase and herbal medicines for docking analysis. According to the study terpenoids from the herbal medicines be able to impede the enzymatic cavity of important amino acids and inhibit the viral protease (<xref rid=\"B40\" ref-type=\"bibr\">Shaghaghi, 2020</xref>). Another study was done in virtual high throughput screening of clinically approved drugs and the structure of SARS-CoV-2 Mpro revealed that Saquinavir and Beclabuvir, Lopinavir, Ritonavir, and Nelfinavir act as the potential candidates for COVID-19 therapy (<xref rid=\"B41\" ref-type=\"bibr\">Sheahan et al., 2020</xref>). Molecular docking study is proved to be an economic method where technology-based ligand-protein interaction for a particularly active site reveals their possibilities as therapeutic candidates before synthesizing them. Various <italic>in silico</italic> studies based on molecular docking has proved US-FDA approved drugs like chloroquine, hydroxychloroquine, remdesivir, and arbidol as potential inhibitors of various viral polymerases and proteases and also known to be the most suitable targets for the SARS-CoV-2 treatment (<xref rid=\"B53\" ref-type=\"bibr\">Wang M. et al., 2020</xref>). Another interesting study on the SARS-CoV-2 spike glycoprotein and membrane CD26 docked complex model reveals a great interface among the proteins. This interaction between the loops of the S1 domain and the CD26 surface establishes CD26 as a potential binding site for S-protein along with ACE2 (<xref rid=\"B48\" ref-type=\"bibr\">Vankadari and Wilce, 2020</xref>). Studies were done on secondary metabolites of various Indian medicinal plants like garlic, curcumin, cardamom, ashwagandha, neem, aloe vera, harsingar, etc., have shown effective inhibition properties against SARS-CoV-2 protease enzyme (<xref rid=\"B44\" ref-type=\"bibr\">Srivastava et al., 2020</xref>). These compounds have an effective affinity toward the key amino acids active site and able to inhibit them during the catalytic process. Although the results obtained from drug repositioning or secondary metabolites of herbal plants by molecular docking provide information regarding suitable drugs candidate for COVID-19 therapy but thorough <italic>in vitro</italic> studies and <italic>in vivo</italic> studies can prove their effectiveness against SARS-CoV-2.</p></sec><sec id=\"S4\"><title>Therapeutic Approaches Against COVID-19</title><p>Providing supportive care to the patients is the best strategy in current situations, as so far no established antiviral treatment is to be useful in controlling an outbreak of novel COVID-19 (<xref rid=\"B14\" ref-type=\"bibr\">Guo et al., 2020</xref>). So far numerous compounds have been checked in animal models or in cell culture to establish them as a potential therapeutic approach against entry or replication of SARS-CoV or MERS-CoV or SARS-CoV-2, but experiments done in animal or <italic>in vitro</italic> does not inevitably translate into efficacy in humans (<xref rid=\"B26\" ref-type=\"bibr\">Li and De Clercq, 2020</xref>). Based on the structural as well as the pathogenesis of COVID-19, continuous extensive investigations are in progress to determine the effective therapeutic agents for SARS-CoV-2 infections. Studies converge on drug discovery against emerging COVID-19 outbreaks can be broadly classified into two different modes of treatment: (A) virus-based therapy and (B) host-based treatment options.</p><sec id=\"S4.SS1\"><title>(A) Virus-Based Therapy</title><p>(i) Viral nucleic acids are made up of nucleosides and nucleotides. Drugs that are capable to target nucleotides or nucleosides and/or viral nucleic acids have a wide-range of activity against CoVs and other viruses. Studies have shown that various nucleoside analog, like Ribavirin, favipiravir, and galidesivir have antiviral activity against some animal CoVs, and in the SARS-CoV epidemic (<xref rid=\"B14\" ref-type=\"bibr\">Guo et al., 2020</xref>). (ii) Major enzymes and proteins involved in viral replication of COVID-19 are potential targets for anti-viral treatment. The PLpro enzymes and papain-like protease of SARS-CoV and MERS-CoV exhibit proteolytic, de-ubiquitylating as well as deISGylating activities. Studies have proven that among various protease inhibitors, lopinavir-ritonavir acts as the most effective one (<xref rid=\"B14\" ref-type=\"bibr\">Guo et al., 2020</xref>). (iii) The key immunogenic antigen of SARS-CoV-2 is membrane-anchored Spike glycoprotein, which plays an essential role in the host cell and virus interaction. Studies have shown that some monoclonal antibodies (mAbs) can target definite epitopes on the RBD of S1 subunit and restrain virus-cell receptor binding; on the other hand, others attach with the S2 subunit and disrupt virus-cell fusion (<xref rid=\"B24\" ref-type=\"bibr\">Jiang et al., 2014</xref>). Studies also suggest that mAbs exhibit neutralizing activities and also able to reduce viral titers <italic>in vitro</italic> as well as in small animal models. As S protein of SARS-CoV-2 displayed high homology to that of SARS-CoV, CR3022 the neutralizing antibody of SARS-CoV was found to bind potently with the RBD domain of SARS-CoV-2 (<xref rid=\"B46\" ref-type=\"bibr\">Tian et al., 2020</xref>). Previous studies also suggest that adoptive transfers of plasma which contain anti-MERS-CoV-S antibodies can block virus attachment and also accelerate viral clearance. Depending upon this idea, scientists are trying to develop convalescent plasma-based therapy against pandemic SARS-CoV-2 infection (<xref rid=\"B29\" ref-type=\"bibr\">Li et al., 2020c</xref>). Studies also imply antiviral peptides as a proposed analog for viral spike protein. Analogous peptides for the pre-transmembrane domain, N terminus, or the loop region which separates the HR1 and HR2 domains of SARS-CoV are competent to inhibit the formation of virus plaque (<xref rid=\"B45\" ref-type=\"bibr\">Tang et al., 2014</xref>). Some studies also prove that some siRNAs have antiviral activities <italic>in vitro</italic> but still they are in preclinical development (<xref rid=\"B55\" ref-type=\"bibr\">Wu et al., 2005</xref>). (iv) Along with E, M, and N proteins some accessory proteins are essential for virion assembly and viral replication by suppressing the host immune response. Anti-viral therapy against such proteins might have effective role in COVID-19 infection (<xref rid=\"B49\" ref-type=\"bibr\">Vigant et al., 2013</xref>). Studies have proved that LJ001 and JL103 (lipophilic thiazolidine derivatives) act as membrane-binding photosensitizers. They are proficient in the production of singlet oxygen molecules which stimulate various changes in lipid membranes results in the prevention of fusion between viral and host cell membranes (<xref rid=\"B64\" ref-type=\"bibr\">Zumla et al., 2016</xref>).</p></sec><sec id=\"S4.SS2\"><title>(B) Host-Based Therapy</title><p>Host-directed therapies are related to the improvement of host immune response, host status, and/or handling of host-related factors coupled with viral replication. (i) Studies have shown that innate interferon response by host cells is important for viral replication within the host cell. Though CoVs are capable to restrain the interferon response to imply immune evasion, <italic>in vitro</italic> studies have revealed that treatment with various recombinant interferons might incline the infection severity (<xref rid=\"B35\" ref-type=\"bibr\">Menachery et al., 2014</xref>). (ii) To suppress dysfunctional systematic inflammation, Corticosteroids have excellent pharmacological effects. These are one of the major imunomodulators which were widely used in the treatment of SERS-CoV and MERS-CoV and are also considered as a helpful agent in the management of the current epidemic of SARS-CoV-2 (<xref rid=\"B28\" ref-type=\"bibr\">Li et al., 2020b</xref>). Another school of study suggests that combined use of both inducers of interferon along with innate immunomodulators might be efficient as antiviral agents against SERS-CoV-2. (iii) Apart from immunomodulators, other substances like metformin, atorvastatin, fibrates, as well as nutritional supplements might play the most important role in treating the current pandemic by boosting immunity (<xref rid=\"B64\" ref-type=\"bibr\">Zumla et al., 2016</xref>). (iv) CoVs generally utilize specific host factors like host receptors or other enzymes and proteins for viral entry as well as viral replication. Thus specific monoclonal or polyclonal antibodies, peptides, or functional inhibitors against host receptors or injecting an excessive liquid form of ACE2 receptor or recombinant technology must use to stop host membrane-viral fusion (<xref rid=\"B20\" ref-type=\"bibr\">Huentelman et al., 2004</xref>). (v) A recent study by <xref rid=\"B17\" ref-type=\"bibr\">Hoffmann et al. (2020)</xref> demonstrated that SARS-CoV-2 spike protein priming depends upon transmembrane protease serine 2 (TMPRSS2) for viral entry. Further studies also revealed that the serine protease inhibitor camostat mesylate, can block TMPRSS2 activity and considered as an attractive candidate for therapy (<xref rid=\"B17\" ref-type=\"bibr\">Hoffmann et al., 2020</xref>). (vi) Another study suggests that the entry of coronavirus into the host cell involved pH- and receptor-dependent endocytosis. A host kinase, AP-2-associated protein kinase 1 (AAK1) regulates clathrin-mediated endocytosis. Molecular docking studies reveal that the Janus kinase inhibitor baricitinib is a potent AAK1-inhibiting drug, is expected to be a suitable drug candidate for COVID-19 (<xref rid=\"B39\" ref-type=\"bibr\">Richardson et al., 2020</xref>; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Possible therapeutic approaches against SARS-CoV-2 infection.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Antiviral agent</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Drug target</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mechanism of action</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Infectious disease</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">References</td></tr></thead><tbody><tr><td valign=\"top\" align=\"justify\" colspan=\"5\" rowspan=\"1\"><bold>(a) Virus based treatment strategy</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ribavirin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RdRp</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits viral RNA synthesis and mRNA capping</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV-2, MERS-CoV, SARS-CoV, RSV, HCV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B14\" ref-type=\"bibr\">Guo et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Favipiravir</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RdRp</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits RdRp</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV-2, Influenza</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B14\" ref-type=\"bibr\">Guo et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Galidesivir</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RdRp</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits viral RNA polymerase function by terminating non-obligate RNA chain</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV, MERS-CoV, IAV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B14\" ref-type=\"bibr\">Guo et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Remdesivir</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RdRp</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Terminates the non-obligate chain</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV-2, MERS-CoV, SARS-CoV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B14\" ref-type=\"bibr\">Guo et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">siRNA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RdRp</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Short chains of dsRNA that interfere</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV, MERS-CoV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B55\" ref-type=\"bibr\">Wu et al., 2005</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lopinavir</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3CLpro</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits 3CLpro</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV-2, MERS-CoV, SARS-CoV, HCoV-229E, HIV, HPV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B14\" ref-type=\"bibr\">Guo et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ritonavir</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3CLpro</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits 3CLpro</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV-2, MERS-CoV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B14\" ref-type=\"bibr\">Guo et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Darunavir and cobicistat</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3CLpro</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits 3CLpro</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV-2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B46\" ref-type=\"bibr\">Tian et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CR3022</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Spike glycoprotein</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">immunogenic antigen against Spike protein</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV-2, SARS-CoV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B46\" ref-type=\"bibr\">Tian et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nafamostat</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Spike glycoprotein</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits spike-mediated membrane fusion A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV-2, MERS-CoV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B34\" ref-type=\"bibr\">Manli et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Griffithsin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Spike glycoprotein</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Griffithsin binds to the SARSCoV spike glycoprotein, thus inhibiting viral entry</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B34\" ref-type=\"bibr\">Manli et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Peptide (P9)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Spike glycoprotein</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits spike protein-mediated cell-cell entry or fusion</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Broad-spectrum (SARS-CoV, MERS-CoV, influenza)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B60\" ref-type=\"bibr\">Zhao et al., 2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">LJ001 and JL103</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lipid membrane</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Membrane-binding photosensitizers that induce singlet oxygen modifications of specific phospholipids</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Enveloped viruses (IAV, filoviruses, poxviruses, arenaviruses, bunyaviruses, paramyxoviruses, flaviviruses and HIV-1)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B64\" ref-type=\"bibr\">Zumla et al., 2016</xref></td></tr><tr><td valign=\"top\" align=\"justify\" colspan=\"5\" rowspan=\"1\"><bold>(b) Host-based treatment strategies</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recombinant interferons</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interferon response</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exogenous interferons</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV-2, SARS-CoV, MERS-CoV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B35\" ref-type=\"bibr\">Menachery et al., 2014</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nitazoxanide</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interferon response</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Induces the host innate immune response to produce interferons by the host’s fibroblasts and protein kinase R (PKR) activation</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Coronaviruses, SARS-CoV-2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B34\" ref-type=\"bibr\">Manli et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chloroquine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Endosomal acidification</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">A lysosomatropic base that appears to disrupt intracellular trafficking and viral fusion events</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV-2, SARS-CoV, MERS-CoV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B34\" ref-type=\"bibr\">Manli et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Corticosteroids</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pulsed methylprednisolone</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Patients with severe MERS who were treated with systemic corticosteroid with or without antivirals and interferons had no favorable response</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV, MERS-CoV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B28\" ref-type=\"bibr\">Li et al., 2020b</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Camostat Mesylate</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surface protease</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TMPRSS2 inhibitor that blocks the TMPRSS2-mediated cell surface entry pathway</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV, MERS-CoV, HCoV-229E</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B17\" ref-type=\"bibr\">Hoffmann et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Baricitinib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Clathrin-mediated endocytosis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AP-2-associated protein kinase 1 (AAK1) regulates clathrin-mediated endocytosis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV, SARS-CoV-2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B39\" ref-type=\"bibr\">Richardson et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Convalescent plasma</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits virus entry to the target cells</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SARS-CoV, SARS-CoV-2, Influenza</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B29\" ref-type=\"bibr\">Li et al., 2020c</xref></td></tr></tbody></table><table-wrap-foot><attrib><italic>RdRp, RNA-dependent RNA polymerase; 3CLpro, 3C-like protease; IAV, influenza A virus; HCV, hepatitis C virus; RSV, respiratory syncytial virus; SARS-CoV, severe acute respiratory syndrome coronavirus; HCoV, human coronavirus; MERSCoV, Middle East respiratory syndrome coronavirus; and RBD, receptor-binding domain.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S4.SS3\"><title>Glycosylation: A New Age Targeted Therapeutic Approach</title><p>Carbohydrate-binding agents are considered as anti-CoV agents that target spike protein and restrain CoV entry. They are capable to bind specifically with the oligosaccharides present on the viral surfaces such as S and HIV glycoprotein (<xref rid=\"B11\" ref-type=\"bibr\">Garcia-Laorden et al., 2008</xref>). In <italic>in vitro</italic> condition as well as in mouse model they inhibit a wide range of CoVs, including HCoV-229E, SARS-CoV, HCoV-NL63, and HCoV-OC43. Glycans have a wide variety of shape, mass, charge, or other physical properties which help them to mediate extensive biological roles. Natural proteins called Lectins target the sugar moieties of a wide variety of glycoproteins which is not involved in cell adhesion. Most lectins belong to glycan families with distinct “carbohydrate-recognition domains” (CRDs) that conserve specific primary amino acid sequences or 3D structure and evolved from shared ancestral genes. In innate immunity, MBL acts as a key pattern-recognition molecule. It mainly functions as an ante-antibody, a humoral factor, crucial for the first-line host defense prior to the production of antibodies. Studies have shown that expression of MBL is associated with the initiation of complement activation via the lectin pathway and also responsible for opsonophagocytosis (<xref rid=\"B9\" ref-type=\"bibr\">Eisen, 2010</xref>). A study by <xref rid=\"B15\" ref-type=\"bibr\">Hartshorn et al. (1993)</xref> demonstrates that MBL function as an opsonin and inhibit hemagglutination as well as infectivity against respiratory viruses, such as influenza A virus. Binding of MBL with the N-linked high-mannose carbohydrate side chain present at the tip of S hemagglutinin protein is able to neutralize the infectivity of the influenza A virus. Studies also revealed that SARS-CoV S-protein contains 23 potential N-linked glycosylation sites. Binding of MBL with the S protein mannose side chains of SARS-CoV can be used as the most useful remedial agent (<xref rid=\"B21\" ref-type=\"bibr\">Ip et al., 2005</xref>). Thus, glycosylation of viral peptides might be considered as a novel therapeutic strategy against current COVID-19 pandemic (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Potential Role of MBL in prevention of SARS-CoV-2. (1) Attachment of MBL at the glycosylation site of Spike Protein by “Lock and Key” mode. (2) Prevent ACE2 mediated entry of viral pathogen. (3) Lectin pathway-mediated phagocytosis of intracellular pathogens.</p></caption><graphic xlink:href=\"fmolb-07-00196-g002\"/></fig></sec></sec><sec id=\"S5\"><title>Discussion</title><p>Since, across the world, COVID-19 infection causes severe public health concern, analysis of the characteristics features of SARS-CoV-2, its interaction with the host receptor and immune responses, the phylogenetic and genomic similarity with other viruses will provide a clearer picture of diseases onset in individuals. Several groups of scientists have postulated that just like SARS-CoV, SARS-CoV-2 also depends upon the ACE2 as a receptor for host cell entry. The interaction of the virus transmembrane spike (S) glycoprotein with host cell receptors act as the determinant of the pathogenesis. A higher degree of disease severity is associated with viral load and age and sex of the patients. In elderly patients, the high viral load is associated with low immunity as well as higher expression of the ACE2 receptor in some hematopoietic cells, in cardiopulmonary tissues, and macrophages and monocytes. At the same time, male patients are more susceptible to the SARS-CoV-2 infection than their female counterparts. “Lymphopenia” (low blood lymphocyte count) is correlated with the clinical severity of COVID-19 related infection. However, lymphopenia can be considered as a biomarker of poor prognosis of COVID-19 which was also correlated with casualty in the influenza A (H1N1) pandemic in the year 2009. The study also, suggests that in one or more host species SARS-CoV-2 attached with integrins as cell receptors, through a conserved sequence RGD (403–405:Arg-Gly-Asp) with the RBD domain of the spike proteins. Pharmacotherapy against integrin can control the association between virus and integrin which is necessary to neutralize pathogenesis.</p><p>The development of SARS-CoV-2 infection majorly depends upon the interaction between the individual’s immune system and the virus. On one hand viral factors like virus type, mutation potential, virus viability along with the immune system factors of an individual like age, gender, nutritional status, genetics, neuroendocrine-immune regulation, etc., contribute to the severity of the disease. An effective immune response majorly depends upon a crucial part, called inflammation. Proper elimination of any infections majorly depends upon inflammation. Initial recognition of pathogens is the primary step for the onset of inflammatory response followed by the recruitment of immune cells. These immune cells help in the elimination of the pathogens and eventually lead to tissue repair and restoration of homeostasis. Though, in some infected individuals, SARS-CoV-2 induces extreme and prolonged cytokine/chemokine responses, called “<italic>cytokine storm.</italic>” This <italic>cytokine storm</italic> is related to multiple organ dysfunction or ARDS and later leads to physiological deterioration and death. An elevated level of serum cytokine IL-6 and C-reactive protein considered as the biomarker of severe β-coronavirus infection. Upon infection with β-coronavirus monocytes, macrophages, and dendritic cells get activated and start the secretion of prominent pro-inflammatory cytokine, like IL-6 along with other inflammatory cytokines. IL-6 can activate either classic <italic>cis</italic>-signaling or <italic>trans</italic>-signaling. Various pleiotropic effects on the acquired immune system (T and B cells) as well as on innate immune system [macrophages, neutrophils, and natural killer (NK) cells], are correlated with activation of <italic>cis</italic>-signaling and leads to cytokine release syndrome (CRS). On the other hand upon activation of <italic>trans</italic>-signaling, elevated IL-6 level creates the “<italic>cytokine storm</italic>” which is related with the secretion of monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), IL-8, and also excessive IL-6. It is also associated with reduced expression of adhesion molecule E-cadherin on endothelial cells. Reduced expression of E-cadherin and secreted VEGF mainly contribute to vascular permeability as well as leakage, which in turn coupled with the pathophysiology of hypotension and pulmonary dysfunction in acute respiratory distress syndrome (ARDS). An antagonist of IL-6 is tocilizumab, previously approved to treat juvenile idiopathic arthritis which is a rheumatic condition, is again “repurposed” for the COVID-19 pandemic. This finding suggests that we can potentially use IL-6 directed therapies not only in COVID-19 but also in other pandemics in the future involving Ebola and influenza viruses.</p><p>In this review, we summaries the different aspect of the therapeutic potential of the various anti-viral derivative. Finally, most reasonable options must be evaluated further in clinical trials against the COVID-19 pandemic. It might consist of either mono-therapy or combinational therapies comprise of interferon beta-1b, lopinavir-ritonavir, and/or mAbs and antiviral peptides. Thorough analyses of glycans are essential for the expansion of glycoprotein-based vaccine which might approach to correlate the immunogenicity with structural variations. In different expression systems, glycosylation act as a measure to evaluate antigen quality. Basic understanding correlated with RBD domain of the spike protein of SARS-CoV-2 consist of complex sialylated N-glycans and sialylated mucin type O-glycans will be useful to design suitable immunogens for vaccine development. MBL is a serum C-type lectin, which can bind SARS-CoV <italic>per se</italic> or infected cell and also capable to inhibit the infectivity of the virus. Studies have shown that “MBL-deficient” individuals are at more risk to SARS infection. We support, MBL as a potent therapeutic and prophylactic strategy in the prevention of SARS-CoV-2 pandemics. Shortly, it will be possible to develop broad-spectrum, novel, antiviral drugs active against a larger array of coronavirus, and also will be the ultimate treatment strategy for circulating and emerging COVID infections.</p></sec><sec id=\"S6\"><title>Author Contributions</title><p>SC contributed to design, editing, and approval of final version of the manuscript. SS drafted and prepared the manuscript, and drew the figures. MM contributed to read and editing the draft of the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>SC and SS were employed by the company Molecular Pharma Pvt., Ltd., Kolkata, India. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Endocrinol.</journal-id><journal-title-group><journal-title>Frontiers in Endocrinology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2392</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849298</article-id><article-id pub-id-type=\"pmc\">PMC7431666</article-id><article-id pub-id-type=\"doi\">10.3389/fendo.2020.00513</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Short-Term Diet Induced Changes in the Central and Circulating IGF Systems Are Sex Specific</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Guerra-Cantera</surname><given-names>Santiago</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/939036/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Frago</surname><given-names>Laura M.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/37366/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Díaz</surname><given-names>Francisca</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/506384/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ros</surname><given-names>Purificacion</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Jiménez-Hernaiz</surname><given-names>Maria</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Freire-Regatillo</surname><given-names>Alejandra</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/156449/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Barrios</surname><given-names>Vicente</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/72437/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Argente</surname><given-names>Jesús</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/36306/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Chowen</surname><given-names>Julie A.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/14136/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Endocrinology, Hospital Infantil Universitario Niño Jesús, Instituto de Investigación La Princesa</institution>, <addr-line>Madrid</addr-line>, <country>Spain</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Pediatrics, Universidad Autónoma de Madrid</institution>, <addr-line>Madrid</addr-line>, <country>Spain</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III</institution>, <addr-line>Madrid</addr-line>, <country>Spain</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Hospital Universitario Puerta de Hierro-Majadahonda</institution>, <addr-line>Madrid</addr-line>, <country>Spain</country></aff><aff id=\"aff5\"><sup>5</sup><institution>IMDEA Food Institute, CEI UAM + CSIC</institution>, <addr-line>Madrid</addr-line>, <country>Spain</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Claire Joanne Stocker, University of Buckingham, United Kingdom</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Nu-Chu Liang, University of Illinois at Urbana-Champaign, United States; David Maridas, Harvard School of Dental Medicine, United States; Deborah Burks, Principe Felipe Research Center (CIPF), Spain</p></fn><corresp id=\"c001\">*Correspondence: Jesús Argente <email>jesus.argente@uam.es</email></corresp><corresp id=\"c002\">Julie A. Chowen <email>julieann.chowen@salud.madrid.org</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Obesity, a section of the journal Frontiers in Endocrinology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>513</elocation-id><history><date date-type=\"received\"><day>26</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>25</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Guerra-Cantera, Frago, Díaz, Ros, Jiménez-Hernaiz, Freire-Regatillo, Barrios, Argente and Chowen.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Guerra-Cantera, Frago, Díaz, Ros, Jiménez-Hernaiz, Freire-Regatillo, Barrios, Argente and Chowen</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Insulin-like growth factor (IGF) 1 exerts a wide range of functions in mammalians participating not only in the control of growth and metabolism, but also in other actions such as neuroprotection. Nutritional status modifies the IGF system, although little is known regarding how diet affects the newest members of this system including pregnancy-associated plasma protein-A (PAPP-A) and PAPP-A2, proteases that liberate IGF from the IGF-binding proteins (IGFBPs), and stanniocalcins (STCs) that inhibit PAPP-A and PAPP-A2 activity. Here we explored if a 1-week dietary change to either a high-fat diet (HFD) or a low-fat diet (LFD) modifies the central and peripheral IGF systems in both male and female Wistar rats. The circulating IGF system showed sex differences in most of its members at baseline. Males had higher levels of both free (<italic>p</italic> < 0.001) and total IGF1 (<italic>p</italic> < 0.001), as well as IGFBP3 (<italic>p</italic> < 0.001), IGFBP5 (<italic>p</italic> < 0.001), and insulin (<italic>p</italic> < 0.01). In contrast, females had higher serum levels of PAPP-A2 (<italic>p</italic> < 0.05) and IGFBP2 (<italic>p</italic> < 0.001). The responses to a short-term dietary change were both diet and sex specific. Circulating levels of IGF2 increased in response to LFD intake in females (<italic>p</italic> < 0.001) and decreased in response to HFD intake in males (<italic>p</italic> < 0.001). In females, LFD intake also decreased circulating IGFBP2 levels (<italic>p</italic> < 0.001). In the hypothalamus LFD intake increased IGF2 (<italic>p</italic> < 0.01) and IGFBP2 mRNA (<italic>p</italic> < 0.001) levels, as well as the expression of NPY (<italic>p</italic> < 0.001) and AgRP (<italic>p</italic> < 0.01), but only in males. In conclusion, short-term LFD intake induced more changes in the peripheral and central IGF system than did short-term HFD intake. Moreover, these changes were sex-specific, with IGF2 and IGFBP2 being more highly affected than the other members of the IGF system. One of the main differences between the commercial LFD employed and the HFD or normal rodent chow is that the LFD has a significantly higher sucrose content, suggesting that this nutrient could be involved in the observed responses.</p></abstract><kwd-group><kwd>obesity</kwd><kwd>IGFs</kwd><kwd>IGFBP2</kwd><kwd>PAPP-A</kwd><kwd>stanniocalcins</kwd><kwd>high fat diet</kwd><kwd>hypothalamus</kwd><kwd>sex differences</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Ministerio de Economía, Industria y Competitividad, Gobierno de España<named-content content-type=\"fundref-id\">10.13039/501100010198</named-content></funding-source></award-group><award-group><funding-source id=\"cn002\">Instituto de Salud Carlos III<named-content content-type=\"fundref-id\">10.13039/501100004587</named-content></funding-source><award-id rid=\"cn002\">CIBEROBN</award-id><award-id rid=\"cn002\">PI1900166</award-id></award-group><award-group><funding-source id=\"cn003\">Ministerio de Educación, Cultura y Deporte<named-content content-type=\"fundref-id\">10.13039/501100003176</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"3\"/><table-count count=\"6\"/><equation-count count=\"0\"/><ref-count count=\"86\"/><page-count count=\"12\"/><word-count count=\"9503\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Insulin-like growth factor (IGF) 1 is involved in a wide range of functions (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B2\" ref-type=\"bibr\">2</xref>) including promotion of systemic growth through actions exerted directly on bone (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>), anabolic effects promoting protein synthesis and glucose uptake in muscle (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>) and stimulation of lipogenesis (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Because of their structural similarity, IGF1 shares metabolic functions with insulin (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>) and elevated levels of this growth factor reduce glycemia (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). In the brain IGF1 is involved in numerous functions including glucose metabolism (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>), neural development (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>), neural activity (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>), synaptogenesis (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>), adult neurogenesis (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>), cognition (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>), and amyloid clearance (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). It also exerts beneficial effects against inflammation (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>) and neurodegeneration (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>).</p><p>The main source of circulating IGF1 is the liver. However, there is also local production in most tissues including the brain, which is largely due to production by astrocytes and microglia (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>). The two ligands of the IGF system, IGF1 and IGF2, are secreted and bound to one of six different IGF-binding proteins (IGFBPs), thus modifying their biological activity. In addition to binding IGF1 or IGF2, IGFBP3, and IGFBP5 bind the acid labile subunit (ALS) to form a trimolecular complex of 150 KDa, which increases the half-life of the ligand (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). In the proximity of the target cell, proteases such as the metalloproteases pregnancy-associated plasma protein-A (PAPP-A) and PAPP-A2, cleave the junction between IGF1 or IGF2 with the IGFBP, allowing the free ligand to bind its receptor (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). Stanniocalcins (STCs) are a third level of regulation of this system, acting as endogenous inhibitors of the activity of both PAPP-A and PAPP-A2 and consequently reducing the release of both IGF1 and IGF2 (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B20\" ref-type=\"bibr\">20</xref>).</p><p>Nutritional status modifies circulating levels of IGF1, as well as of other members of the IGF system (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>–<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). High fat diet (HFD)-induced obesity has been shown to increase the expression of IGF2 in adipose tissue (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>) and to inhibit the effects of IGF1 in chondrocytes (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>), while IGF1 stimulates adipose tissue proliferation (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Moreover, IGFBP2 is reported to participate in glucose metabolism and to be a target of leptin (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). The more recently described members of this family are also involved in metabolism as, for example, adult female PAPP-A knockout mice have been shown to be resistant to high fat/high sugar intake (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). High fat diet-induced and leptin-deficient obesity is associated with reduced STC2 synthesis in liver, with STC2 administration attenuating hyperlipidemia and steatosis (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). Recently, circulating levels of PAPP-A and PAPP-A2, as well as STC-2, were reported to be unchanged in response to metreleptin treatment in adult men and women (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Thus, more information is required regarding the metabolic implications of the IGF system including the pappalysins and stanniocalcins.</p><p>Central IGF1 also modulates the neuroendocrine control of metabolism (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>), but less is known regarding the participation of other members of this system at the central level in response to metabolic changes or to specific nutrients. Moreover, in recent years the important role of hypothalamic inflammation in obesity and its secondary complications has been obviated. As IGF1 exerts neuroprotective and anti-inflammatory effects (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>), obesity or nutrition-induced modifications in this system could be involved in hypothalamic inflammation and central metabolic control.</p><p>In this study we aimed to determine the possible changes in the central and circulating IGF systems in response to short-term high-fat diet (HFD) or low-fat diet (LFD) consumption. Low fat diets have been commercialized as controls for the HFD, but their use as such is questionable and thus, we also compared the metabolic response between chow and LFD.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Ethical Statement</title><p>All experiments were designed according to the European Communities Council Directive (2010/63/UE) and the Royal Decree 53/2013 pertaining to the protection of experimental animals. This study was also approved by the Ethical Committee of Animal Experimentation of the Hospital Puerta de Hierro de Madrid and the Animal Welfare Organ of the Comunidad Autónoma de Madrid.</p></sec><sec><title>Animals and Diets</title><p>Male and female postnatal day (PND) 50 Wistar rats were purchased from Charles River Laboratories and acclimated to the new environment for 13 days before dietary challenge. They were then randomly distributed between the three experimental groups for each sex (<italic>n</italic> = 6/group). Starting at PND 63, animals were fed <italic>ad libitum</italic> with either a HFD (62% kcal from fat, 18% kcal from proteins, 20% kcal from carbohydrates, 5.1 kcal/g, LabDiet), a LFD (10% kcal from fat, 18% kcal from proteins, 72% kcal from carbohydrates, 3.76 kcal/g, LabDiet) or standard rodent chow (6% kcal from fat, 17% kcal from proteins, 77% from carbohydrates, 3.41 kcal/g, Panlab) for 1 week (more information regarding the diets is shown in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). The rats were given free access to tap water. Body weight was measured every 3 days until sacrifice on PND70. Food intake was determined throughout the study. Total kcals and the amount of energy contributed by fat were calculated. Energy efficiency was calculated as total weight gain (g) divided by total energy intake (kcal) during the week of study.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Composition of the diets employed; normal rat chow, low fat diet (LFD), high fat diet (HFD).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Chow</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>LFD</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>HFD</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"4\" style=\"background-color:#bbbdc0\" rowspan=\"1\"><bold>ENERGY PROVIDED BY (kcal/g)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Protein</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18.1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Fat</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">61.6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Carbohydrates</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">76.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">71.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20.3</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"4\" style=\"background-color:#bbbdc0\" rowspan=\"1\"><bold>INGREDIENTS (%)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Protein</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Fat</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">34.9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lard</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.66</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cholesterol (ppm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">301</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Omega-3 FA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.39</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Saturated FA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.52</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13.68</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Monounsaturated FA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.53</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polyunsaturated FA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.59</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Carbohydrates</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">65.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">67.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25.9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sucrose</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.94</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.85</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Starch/maltodextrin/dextrin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">34.16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.15</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Fiber</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.5</td></tr></tbody></table></table-wrap></sec><sec><title>Tissues and Sacrifices</title><p>Twelve hours before sacrifice, animals were weighed and then fasted. The animals were killed between 09:00 and 11:00 by decapitation. Trunk blood was collected and after clotting it was centrifuged at 3,000 rpm for 15 min. The serum was aliquoted and stored at −80°C avoiding freeze-thaw cycles. Peripheral glucose levels were measured by using a Freestyle Optimum Neo glucometer (Abbott, Witney, UK).</p><p>After decapitation, hypothalami, defined rostrally by the optic chiasm and caudally by the anterior margin of the mammillary bodies, were dissected and then frozen at −80°C. The inguinal adipose depot (subcutaneous adipose tissue; SCAT) and the perigonadal adipose depot (visceral adipose tissue; VAT) were dissected and weighed. The amount of each adipose tissue depot is expressed as percentage of body weight [weight (mg) relative to body weight (g)].</p></sec><sec><title>ELISA and Colorimetric Assays</title><p>Serum levels of free IGF1 (AnshLabs, Webster, Texas, USA), total IGF1 (Mediagnost, Reutlingen, Germany), IGF2 (BlueGene, Shangai, China), IGFBP2 (Mediagnost), IGFBP3 (Mediagnost), IGFBP5 (BlueGene), PAPP-A2 (BlueGene), insulin (Millipore, Burlington, Massachusetts, USA), and leptin (Millipore) were quantified by ELISA following the manufacturer's instructions. Non-esterified fatty-acids (NEFA) (Wako Diagnostics, Richmond, Virginia, USA) and triglycerides (Spin React, Girona, Spain) were measured by colorimetric assays as described by the manufacturers.</p></sec><sec><title>Protein and RNA Extraction</title><p>Protein and RNA extraction from hypothalami was performed by using an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. After tissue lysis and DNA elimination, 70% ethanol was mixed with the sample, and then placed in an RNeasy spin column and centrifuged. Protein was isolated from the same tissues by collecting the first elution from the RNeasy® Mini Spin columns. The eluted volume was mixed with 4 volumes of cold acetone and stored O/N at −20°C. Samples were centrifuged at 3,000 rpm at room temperature for 10 min and the acetone removed. The pellets were resuspended in a CHAPS hydrate (Sigma-Aldrich, Darmstadt, Germany) solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 0.5% 1 M Tris pH 8.8, in distilled water and stored at −80°C until used. The Bradford method was employed for protein quantification by using Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Hercules, California, USA).</p></sec><sec><title>Western Blotting</title><p>For Western blotting, 20–40 μg of protein, depending upon the target protein to be analyzed, were resolved in SDS-denaturing polyacrylamide gels. Proteins were transferred to previously activated PVDF membranes at 350 mA for 90 min.</p><p>Non-specific binding was blocked by incubating with 5% non-fat dried milk or bovine serum albumin (BSA, phosphorylated proteins) in TBS-T [Tris-buffered saline and 0.1% (v/v) Tween 20], which was also used for preparing the primary and secondary antibody solutions. The antibodies and their dilutions are shown in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>. Clarity Western ECL Substrate (Bio-Rad Laboratories) was employed to visualize the chemiluminiscent signal by ImageQuant Las 4000 Software (GE Healthcare Life Sciences, Barcelona, Spain). Each protein was normalized to actin levels, or total protein levels for phosphorylated proteins, in the same sample.</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Antibodies used for Western blotting.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Antibody</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Type</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Dilution</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Host</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Commercial source</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Reference</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Actin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Monoclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1:5,000</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mouse</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NeoMarkers</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1295-P1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AKT</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polyclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1:1,000</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Goat</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Santa Cruz</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">sc-1619</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ERK</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Monoclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1:1,000</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mouse</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Santa Cruz</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">sc-135900</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GAPDH</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polyclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1:4,000</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rabbit</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sigma-Aldrich</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN1\"><sup>#</sup></xref>G9545</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GFAP</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Monoclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1:3,000</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mouse</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sigma-Aldrich</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">G-3893</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Iba1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polyclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1:1,000</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rabbit</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Synaptic Systems</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">234003</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IRS1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polyclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1:500</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rabbit</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Millipore</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN1\"><sup>#</sup></xref>06-248</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JNK</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Monoclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/1,000</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mouse</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Santa Cruz</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">sc-1648</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pAKT (Ser 473)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polyclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1:1,000</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rabbit</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Promega</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">G7441</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pERK</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polyclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1:1,000</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rabbit</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cell Signaling</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN1\"><sup>#</sup></xref>9101</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PI3K p110β</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polyclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1:1,000</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rabbit</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Santa Cruz</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">sc-602</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pIRS1 (Ser 789)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polyclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1:750</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rabbit</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cell Signaling</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN1\"><sup>#</sup></xref>2389</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pJNK</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polyclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1:3,000</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rabbit</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Promega</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">V7932</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">α-goat HRP conjugated</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polyclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1:2,000</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rabbit</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Thermo Fisher</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN1\"><sup>#</sup></xref>31402</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">α-mouse HRP conjugated</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polyclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1:2,000</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Goat</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Invitrogen</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN1\"><sup>#</sup></xref>31430</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">α-rabbit HRP conjugated</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polyclonal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1:2,000</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Goat</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dako</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">P0448</td></tr></tbody></table><table-wrap-foot><p><italic>AKT, protein kinase B; ERK, extracellular signal-regulated kinases; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GFAP, Glial fibrillary acidic protein; Iba1, Ionized calcium binding adaptor molecule 1; IRS1, insulin receptor substrate 1; PI3K, phosphatidylinositol 3-kinases; JNK, c-Jun N-terminal kinases; HRP, horseradish peroxidase</italic>.</p></table-wrap-foot></table-wrap></sec><sec><title>Real Time qPCR</title><p>For RT-PCR, 0.5–1 μg of RNA was retro-transcribed to cDNA by using a High-capacity cDNA reverse transcriptase kit (Applied Biosystems, Carlsbad, California, USA) or NZY First-Strand cDNA Synthesis Kit (NZY Tech, Lisbon, Portugal). TaqMan probes (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>) were used for RT-PCR in an ABI PRISM 7000 or QuantStudio 3 Real-Time PCR System (both from Applied Biosystems). Phosphoglycerate kinase 1 (Pgk1) and 18S (Rps18) were used as endogenous housekeeping controls.</p><table-wrap id=\"T3\" position=\"float\"><label>Table 3</label><caption><p>List of Taqman probes used for RT-PCR.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Gene</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Reference</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Agouti-related peptide (<italic>Agrp</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn01431703_g1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cocaine- and amphetamine-regulated transcript (<italic>Cart</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn00567382_m1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insulin-like growth factor 1 (<italic>Igf1</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn99999087_m1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insulin-like growth factor 1 receptor (<italic>Igf1r</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn01477918_m1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insulin-like growth factor 2 (<italic>Igf2</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn01454518_m1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insulin-like growth factor 2 receptor (<italic>Igf2r</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn01636937_m1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insulin-like growth factor-binding protein 1 (<italic>Igfbp1</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn00565713_m1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insulin-like growth factor-binding protein 2 (<italic>Igfbp2</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn00565473_m1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insulin-like growth factor-binding protein 3 <italic>(Igfbp3</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn00561416_m1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insulin-like growth factor-binding protein 4 (<italic>Igfbp4</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn01464112_m1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insulin-like growth factor-binding protein 5 (<italic>Igfbp5</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn00563116_m1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neuropeptide Y (<italic>Npy</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn01410145_m1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pregnancy-associated plasma protein A (<italic>Pappa</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn01458295_m1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phosphoglycerate kinase 1 (<italic>Pgk1</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn00821429_g1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pro-opiomelanocortin (<italic>Pomc</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn00595020_m1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">18S <italic>(Rps18)</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn01428915_g1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Stanniocalcin 1 (<italic>Stc1</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn00579636_m1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Stanniocalcin 2 (<italic>Stc2</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rn00573702_m1</td></tr></tbody></table></table-wrap><p>For the mathematical analysis, the ΔΔCT method was employed with expression of the housekeeping gene used as the endogenous control. Relative levels of expression were determined by normalizing the results to levels in the male chow group.</p></sec><sec><title>Statistical Analysis</title><p>Data are presented as mean ± SEM. Statistics was performed using SPSS 15.0 software. Two-way ANOVA with Bonferroni as the <italic>post-hoc</italic> test was used in each case, with sex and diet used as factors. Pearson correlation coefficient was also calculated to assess the linear correlation between variables. Values of <italic>p</italic> < 0.05 were considered significant.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>Body Composition</title><p>There was an effect of sex [<italic>F</italic><sub>(1, 35)</sub> = 575.9, <italic>p</italic> < 0.001], with males weighing more than females regardless of diet. Short-term HFD intake induced body weight gain, but exclusively in males [<italic>F</italic><sub>(2, 17)</sub> = 4.9, <italic>p</italic> < 0.05; <xref rid=\"T4\" ref-type=\"table\">Table 4</xref>].</p><table-wrap id=\"T4\" position=\"float\"><label>Table 4</label><caption><p>Effects of 1 week on a high fat diet (HFD), low fat diet (LFD), or normal rat chow on body composition, glycemia, serum levels of insulin, Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), leptin, non-esterified fatty-acids (NEFA) and triglycerides, and energy intake in male and female rats.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Chow males</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>HFD males</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>LFD males</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Chow females</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>HFD females</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>LFD females</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Sig</bold>.</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Body weight (g)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">348.3 ± 12.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">359.5 ± 9.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">340.5 ± 2.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">186.7 ± 6.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">202.2 ± 6.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">199.0 ± 6.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">a, <italic>p</italic> < 0.001</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weight gain (% baseline)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.8 ± 0.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.5 ± 0.3<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.2 ± 0.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.2 ± 0.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.3 ± 1.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.7 ± 1.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">a, <italic>p</italic> < 0.01</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total kcal/rat</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">482.5 ± 31.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">621.4 ± 80.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">556.5 ± 10.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">328.5 ± 17.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">398.7 ± 27.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">394.8 ± 20.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">a, <italic>p</italic> < 0.001 b, <italic>p</italic> = 0.05</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Kcal/rat/day/100 g body weight</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19.4 ± 1.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.9 ± 2.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22.5 ± 0.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24.1 ± 0.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.1 ± 0.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.2 ± 0.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">a, <italic>p</italic> < 0.01 b, <italic>p</italic> < 0.05</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Kcal from fat (total)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.5 ± 1.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">378.4 ± 49.5<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">56.1 ± 0.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20.1 ± 0.9<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>@</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">256.2 ± 7.3<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">41.1 ± 1.8<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>#</sup></xref><sup>;</sup><xref ref-type=\"table-fn\" rid=\"TN2\"><sup>@</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic> < 0.001</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Energy efficiency (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.5 ± 0.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.4 ± 0.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.9 ± 0.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.5 ± 0.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.0 ± 0.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.7 ± 0.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">a, <italic>p</italic> < 0.001</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Visceral adipose tissue (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.42 ± 0.18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.74 ± 0.31</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.57 ± 0.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.70 ± 0.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.03 ± 0.14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.96 ± 0.13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">a, <italic>p</italic> < 0.01</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Subcutaneous adipose tissue (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.89 ± 0.10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.01 ± 0.10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.96 ± 0.09</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.62 ± 0.05</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.80 ± 0.12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.74 ± 0.06</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">a, <italic>p</italic> < 0.01</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Glycemia (mg/dl)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">77.5 ± 3.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">74.7 ± 3.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">74.8 ± 3.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">78.5 ± 5.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">67.0 ± 3.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">76.0 ± 2.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insulin (ng/ml)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.25 ± 0.75</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.33 ± 0.33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.09 ± 0.37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.28 ± 0.25</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.14 ± 0.11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.04 ± 0.22</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">a, <italic>p</italic> < 0.001</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HOMA-IR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.2 ± 4.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14.7 ± 2.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17.6 ± 2.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13.1 ± 2.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.3 ± 0.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11.1 ± 0.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">a, <italic>p</italic> < 0.01</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Leptin (ng/ml)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.25 ± 0.79</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.91 ± 1.14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.99 ± 0.75</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.37 ± 0.11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.88 ± 0.45</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.80 ± 0.46</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">a, <italic>p</italic> < 0.001</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NEFA (mmol/l)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.95 ± 0.09</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.04 ± 0.13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.19 ± 0.12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.00 ± 0.09</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.94 ± 0.06</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.19 ± 0.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Triglycerides (mg/dl)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">74.4 ± 15.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53.8 ± 4.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">69.4 ± 12.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">44.1 ± 9.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20.0 ± 1.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35.8 ± 9.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">a, <italic>p</italic> < 0.001</td></tr></tbody></table><table-wrap-foot><p><italic>a, overall sex effect; b, overall diet effect</italic>;</p><fn id=\"TN1\"><label>#</label><p><italic>different compared to chow rats of the same sex</italic>;</p></fn><fn id=\"TN2\"><label>@</label><p><italic>different between sexes on the same diet. ns, non-significant. n = 6, except for energy intake where n = 3</italic>.</p></fn></table-wrap-foot></table-wrap><p>The amount of VAT [<italic>F</italic><sub>(1, 35)</sub> = 24.1, <italic>p</italic> < 0.01] and SCAT [<italic>F</italic><sub>(1, 35)</sub> = 10.2, <italic>p</italic> < 0.01] were affected by sex, with males having a greater percentage of adipose tissue in both depots compared to females. No dietary influence was seen on either of these parameters.</p><p>An effect of sex was observed on the number of kcal consumed per rat [<italic>F</italic><sub>(1, 17)</sub> = 31.8, <italic>p</italic> < 0.001; <xref rid=\"T4\" ref-type=\"table\">Table 4</xref>], with males consuming more energy than females regardless of diet. In addition, diet had an overall effect on this parameter [<italic>F</italic><sub>(2, 17)</sub> = 3.7, <italic>p</italic> = 0.05]. When caloric intake was adjusted according to body weight there continued to be a sex effect [<italic>F</italic><sub>(1, 17)</sub> = 13.3, <italic>p</italic> < 0.01], but in this case females had a higher energy intake. There was also an effect of diet [<italic>F</italic><sub>(2, 17)</sub> = 4.0, <italic>p</italic> < 0.05]. Energy efficiency, expressed as grams of weight gained per kcal consumed, was sex dependent [<italic>F</italic><sub>(1, 17)</sub> = 44.1, <italic>p</italic> < 0.001] with males having a higher index of energy efficiency than females.</p><p>Kilocalories from fat were affected both by sex [<italic>F</italic><sub>(1, 17)</sub> = 8.4, <italic>p</italic> < 0.05] and diet [<italic>F</italic><sub>(2, 17)</sub> = 126.5, <italic>p</italic> < 0.001], with an interaction between these factors [<italic>F</italic><sub>(2, 17)</sub> = 4.9, <italic>p</italic> < 0.05]. As expected, HFD rats consumed more kcal from fat compared to chow and LFD animals (<italic>p</italic> < 0.001). Moreover, fat consumption on the LFD was also higher than on the chow diet in both sexes (<italic>p</italic> < 0.001).</p></sec><sec><title>Serum Levels of Metabolic Factors and IGF Family Members</title><p>There was an overall effect of sex on serum leptin [<italic>F</italic><sub>(1, 35)</sub> = 22.4, <italic>p</italic> < 0.001], insulin [<italic>F</italic><sub>(1, 34)</sub> = 18.6, <italic>p</italic> < 0.001], and triglyceride [<italic>F</italic><sub>(1, 35)</sub> = 15.9, <italic>p</italic> < 0.001] levels, as well as HOMA-IR index [<italic>F</italic><sub>(1, 33)</sub> = 15.3, <italic>p</italic> < 0.01], with males having overall higher values than females in all cases (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). There was no effect of either sex or diet on glycemia or non-esterified fatty acids (NEFA) levels (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>).</p><p>Males had overall higher serum levels of free IGF1 [<italic>F</italic><sub>(1, 35)</sub> = 67.6, <italic>p</italic> < 0.001; <xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>] and total IGF1 [<italic>F</italic><sub>(1, 35)</sub> = 15.7, <italic>p</italic> < 0.001; <xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>] than females, with no dietary effect found. On a chow diet males had higher levels of IGF2 than females on the same diet [<italic>F</italic><sub>(1, 9)</sub> = 6.2, <italic>p</italic> < 0.05; <xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref>]. In males, IGF2 levels were lower on the HFD compared to chow [<italic>F</italic><sub>(2, 15)</sub> = 3.6, <italic>p</italic> < 0.05]. In contrast, in females IGF2 levels were increased after LFD consumption compared to both chow and HFD [<italic>F</italic><sub>(2, 14)</sub> = 5.9, <italic>p</italic> < 0.05], with this resulting in females having higher levels than males when on the LFD [<italic>F</italic><sub>(1, 10)</sub> = 5.2, <italic>p</italic> < 0.05].</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Serum levels of free insulin-like growth factor (IGF)-1 <bold>(A)</bold>, total IGF-1 <bold>(B)</bold>, IGF 2 <bold>(C)</bold>, IGF binding protien (IGFBP) 2 <bold>(D)</bold>, IGFBP3 <bold>(E)</bold>, IGFBP5 <bold>(F)</bold>, and pregnancy-associated plasma protein (PAPP-A)2 <bold>(G)</bold> in rats on a high fat diet (HFD), low fat diet (LFD) or a chow diet for 1 week. **<italic>p</italic> < 0.01; ***<italic>p</italic> < 0.001. a, overall effect of sex. <italic>n</italic> = 6.</p></caption><graphic xlink:href=\"fendo-11-00513-g0001\"/></fig><p>There was an effect of diet [<italic>F</italic><sub>(2, 35)</sub> =3.6, <italic>p</italic> < 0.05; <xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>] and sex [<italic>F</italic><sub>(1, 35)</sub> = 24.9, <italic>p</italic> < 0.001] on serum IGFBP2 levels, as well as an interaction between sex and diet [<italic>F</italic><sub>(2, 35)</sub> = 7.0, <italic>p</italic> < 0.01]. Females had higher levels than males when on chow [<italic>F</italic><sub>(1, 11)</sub> = 16.6, <italic>p</italic> < 0.01] or a HFD [<italic>F</italic><sub>(1, 11)</sub> = 13.4, <italic>p</italic> < 0.01]. In females, serum IGFBP2 levels were decreased after LFD consumption compared to both chow and HFD [<italic>F</italic><sub>(2, 17)</sub> = 5.6, <italic>p</italic> < 0.05].</p><p>Males had overall higher circulating IGFBP3 [<italic>F</italic><sub>(1, 35)</sub> = 53.2, <italic>p</italic> < 0.001; <xref ref-type=\"fig\" rid=\"F1\">Figure 1E</xref>] and IGFBP5 [<italic>F</italic><sub>(1, 35)</sub> = 15.4, <italic>p</italic> < 0.001; <xref ref-type=\"fig\" rid=\"F1\">Figure 1F</xref>] levels than females, with no dietary effect found. On the contrary, females had higher PAPP-A2 levels [<italic>F</italic><sub>(1, 30)</sub> = 4.3, <italic>p</italic> < 0.05; <xref ref-type=\"fig\" rid=\"F1\">Figure 1G</xref>] in serum compared to males.</p></sec><sec><title>Hypothalamic Response to Dietary Changes</title><p>We found no effect of sex or diet on hypothalamic IGF1 mRNA levels (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>). Hypothalamic IGF2 mRNA levels were affected by diet [<italic>F</italic><sub>(2, 35)</sub> = 12.6, <italic>p</italic> < 0.01; <xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>], with an increase in response to LFD, reaching significance in males. Diet also affected IGFBP2 mRNA levels [<italic>F</italic><sub>(2, 35)</sub> = 12.4, <italic>p</italic> < 0.001; <xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>], which were increased by LFD intake with this being significant in males. Despite no sex differences being observed on a chow diet, relative expression of IGFBP2 in response to LFD was higher in males than females (<italic>p</italic> < 0.05). There was a positive correlation between the relative levels of hypothalamic IGF2 and IGFBP2 mRNA (r = 0.882, <italic>p</italic> < 0.001; <xref ref-type=\"fig\" rid=\"F2\">Figure 2D</xref>).</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Relative mRNA levels of the insulin-like growth factor (IGF) system in the hypothalamus: IGF1 <bold>(A)</bold>, IGF2 <bold>(B)</bold>, IGF binding protien (IGFBP)2 <bold>(C)</bold>, and the correlation of relative hypothalamic mRNA levels of IGF2 and IGFBP2 <bold>(D)</bold>. **<italic>p</italic> < 0.01; ***<italic>p</italic> < 0.001; ns, non-significant; HFD, high fat diet; LFD, low fat diet. <italic>n</italic> = 6.</p></caption><graphic xlink:href=\"fendo-11-00513-g0002\"/></fig><p>There were no differences between groups in the relative hypothalamic mRNA levels of IGF-1R, IGF-2R, IGFBP1, IGFBP3, IGFBP4, IGFBP5, PAPP-A, STC-1, or STC-2 (<xref rid=\"T5\" ref-type=\"table\">Table 5</xref>).</p><table-wrap id=\"T5\" position=\"float\"><label>Table 5</label><caption><p>Relative gene expression of members of the IGF system in the hypothalamus.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Chow males</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>HFD males</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>LFD males</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Chow females</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>HFD females</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>LFD females</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Sig</bold>.</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IGF-1R</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 12.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">106.2 ± 11.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">113.8 ± 10.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110.6 ± 15.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.2 ± 9.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">133.1 ± 23.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IGF-2R</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 14.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">95.5 ± 17.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">116.6 ± 12.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.6 ± 8.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">89.7 ± 16.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">109.2 ± 17.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IGFBP1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 10.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">122.3 ± 24.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">132.7 ± 17.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">126.5 ± 14.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">105.9 ± 15.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">120.7 ± 16.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IGFBP3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 13.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">97.7 ± 11.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">120.4 ± 14.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">105.7 ± 9.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">117.5 ± 14.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">128.5 ± 14.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IGFBP4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 23.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91.4 ± 24.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">145.8 ± 25.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">94.7 ± 27.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91.1 ± 13.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">119.0 ± 24.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IGFBP5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 19.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91.6 ± 13.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">128.2 ± 16.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">86.2 ± 7.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">89.9 ± 12.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">121.9 ± 26.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PAPP-A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 6.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">104.4 ± 20.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">125.7 ± 21.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">109.8 ± 17.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">120.7 ± 22.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">116.8 ± 15.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">STC-1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 12.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">101.3 ± 12.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">123.1 ± 21.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">121.6 ± 12.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">94.5 ± 13.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">115.2 ± 17.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">STC-2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 18.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">105.8 ± 11.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">129.3 ± 15.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">98.9 ± 9.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">98.0 ± 11.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">121.3 ± 19.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ns</td></tr></tbody></table><table-wrap-foot><p><italic>Data are represented as mean ± SEM. ns, non-significant. HFD, high fat diet; LFD, low fat diet. n = 6</italic>.</p></table-wrap-foot></table-wrap><p>The relative mRNA levels of neuropeptide Y [NPY; <italic>F</italic><sub>(2, 34)</sub> = 9.6, <italic>p</italic> < 0.01; <xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>] and Agouti-related protein [AgRP; <italic>F</italic><sub>(2, 34)</sub> = 3.9, <italic>p</italic> < 0.01; <xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>] were affected by diet, with an interaction between sex and diet [NPY: <italic>F</italic><sub>(2, 34)</sub> = 5.1, <italic>p</italic> < 0.05; AgRP: <italic>F</italic><sub>(2, 34)</sub> = 4.9, <italic>p</italic> < 0.05]. These orexigenic neuropeptides increased in response to LFD, but only in males [NPY: <italic>F</italic><sub>(2, 16)</sub> = 10.4, <italic>p</italic> < 0.01; AgRP: <italic>F</italic><sub>(2, 16)</sub> = 5.8, <italic>p</italic> < 0.05]. On a LFD, males had higher levels of both NPY [<italic>F</italic><sub>(1, 11)</sub> = 6.4, <italic>p</italic> < 0.05] and AgRP [<italic>F</italic><sub>(1, 11)</sub> = 9.5, <italic>p</italic> < 0.05] compared to females.</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Relative mRNA levels of neuropeptide Y (NPY; <bold>A</bold>), Agouti related protein (AgRP; <bold>B</bold>), proopiomelanocortin (POMC; <bold>C</bold>) and cocaine and amphetamine regulated transcript (CART; <bold>D</bold>) and protein levels of glial fibrillary acidic protein (GFAP; <bold>E</bold>) and phophorylated extracellular signal-regulated kinase (pERK; <bold>F</bold>) in the hypothalamus of rats on a (HFD), low fat diet (LFD) or a chow diet for 1 week. These images are all from the same blot, but were not contiguous and for this reason they are individualy placed in order of the experimental groups in the graph. **<italic>p</italic> < 0.01; ***<italic>p</italic> < 0.001; b, effect of diet, ns, non-significant. <italic>n</italic> = 6.</p></caption><graphic xlink:href=\"fendo-11-00513-g0003\"/></fig><p>Proopiomelanocortin (POMC) mRNA levels showed no significant changes (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>). There was an overall dietary effect on cocaine and amphetamine-regulated transcript (CART) mRNA levels [<italic>F</italic><sub>(2, 34)</sub> = 4.7, <italic>p</italic> < 0.05; <xref ref-type=\"fig\" rid=\"F3\">Figure 3D</xref>], with levels increasing in animals of both sexes on a LFD.</p><p>To assess if gliosis and hypothalamic inflammation were present, we analyzed glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba1) and c-jun N-terminal kinase (JNK) activation. There was effect of diet on GFAP levels [<italic>F</italic><sub>(2, 35)</sub> = 4.1, <italic>p</italic> < 0.05; <xref ref-type=\"fig\" rid=\"F3\">Figure 3E</xref>], with an increase in animals after LFD consumption. There was no effect on Iba1 or pJNK levels (<xref rid=\"T6\" ref-type=\"table\">Table 6</xref>).</p><p>To determine whether activation of the insulin/IGF signaling pathways in the hypothalamus was altered pIRS1, PI3K, pAKT (Ser473), and pERK were analyzed. There was an effect of diet on pERK levels [<italic>F</italic><sub>(2, 34)</sub> = 3.9, <italic>p</italic> < 0.05; <xref ref-type=\"fig\" rid=\"F3\">Figure 3F</xref>], with an overall increase in animals on a LFD. No differences in pIRS1, PI3K, or pAKT were observed (<xref rid=\"T6\" ref-type=\"table\">Table 6</xref>).</p><table-wrap id=\"T6\" position=\"float\"><label>Table 6</label><caption><p>Effects of 1 week on a high fat diet (HFD), low fat diet (LFD) or chow diet on the phosphorylation of proteins involved in insulin and IGF signaling in the hypothalamus of male and female rats (<italic>n</italic> =6), as well as, Iba1: ionized calcium binding adaptor molecule 1 (Iba1), a marker of microglia and cell stress markers (JNK: c-Jun N-terminal kinases).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Chow males</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>HFD males</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>LFD males</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Chow females</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>HFD females</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>LFD females</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Sig</bold>.</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Iba1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 10.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">88.6 ± 12.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">97.1 ± 15.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">93.0 ± 14.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">90.9 ± 15.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">95.1 ± 18.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">n.s.</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pAKT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 9.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.4 ± 8.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">103.6 ± 13.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91.6 ± 11.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">87.8 ± 12.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.9 ± 20.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">n.s.</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PI3KI</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 13.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">151.9 ± 18.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">140.1 ± 14.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">155.3 ± 23.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">142.3 ± 15.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">119.1 ± 19.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">n.s.</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pIRS1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 22.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">148.3 ± 21.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">183.0 ± 30.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">152.4 ± 31.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">132.4 ± 33.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">196.7 ± 55.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">n.s.</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pJNK</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100.0 ± 1.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">104.9 ± 3.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">104.9 ± 5.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">101.9 ± 2.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.0 ± 1.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">98.6 ± 4.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">n.s.</td></tr></tbody></table><table-wrap-foot><p><italic>Phosphorylated proteins were normalized with the total form of the protein. ns, non-significant</italic>.</p></table-wrap-foot></table-wrap></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Here we show that there were not only sex differences in the IGF system's response to a short-term dietary change, but baseline serum levels of all members of the IGF system studied were significantly different between male and female rats. We observed higher levels of free IGF1, total IGF1, IGFBP3, and IGFBP5, as well as insulin in males compared to females. The GH secretory pattern differs between male and female rodents (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B33\" ref-type=\"bibr\">33</xref>) and could underlie some of these sex differences in the IGF system. Males have been reported to have higher IGF1 and IGFBP3 levels than females (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>, <xref rid=\"B35\" ref-type=\"bibr\">35</xref>), as found here. However, Frystyk and colleagues reported no sex differences in free IGF1 levels. It is possible that the employment of different methodologies to determine free IGF1 levels underlies this discrepancy in results. Although males were found to have higher circulating levels of most members of the IGF system, females had higher circulating levels of PAPP-A2, which could promote the availability of IGF1 for the tissues. The sex differences in the GH-IGF system are at least in part due to differences in sex steroid levels both during development and adulthood (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>–<xref rid=\"B38\" ref-type=\"bibr\">38</xref>) and underlie the differences between males and females in growth and body size. More recently, the IGF system has also been implicated in the sex differences in the response to or propensity to develop different pathologies, as well as longevity (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>).</p><p>Baseline sex differences in body weight and metabolism were observed, with males having a greater body weight, weight gain, energy intake, energy efficiency, and circulating leptin levels compared to females, as previously reported (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>–<xref rid=\"B42\" ref-type=\"bibr\">42</xref>), as well as higher serum triglycerides levels. After 1 week on a HFD we found few effects on body weight or body composition, which is in accordance with some previous studies in rodents (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>, <xref rid=\"B43\" ref-type=\"bibr\">43</xref>), but not others (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>). The LFD group was included in this study as this diet has been widely used as a control group in diet-induced obesity (DIO) models (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>, <xref rid=\"B45\" ref-type=\"bibr\">45</xref>, <xref rid=\"B46\" ref-type=\"bibr\">46</xref>), but the higher content of carbohydrates, which is largely composed of sucrose, compared to HFD may also have metabolic effects. Neither diet affected final body weight in either sex, although the percentage weight gain in males on the HFD was significantly greater compared to the other groups. This is in accordance with their higher energy intake, as well as greater intake of kcals from fat compared to the other groups. Circulating levels of leptin are directly correlated with the amount of adipose tissue (<xref rid=\"B47\" ref-type=\"bibr\">47</xref>) and neither serum leptin levels nor adipose content were modified here on this short-term HFD. No changes in circulating levels of triglycerides, insulin or leptin levels were seen on the short-term HFD, as reported by others in mice (<xref rid=\"B48\" ref-type=\"bibr\">48</xref>).</p><p>The circulating IGF system is modified in human subjects with obesity. Serum levels of free IGF1 and IGFBP3, but not of total IGF1, are reported to be higher and IGFBP2 lower (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>, <xref rid=\"B49\" ref-type=\"bibr\">49</xref>) in children with obesity compared to control children. Serum IGF1 levels have also been reported to be higher when visceral adipose tissue content is elevated although a direct correlation of circulating IGF levels and BMI was not observed (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>), suggesting the possible relevance of adipose distribution. In the study reported here, the lack of changes in IGF1 and IGFBP3 is probably due to the limited time of exposure to this diet and the lack of adipose accumulation. Indeed, the observed changes in the IGF system in obese subjects are most likely explained by their overall metabolic status rather than a direct response to specific nutrients or a specific diet as studied here.</p><p>Circulating IGF2 levels were affected by the short-term dietary changes, even though there was no significant modification in body weight; moreover, these changes were different between the sexes. In males, circulating IGF2 levels were reduced during HFD consumption, with HFD having no effect on this parameter in females. The response of this growth factor to metabolic changes is less clear with circulating IGF2 levels reported to being both reduced (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>) and increased (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>, <xref rid=\"B53\" ref-type=\"bibr\">53</xref>) in obese compared to non-obese men and women. Shandu et al. found that serum IGF2 levels were reduced in subjects who gained weight during the study compared to those who maintained or lost weight and that lower serum IGF2 levels are negatively correlated with the risk to gain weight (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>). Thus, the observed reduction in circulating IGF2 levels in males on the HFD could be a predictor of potential weight gain and metabolic risk. This reduction in IGF2 was not observed in females and this could be related to the observation that young adult female rodents tend be more resistant to HFD-induced weight gain (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>, <xref rid=\"B55\" ref-type=\"bibr\">55</xref>), which is in concordance with this growth factor possibly being an indicator of early metabolic changes.</p><p>Circulating levels of IGF2 were also affected by LFD intake and this also occurred in a sex specific manner. Rats of both sexes given the LFD had a higher energy intake compared to those on the chow diet, which is possibly due to the novelty and/or palatability of the diet and thus increased consumption, although increased energy intake of LFD compared to chow over a longer time-period has also been reported in male C57 mice (<xref rid=\"B56\" ref-type=\"bibr\">56</xref>) suggesting that this increase is not due only to novelty. In contrast to the HFD, LFD increased circulating levels of IGF2 and this effect was only observed in females. IGF2 participates in bone growth, adipose tissue accumulation and glucose metabolism, stimulating glucose uptake by adipocytes and acting directly at the level of the pancreas (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>–<xref rid=\"B59\" ref-type=\"bibr\">59</xref>). Thus, it is possible that the high sucrose content of the LFD is involved in stimulating this rise in IGFBP2. LFD intake also decreased serum IGFBP2 levels, and again this effect of LFD was only found in females. Plasma IGFBP2 levels are reported to be negatively correlated with BMI (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>, <xref rid=\"B60\" ref-type=\"bibr\">60</xref>), as well as with adipogenesis and lipogenesis (<xref rid=\"B61\" ref-type=\"bibr\">61</xref>). IGFBP2 is the second most abundant IGF-binding protein in the circulation (<xref rid=\"B62\" ref-type=\"bibr\">62</xref>, <xref rid=\"B63\" ref-type=\"bibr\">63</xref>) and has been suggested to be protective against obesity and to improve glucose tolerance on a HFD (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B64\" ref-type=\"bibr\">64</xref>). IGFBP2 binds IGF2 with a slight preference over IGF1 [reviewed by (<xref rid=\"B65\" ref-type=\"bibr\">65</xref>)] and the decrease in IGFBP2 in LFD females may lead to increased IGF2 availability. These changes in circulating IGF2 and IGFBP2 in response to LFD are sex-dependent and could be involved in the differential impact of poor dietary habits on the homeostatic circuitry regulating metabolism (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>, <xref rid=\"B66\" ref-type=\"bibr\">66</xref>).</p><p>In the hypothalamus we found no effect of short-term dietary challenge on the mRNA levels of IGF1. In contrast, both IGF2 and IGFBP2 mRNA levels were increased in males after LFD intake. The metabolic effects of IGF2 and IGFBP2 in the brain are largely unknown, but our results suggest that these factors may also participate in metabolic control in the hypothalamus. The fact that the LFD had more effects on the IGF system than did the HFD, at least during short-term intake, raises two important considerations. First, studies where a LFD is used as a control diet for HFD intake do not necessarily reflect changes in response to high fat intake, but could reflect what is occurring in response to the LFD. Thus, comparison to a normal chow diet is important. Secondly, the question becomes why does the LFD induce these changes? Although the LFD and chow diet used here have similar percentages of carbohydrates, proteins and fats, the carbohydrate composition is quite different. The amount of sucrose is considerably higher (33.1%) in the LFD compared to the chow (0.9%) or HFD (8.9%). Thus, the possibility that the changes in IGF2 and IGFBP2 are related to specific nutrients, such as sucrose, deserves further investigation. Previous studies indicate sex specific metabolic responses to sucrose intake (<xref rid=\"B67\" ref-type=\"bibr\">67</xref>) and even though no effect was observed on body weight in either sex after 2 weeks of a high-sucrose diet, Busserolles and colleagues found that females were more resistant to the pro-oxidant effects of this diet (<xref rid=\"B68\" ref-type=\"bibr\">68</xref>).</p><p>Excess HFD consumption can lead to important changes in the brain, promoting gliosis and inflammatory responses (<xref rid=\"B69\" ref-type=\"bibr\">69</xref>, <xref rid=\"B70\" ref-type=\"bibr\">70</xref>). This involves astrocyte and microglia activation, which is initially protective (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>, <xref rid=\"B72\" ref-type=\"bibr\">72</xref>) but when prolonged can become damaging (<xref rid=\"B73\" ref-type=\"bibr\">73</xref>), leading to neuronal death in the arcuate nucleus (<xref rid=\"B74\" ref-type=\"bibr\">74</xref>). There was an overall effect of diet on hypothalamic GFAP levels, with LFD inducing a slight increase in both males and females, but no changes in the levels of the microglial marker Iba1 or activation of inflammatory pathways were found in response to either diet. Although HFD is reported to induce hypothalamic gliosis/inflammation in less than a week, which then wains only to reappear a couple of weeks later (<xref rid=\"B70\" ref-type=\"bibr\">70</xref>). Other studies report that at 1 week of HFD intake no signs of hypothalamic gliosis/inflammation can be detected (<xref rid=\"B75\" ref-type=\"bibr\">75</xref>, <xref rid=\"B76\" ref-type=\"bibr\">76</xref>), similar to that observed here. It is possible that the initial protective glial/inflammatory reaction to excess fat intake begins to switch after ~1 week of continuous exposure to this toxic diet, transitioning from a protective to a harmful response. This hypothesis obviously needs further investigation.</p><p>The mRNA levels of NPY and AgRP increased after LFD consumption, but only in males and with no effect of HFD. There were no changes in POMC mRNA levels in response to either diet. Insulin suppresses the hypothalamic orexigenic circuitry to reduce food intake (<xref rid=\"B77\" ref-type=\"bibr\">77</xref>) and has been shown to decrease both NPY and AgRP mRNA levels in hypothalamic cells <italic>in vitro</italic> (<xref rid=\"B78\" ref-type=\"bibr\">78</xref>). As IGF1 and IGF2 have “insulin-like” effects it is possible that they are involved in the modulation of metabolic neuropeptides. Indeed, IGF1 has been shown to modulate POMC mRNA levels (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>), but whether IGF2 has metabolic effects at the hypothalamic levels remains unknown. It is possible that the higher expression of both orexigenic neuropeptides after LFD consumption in males is due to the higher sucrose content of this diet. We previously reported that normal male Wistar rats given a 33% sucrose solution instead of water for 2 months had increased NPY and AgRP mRNA levels, with no changes in POMC or CART mRNA expression (<xref rid=\"B79\" ref-type=\"bibr\">79</xref>).</p><p>One of the novel observations reported here is that there are sex specific changes in IGF2 and IGFBP2, both systemically and centrally, in response to short-term dietary changes. Sex differences in the response to metabolic challenges and the propensity to become obese, as well as to develop complications associated with obesity, have been widely reported (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>, <xref rid=\"B66\" ref-type=\"bibr\">66</xref>, <xref rid=\"B80\" ref-type=\"bibr\">80</xref>). The metabolic responses to manipulations of both IGF2 and IGFBP2 have also been shown to be different between males and females. For example, in IGFBP2 KOs males becoming overweight in adulthood while this does not occur in females, at least in young adults (<xref rid=\"B81\" ref-type=\"bibr\">81</xref>). Hypothalamic IGF2 expression is upregulated in the female offspring of mothers ingesting a HFD, while this is not observed in males (<xref rid=\"B82\" ref-type=\"bibr\">82</xref>). Both IGF2 and IGFBP2 expression levels are modified by estrogens in various tissues including the brain (<xref rid=\"B83\" ref-type=\"bibr\">83</xref>–<xref rid=\"B85\" ref-type=\"bibr\">85</xref>), suggesting that the sex steroid environment participates in the control of these two factors. Moreover, there is a clear interaction between estrogens and the IGF system (<xref rid=\"B86\" ref-type=\"bibr\">86</xref>) and these two hormonal systems are sexually dimorphic, as well as their interactions. The physiological meaning or outcome of these sex differences in the IGF system and their effects on metabolism have yet to be determined, but it is clear that studies aimed to understand metabolic disarray and in the search for treatments of obesity must take this into consideration.</p><p>In conclusion, short-term LFD intake induced more changes in both the central and peripheral IGF system than did HFD, with these effects being different in males and females. As bodyweight was not changed in response to either diet, it is possible that the observed changes in the IGF system are related to the dietary composition. This possibility deserves further investigation.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>All experiments were designed according to the European Communities Council Directive (2010/63/UE) and the Royal Decree 53/2013 pertaining to the protection of experimental animals. This study was also approved by the Ethical Committee of Animal Experimentation of the Hospital Puerta de Hierro de Madrid and the Animal Welfare Organ of the Comunidad Autónoma de Madrid.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>JA and JC: conception and design of study. SG-C, FD, PR, and AF-R: animal handling. SG-C, MJ-H, and VB: biochemical analysis. SG-C, LF, and JC: data analysis. SG-C, LF, and JC: redaction of manuscript. SG-C, LF, FD, PR, MJ-H, AF-R, VB, JA, and JC: revision of manuscript. All authors: contributed to the article and approved the submitted version.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> The authors declare that this study received public research funding from the Spanish Government. The authors are funded by grants from the Spanish Ministry of Science and Innovation (BFU2017-82565-C21-R2 to JC and LF), Spanish Ministry of Education, Culture and Sports (university training grant PU13/00909 to AF-R), Fondo de Investigación Sanitaria (PI1900166 to JA) and Fondos FEDER. Centro de Investigación Biomédica en Red Fisiopatología de Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III (JA). 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Cell Dev. Biol.</journal-id><journal-title-group><journal-title>Frontiers in Cell and Developmental Biology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-634X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850838</article-id><article-id pub-id-type=\"pmc\">PMC7431667</article-id><article-id pub-id-type=\"doi\">10.3389/fcell.2020.00722</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cell and Developmental Biology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Olfactomedin-3 Enhances Seizure Activity by Interacting With AMPA Receptors in Epilepsy Models</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Tang</surname><given-names>Shirong</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Tiancheng</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Xiaogang</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Guo</surname><given-names>Yi</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/569360/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Xu</surname><given-names>Ping</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zeng</surname><given-names>Junwei</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Luo</surname><given-names>Zhong</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Dongxu</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zheng</surname><given-names>Yongsu</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Luo</surname><given-names>Yuemei</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Yu</surname><given-names>Changyin</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Xu</surname><given-names>Zucai</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff7\"><sup>7</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/963324/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Neurology, The Affiliated Hospital of Zunyi Medical University</institution>, <addr-line>Zunyi</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Neurology, The Thirteenth People’s Hospital of Chongqing</institution>, <addr-line>Chongqing</addr-line>, <country>China</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Neurology, Lanzhou University Second Hospital</institution>, <addr-line>Lanzhou</addr-line>, <country>China</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Neurology, Chongqing General Hospital, University of Chinese Academy of Sciences</institution>, <addr-line>Chongqing</addr-line>, <country>China</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Department of Neurology, The First Affiliated Hospital of Chongqing Medical University</institution>, <addr-line>Chongqing</addr-line>, <country>China</country></aff><aff id=\"aff6\"><sup>6</sup><institution>Department of Physiology, Zunyi Medical University</institution>, <addr-line>Zunyi</addr-line>, <country>China</country></aff><aff id=\"aff7\"><sup>7</sup><institution>Key Laboratory of Brain Science, Zunyi Medical University</institution>, <addr-line>Zunyi</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Igor Jakovcevski, Helmholtz-Gemeinschaft Deutscher Forschungszentren (HZ), Germany</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Lidija Radenovic, University of Belgrade, Serbia; Thomas Fath, Macquarie University, Australia</p></fn><corresp id=\"c001\">*Correspondence: Changyin Yu, <email>yuchangyin68@163.com</email></corresp><corresp id=\"c002\">Zucai Xu, <email>doctorxzc@126.com</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>These authors have contributed equally to this work</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Cell Adhesion and Migration, a section of the journal Frontiers in Cell and Developmental Biology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>722</elocation-id><history><date date-type=\"received\"><day>29</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>14</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Tang, Wang, Zhang, Guo, Xu, Zeng, Luo, Li, Zheng, Luo, Yu and Xu.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Tang, Wang, Zhang, Guo, Xu, Zeng, Luo, Li, Zheng, Luo, Yu and Xu</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p><bold>Background:</bold> OLFM3 (olfactomedin-3) is a member of the olfactomedin domain family, which has been found to stimulate the formation and adhesion of tight cell connections and to regulate cytoskeleton formation and cell migration. Differences in the gene coding for OLFM3 have been found between patients with epilepsy and controls. However, the exact role of OLFM3 in epilepsy has not been thoroughly investigated.</p><p><bold>Methods:</bold> Biochemical methods were used to assess OLFM3 expression and localization in the cortex of patients with temporal lobe epilepsy and in the hippocampus and cortex of epileptic mice. Electrophysiological recordings were used to measure the role of OLFM3 in regulating hippocampal excitability in a model of magnesium-free-induced seizure <italic>in vitro</italic>. Behavioral experiments were performed in a pentylenetetrazol (PTZ)-induced seizure model, and electroencephalograms (EEGs) were recorded in the chronic phase of the kainic acid (KA)-induced epilepsy model <italic>in vivo</italic>. OLFM3 and its interaction with AMPAR (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor) subunits were analyzed by co-immunoprecipitation.</p><p><bold>Results:</bold> The expression of OLFM3 was increased in the cortex of patients with temporal lobe epilepsy and in the hippocampus and cortex of epileptic mice compared with controls. Interestingly, lentivirus-mediated overexpression of OLFM3 in the hippocampus increased the susceptibility of mice to PTZ-induced seizures, and OLFM3 knockdown had the opposite effect. OLFM3 affected AMPAR currents in a brain-slice model of epileptiform activity induced by Mg2+-free medium. We found that OLFM3 co-immunoprecipitation with GluA1 and GluA2. Furthermore, downregulation or overexpression of OLFM3 in the hippocampus affected the membrane expression of GluA1 and GluA2 in epileptic mice.</p><p><bold>Conclusion:</bold> These findings reveal that OLFM3 may enhance seizure activity by interacting with GluA1 and GluA2, potentially indicating a molecular mechanism for new therapeutic strategies.</p></abstract><kwd-group><kwd>epilepsy</kwd><kwd>GluA1</kwd><kwd>GluA2</kwd><kwd>OLFM3</kwd><kwd>epilepsy model</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Natural Science Foundation of China<named-content content-type=\"fundref-id\">10.13039/501100001809</named-content></funding-source></award-group><award-group><funding-source id=\"cn002\">Guizhou Science and Technology Department<named-content content-type=\"fundref-id\">10.13039/501100004001</named-content></funding-source></award-group><award-group><funding-source id=\"cn003\">Department of Health of Guizhou Province<named-content content-type=\"fundref-id\">10.13039/501100010891</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"6\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"50\"/><page-count count=\"13\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Epilepsy is a common chronic neurological disease, and temporal lobe epilepsy (TLE) is one of the most common types of epilepsy in adult patients (<xref rid=\"B12\" ref-type=\"bibr\">Hauser et al., 1993</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Jobst and Cascino, 2015</xref>). At present, there are approximately 70 million people worldwide suffering from epilepsy, most of whom are from low- and middle-income countries (<xref rid=\"B7\" ref-type=\"bibr\">Espinosa-Jovel et al., 2018</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Hafele et al., 2018</xref>). Recurrent epileptic seizures seriously affect the physical and mental health of patients (<xref rid=\"B30\" ref-type=\"bibr\">Olesen et al., 2012</xref>; <xref rid=\"B44\" ref-type=\"bibr\">Wiglusz et al., 2018</xref>) and increase the public health burden on patients, family members and society. In recent years, the public has become increasingly concerned about epilepsy (<xref rid=\"B29\" ref-type=\"bibr\">Novotna and Rektor, 2017</xref>). The specific occurrence of epilepsy and the molecular mechanisms of its development are still not completely clear (<xref rid=\"B32\" ref-type=\"bibr\">Perucca and Perucca, 2019</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Wang and Chen, 2019</xref>).</p><p>OLFM3 (olfactomedin-3) is a member of the olfactomedin domain family (OLFM) (<xref rid=\"B33\" ref-type=\"bibr\">Pronker et al., 2015</xref>). Currently, 13 members of this family have been identified in mammals, while there are only 5 in humans: OLFM1, OLFM2, OLFM3, OLFM4, and MYOC (<xref rid=\"B38\" ref-type=\"bibr\">Shi et al., 2016</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Koch et al., 2017</xref>). OLFM members are a group of mediators that interact with extracellular proteins, playing an important role in nervous system growth and development, cell adhesion, and cell cycle regulation (<xref rid=\"B11\" ref-type=\"bibr\">Han and Kursula, 2015</xref>). OLFM3 is expressed in the retina and brain and plays an important role in the normal development of eyes (<xref rid=\"B28\" ref-type=\"bibr\">Nakaya et al., 2013</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Sultana et al., 2014</xref>). In recent years, OLFM3 has been found to be a neuronal expression protein that can stimulate the formation and adhesion of tight cell connections and regulate cytoskeleton formation and cell migration (<xref rid=\"B2\" ref-type=\"bibr\">Anholt, 2014</xref>). The AMPAR (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor) is an ionic glutamate receptor consisting of four subunits of GluA1, GluA2, GluA3, and GluA4 (<xref rid=\"B25\" ref-type=\"bibr\">Mayer, 2016</xref>). In adult brains, GluA1-GluA2 and GluA2-GluA3 are dominant, and the role of GluA1-GluA2 is particularly important (<xref rid=\"B25\" ref-type=\"bibr\">Mayer, 2016</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Gupta, 2018</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Liu et al., 2018</xref>), mediating rapid excitatory synaptic transmission (<xref rid=\"B14\" ref-type=\"bibr\">Herguedas et al., 2019</xref>). The concentration of the AMPA receptor subunits GluA1 and GluA2 has been shown to be significantly reduced in hippocampal synaptosomes in the postsynaptic density of a kainic acid (KA)-based rat model of chronic TLE. These changes in synaptic AMPAR subunits may contribute to further aggravation of the excitotoxic vulnerability of neurons and have significant implications for hippocampal cognition (<xref rid=\"B6\" ref-type=\"bibr\">Egbenya et al., 2018</xref>). When a stimulus reaches the threshold, the type 1 membrane vesicular glutamate transporter (vesicular glutamate transporter 1, vGlut1) transports vesicles to the presynaptic site to stimulate the release of the excitatory neurotransmitter glutamate (<xref rid=\"B46\" ref-type=\"bibr\">Yu et al., 2018</xref>). Postsynaptic density 95 (PSD-95) plays an important role in anchoring postsynaptic AMPARs through the skeleton proteins in the postsynaptic membrane. Finally, glutamic acid interacts with AMPARs and has a fast excitatory synaptic transmission effect (<xref rid=\"B24\" ref-type=\"bibr\">Matt et al., 2018</xref>).</p><p>In recent years, differences in the gene encoding OLFM3 have been found between patients with epilepsy and controls (<xref rid=\"B13\" ref-type=\"bibr\">Heinzen et al., 2010</xref>). In addition, proteomics and mass spectrometry have revealed that OLFM3 may be involved in AMPAR complex formation (<xref rid=\"B36\" ref-type=\"bibr\">Schwenk et al., 2012</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Shanks et al., 2012</xref>). Previous studies have confirmed that the AMPAR complex plays a crucial role in the occurrence and development of epilepsy (<xref rid=\"B25\" ref-type=\"bibr\">Mayer, 2016</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Lorgen et al., 2017</xref>). However, how OLFM3 participates in the formation of the AMPAR complex and whether it is involved in the occurrence of epilepsy have not been studied. Therefore, we investigated whether OLFM3 interacts with AMPARs and participates in the pathogenesis of epilepsy.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Human Samples</title><p>In our study, all brain tissue samples were randomly selected from our brain tissue bank (<xref rid=\"B45\" ref-type=\"bibr\">Yang et al., 2018</xref>). The tissues were collected from 24 patients [13 males and 11 females; mean age 34.54 ± 1.89 years (18–53 years); mean disease course 11.33 ± 5.47 years (4–25 years)] who had been diagnosed with TLE and had subsequently undergone surgical resection. Each presurgical assessment included a detailed history and neurological examination to ensure that these patients were suitable candidates for surgery. All patients were unresponsive to three or more antiepileptic drugs (AEDs). Twelve histologically normal samples were randomly collected for use as a control group; these tissues were collected from 6 males and 6 females, with a mean age of 32.92 ± 2.20 years (range, 23–50 years). The control samples were obtained from patients who had undergone cranial surgery due to increased intracranial pressure caused by head trauma and who had no history of epilepsy, had no apparent signs of central nervous system disease and had not been exposed to AEDs. In addition, no significant differences in gender or age were observed between the patients and the control group. The clinical features of the patients with TLE and the controls are summarized in <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Tables S1</xref>, <xref ref-type=\"supplementary-material\" rid=\"TS1\">S2</xref>, respectively. Given practical and ethical considerations, we were unable to obtain normal hippocampal specimens from control patients. Therefore, we only studied the (neo)cortex specimens from patients.</p><p>All clinical samples were collected after being obtained from the patients in the operating room. The samples were immediately frozen in liquid nitrogen and stored at −80°C until use for Western blotting to survey the difference between the TLE and control groups. The remaining samples were sectioned at a thickness of 10 μm and stored at −20°C for immunofluorescence analysis.</p></sec><sec id=\"S2.SS2\"><title>Mouse Models of Epilepsy</title><p>Specific-pathogen-free (SPF) male C57BL/6 mice (20–24 g, 8–10 weeks old) were used for the study. The mice were obtained from the Experimental Animal Center of Chongqing Medical University and were randomly divided into a normal control group and an experimental group. All animals were housed in cages under standard conditions (12-h/12-h light/dark cycles at 23 ± 1°C, with access to food and water <italic>ad libitum</italic>). The pentylenetetrazol (PTZ) kindling model, the classical model for the study of epileptic susceptibility and drug screening, was generated according to the methods described in a previous study (<xref rid=\"B50\" ref-type=\"bibr\">Zhu et al., 2016</xref>). Mice were intraperitoneally injected with PTZ (35 mg/kg, Sigma-Aldrich, St. Louis, MO, United States) once every other day for a total of 15 injections (from day 1 to day 30). After each injection, all the mice were immediately observed for 30 min. The evoked behavioral seizures were rated based on Racine’s standard scale (1972). Mice with at least three consecutive seizures scoring 4 or 5 were considered successfully established kindling models. To prevent false positives in one model experiment, we used another classical model for further verification. The KA-induced chronic epilepsy model, a classical model used for research on epilepsy mechanisms, was generated as previously studied (<xref rid=\"B35\" ref-type=\"bibr\">Sada et al., 2015b</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Moghbelinejad et al., 2016</xref>). Briefly, after the mice were deeply anesthetized, stereotaxic injections were performed into the dorsal blade of the CA1 area at the following coordinates with respect to the bregma [anteroposterior (AP), -1.6 mm; mediolateral (ML), -1.5 mm; dorsoventral (DV), -1.5 mm]. Mice were unilaterally injected with 1.0 nmol of KA (Sigma-Aldrich Co., St. Louis, MO, United States) in 50 nl saline, and 50 nl of saline was injected into the hippocampus in the control group. Once the mice were awake, we monitored them for stage 3–5 status epilepticus (SE) on the Racine scale (<xref rid=\"B49\" ref-type=\"bibr\">Zheng et al., 2017</xref>), and only mice with stage 3–5 SE were included in the subsequent behavioral monitoring tests. Mortality rates for PTZ- and KA-induced mouse models of epilepsy were 10–20% and 5%, respectively.</p></sec><sec id=\"S2.SS3\"><title>Lentiviral Vector Injections</title><p>All recombinant lentiviral products used in this study were designed and synthesized by Hanbio Biotechnological Co., Ltd. (Shanghai, China), and the lentiviral titre was calculated as 3 × 10<sup>8</sup> TU/ml. Lentiviral vectors (pHBLV-U6-Scramble-ZsGreen-Puro) containing OLFM3-short hairpin RNA (shRNA; CACTTAACAGGAGCCAAAGTGTATT) and a transgene encoding green fluorescent protein (GFP) (OLFM3-shRNA) were injected into the mouse hippocampus to locally knock down OLFM3, and lentiviral vectors (pHBLV-CMVIE-ZsGreen-Puro) encoding an amplified sequence of OLFM3 (NM_153458.3, OLFM3-LV) and GFP were injected into the mouse hippocampus to locally overexpress OLFM3. Moreover, identical lentiviral vectors containing either a non-sense control shRNA and GFP (Con-sh) or only GFP (Con-LV) were used as negative controls for the knockdown and overexpression experiments, respectively.</p><p>Intrahippocampal injection of lentiviral vectors was performed as in previous studies (<xref rid=\"B15\" ref-type=\"bibr\">Jagirdar et al., 2016</xref>; <xref rid=\"B48\" ref-type=\"bibr\">Zhang et al., 2016</xref>). Briefly, the mice were deeply anesthetized and then positioned in a stereotaxic frame (Stoelting Co., Ltd., United States). Three microliters of lentiviral particles were bilaterally injected into the dorsal blade of the CA1 area through a glass pipette (0.2 μl/min). The mice were randomly divided into the following 4 groups: OLFM3-LV, Con-LV, OLFM3-shRNA and Con-sh. Successful lentivirus infection was detected at 14, 30, and 45 days after injection by Western blotting (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Figure S2</xref>).</p></sec><sec id=\"S2.SS4\"><title>Behavioral Analysis</title><p>Two weeks after intrahippocampal injections of lentiviral vectors in mice, the epilepsy models were induced as described above, and the seizures were analyzed by two researchers who were blinded to the treatment conditions. For behavioral analysis in the PTZ kindling epileptic model, evoked seizures were assessed after PTZ injection according to Racine’s standard scale. Moreover, video monitoring was used to observe spontaneous recurrent seizures (SRSs) for 1 month, and the latency and frequency of SRSs of stages 3–5 (Racine’s standard scale) were recorded in the KA-induced epilepsy model. Seizure times of 24 h/day were measured for 21 consecutive days, beginning on the 14th day after SE. Mice were sacrificed on the 35th day after SE, and the neocortex and hippocampus were collected for the following experiments.</p><p>For immunofluorescence analysis, the brain tissues were fixed with 4% paraformaldehyde and then successively incubated in 20% and 30% sucrose in PBS for 24 h each. The tissues were then sectioned at 10 μm and stored at −20°C for later experimentation. For Western blotting, the neocortex and hippocampus were immediately frozen in liquid nitrogen and then stored at −80°C for later use.</p></sec><sec id=\"S2.SS5\"><title><italic>In vivo</italic> Multichannel Electroencephalogram (EEG) Recordings</title><p>The local field potential recording was conducted to prevent false positives in the behavior experiment. Following anesthesia, we implanted a multichannel microwire array into the hippocampus of each mouse and performed multichannel EEG recordings as previously described (<xref rid=\"B34\" ref-type=\"bibr\">Sada et al., 2015a</xref>). EEGs were recorded <italic>in vivo</italic> from mice in the chronic phase of the KA-induced epilepsy model using an OmniPlex D Neural Data Acquisition System (Plexon, Dallas, TX, United States). An electrophysiological seizure was defined as a seizure with a high frequency (>5 Hz), a high amplitude (>2 times the baseline), and a duration longer than 5 s (<xref rid=\"B49\" ref-type=\"bibr\">Zheng et al., 2017</xref>).</p></sec><sec id=\"S2.SS6\"><title>Immunofluorescence Staining</title><p>Immunofluorescence staining was conducted as described previously (<xref rid=\"B8\" ref-type=\"bibr\">Fachim et al., 2016</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Zhang et al., 2017</xref>). The following primary antibodies were used: rabbit anti-OLFM3 (1:50, Proteintech, Wuhan, China), mouse anti-glial fibrillary acidic protein (GFAP) (1:50, Boster Bioengineering, Wuhan, China), guinea pig anti-microtubule-associated protein 2 (MAP2) (1:200, Sysy, Göttingen, Germany), guinea pig anti-vGlut1(1:200, Sysy, Göttingen, Germany), mouse anti-PSD95 (1:100, Abcam, United States), mouse anti-GluA1 (1:50, Santa Cruz Biotechnology, United States), mouse anti-GluA2 (1:100, Abcam, United States), Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (1:50, Zhongshan Golden Bridge, Inc., Beijing, China), Alexa Fluor 594-conjugated goat anti-mouse IgG antibody (1:200, Zhongshan Golden Bridge Inc., Beijing, China), and Alexa Fluor 633-conjugated goat anti-guinea pig IgG antibody (1:50, Abcam, United States). Finally, the samples were treated with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma, St. Louis, MO, United States) for 10 min to identify the nuclei. Immunofluorescently labeled sections were imaged using a laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) and an Olympus IX 70 inverted microscope (Olympus America, Melville, NY, United States).</p></sec><sec id=\"S2.SS7\"><title>Western Blotting and Co-immunoprecipitation</title><sec id=\"S2.SS7.SSS1\"><title>Western Blotting</title><p>Tissue samples were collected from humans and mice for Western blotting analysis according to a previously described procedure (<xref rid=\"B48\" ref-type=\"bibr\">Zhang et al., 2016</xref>). Briefly, total protein and membrane proteins were extracted with commercial extraction kits (from Beyotime Institute of Biotechnology, Shanghai, China, and Thermo Fisher Scientific Corporation, United States, respectively) according to the manufacturers’ instructions. SDS-PAGE gels were used to separate total protein lysates, which were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, United States). The PVDF membranes were incubated with 5% skim milk for 1 h at 37°C to block non-specific binding. Later, the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-OLFM3 (1:500, Proteintech, Wuhan, China), mouse anti-GluA1 (1:200, Santa Cruz Biotechnology, United States), mouse anti-GluA2 (1:1,000, Abcam, United States), rabbit anti-GluA3 (1:1,000, Cell Signaling Technology, MA, United States), rabbit anti-GluA4 (1:500, Proteintech, Wuhan, China), mouse anti-PSD95 (1:1,000, Abcam, United States), rabbit anti-GAPDH (1:3,000, Proteintech, Wuhan, China) and rabbit anti-Na-K-ATPase (ATP1A1, 1:500, Proteintech, Wuhan, China). The secondary antibodies were incubated with the PVDF membranes for 1 h at 37°C on the next day. Enhanced chemiluminescence (ECL) reagent (Thermo, Marina, CA, United States) and a Fusion FX5 image analysis system (Vilber Lourmat Sté, Marne-la-Vallée, France) were used to visualize the bands. Finally, Quantity One software (Bio-Rad, CA, United States) was used to quantify the resulting optical density (OD) values, which were normalized to GAPDH or ATP1A1 expression.</p></sec><sec id=\"S2.SS7.SSS2\"><title>Co-immunoprecipitation</title><p>Protein extracts from mouse hippocampal tissues were homogenized and then mixed with immunoprecipitation (IP) lysis buffer. Equal amounts of protein were incubated with 2 μl of rabbit IgG (Abcam, Cambridge, MA, United States) as a polyclonal isotype control, 4 μl of OLFM3, 2 μl of GluA1, 2 μl of GluA2, 2 μl of GluA3, 2 μl of GluA4, or 2 μl of PSD95 antibodies for 4 h at 4°C; then, Protein A/G agarose beads (20 μl; Santa Cruz Biotechnology, Dallas, TX, United States) were incubated with the samples overnight at 4°C. The protein-bead complexes were washed 5 times and pelleted by centrifugation. Next, the supernatants were mixed with 1 × loading buffer and heated for Western blotting, which was conducted with the same antibodies as mentioned above.</p></sec></sec><sec id=\"S2.SS8\"><title>Electrophysiology</title><p>The hippocampal slices were prepared, and whole-cell patch-clamp recordings and electrophysiological analysis were conducted as described in a previous publication (<xref rid=\"B27\" ref-type=\"bibr\">Mouro et al., 2018</xref>). First, the mice were anesthetized 14 days after intrahippocampal lentiviral injection. Glass electrodes (3–5 MΩ) were placed in the CA1 cell layer of brain slices perfused with artificial cerebral spinal fluid (ACSF; 124 mM NaCl, 3 mM KCl, 1.23 mM NaH<sub>2</sub>PO<sub>4</sub>, 2 mM MgCl<sub>2</sub>, 2 mM CaCl<sub>2</sub>, 26 mM NaHCO<sub>3</sub>, 10 mM glucose, and pH 7.4) to record cellular electrophysiological changes. Spontaneous epilepsy was induced using Mg<sup>2+</sup>-free ACSF. A whole-cell current clamp was used to record the action potentials (APs) in the CA1 region to detect cellular excitability as previously reported. A whole-cell voltage-clamp recording technique was used to record miniature excitatory postsynaptic currents (mEPSCs). The mEPSCs were recorded at −70 mV, and the brain slices were bathed with ACSF containing 1 μM tetrodotoxin (TTX) and 10 μM bicuculline. To evaluate AMPAR-mediated EPSCs, we generated evoked currents using a 400-s pulse at a rate of 0.1 Hz (intensity, 50 to 200 A) delivered by a stimulation isolation unit using an S48 pulse generator (AstroMed). A bipolar stimulating electrode was positioned in the Schaffer collaterals. The evoked currents were measured in the presence of 100 M PTX and collected at two holding potentials. At -70 mV, with application of the N-methyl-D-aspartate receptor (NMDAR)-selective antagonist D-APV (50 M), the peak amplitude of the evoked EPSCs was identified as the AMPAR-mediated current. To record the paired pulse ratio (PPR) of the AMPA-EPSCs, a bipolar stimulating electrode was positioned in the Schaffer collaterals. EPSCs were evoked at a holding potential of -70 mV in the presence of 100 M PTX and 50 M D-APV. The intervals of paired stimulations were set at 25, 50, and 100 ms. The values of the ratios were defined as [p2/p1], where p1 and p2 are the amplitudes of the EPSCs evoked by the first and second pulses, respectively (<xref rid=\"B3\" ref-type=\"bibr\">Beaudoin et al., 2018</xref>; <xref rid=\"B40\" ref-type=\"bibr\">Wang et al., 2018</xref>). All recordings were monitored with a Multiclamp 700B amplifier (Axon, United States), digitized at 10 kHz and filtered at 2 kHz. Data were collected after the currents had been stable without rundown for at least 5 min (5 neurons from 5 mice per group). Mini Analysis 6.0.1 and pClamp 9.2 software (Axon, United States) were used to analyse the recording data.</p></sec><sec id=\"S2.SS9\"><title>Statistical Analysis</title><p>After verifying that the experimental data exhibited normal distributions and equal variances, we analyzed the data in SPSS 18.0 (SPSS, Inc., Chicago, IL, United States) using the Student’s <italic>t</italic>-test to evaluate differences between two groups and one-way ANOVA to evaluate differences among more than two groups. Each experiment was repeated three times. The χ<sup>2</sup> test was used to compare gender-specific differences between patients with TLE and controls. <italic>P</italic> < 0.05 and <italic>P</italic> < 0.01 indicate statistically significant differences. These data were presented as the means ± standard error of the mean (SEM) and were analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA, United States).</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Expression of OLFM3 in Patients With TLE and in Mouse Epilepsy Models</title><p>The detailed clinical features of TLE patients and control subjects are presented in <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Tables S1</xref>, <xref ref-type=\"supplementary-material\" rid=\"TS1\">S2</xref>, respectively (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Material</xref> and <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Tables S1</xref>, <xref ref-type=\"supplementary-material\" rid=\"TS1\">S2</xref>). No significant difference in age or gender was found between the TLE and control groups (<italic>P ></italic> 0.05, <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table S2</xref>). First, Western blotting was applied to survey OLFM3 expression levels in the cortices of patients with TLE (<italic>n</italic> = 24) and controls (<italic>n</italic> = 12). The expression of OLFM3 was significantly higher in the TLE group than the control group (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>) (<italic>P</italic> < 0.01). Next, OLFM3 expression in mouse models of epilepsy induced by PTZ (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>) and KA (<xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref>) was assessed by Western blotting. As in human samples, the OLFM3 expression levels were significantly upregulated in the hippocampus and cortex of the epileptic compared with the control mice (<italic>n</italic> = 5 per group; <italic>P</italic> < 0.01).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Increased OLFM3 expression in patients with TLE and the two mouse models. <bold>(A)</bold> Representative Western blots showing significantly increased OLFM3 expression in the cortex of patients with TLE (<italic>n</italic> = 24) compared with controls (<italic>n</italic> = 12) (**<italic>P</italic> < 0.01). <bold>(B)</bold> OLFM3 expression levels were significantly increased in the cortex and hippocampus of PTZ-kindled epileptic mice compared with control mice (<italic>n</italic> = 5 per group; **<italic>P</italic> < 0.01). <bold>(C)</bold> OLFM3 expression was significantly higher in the cortex and hippocampus of the spontaneous seizure group induced by KA than the non-spontaneous seizure group (<italic>n</italic> = 5 per group; **<italic>P</italic> < 0.01). All expression levels of OLFM3 were normalized by calculating the OD ratio of OLFM3 to GAPDH (OLFM3/GAPDH).</p></caption><graphic xlink:href=\"fcell-08-00722-g001\"/></fig></sec><sec id=\"S3.SS2\"><title>Localization of OLFM3 in the Human Neocortex and in the Mouse Cortex and Hippocampus</title><p>Immunofluorescence staining showed that OLFM3 was co-localized with the neuronal dendrite marker MAP2 (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>) but was not co-expressed with GFAP (a marker of astrocytes) in the human neocortex (<xref ref-type=\"fig\" rid=\"F2\">Figure 2D</xref>). Furthermore, the localization of OLFM3 was examined in mice, and similar results showed that OLFM3 was co-expressed with MAP2 (<xref ref-type=\"fig\" rid=\"F2\">Figures 2B,C</xref>) but not GFAP in both the cortex and hippocampus (<xref ref-type=\"fig\" rid=\"F2\">Figures 2E,F</xref>). Additionally, the expression of OLFM3 overlapped with that of the excitatory postsynaptic marker PSD95 but not the excitatory presynaptic marker vGlut1 in the human neocortex and in the mouse cortex and hippocampus (<xref ref-type=\"fig\" rid=\"F2\">Figures 2G–I</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Localization of OLFM3 in the human neocortex and in the mouse cortex and hippocampus. <bold>(A–C)</bold> OLFM3 (green) and MAP2 (red) were co-expressed (yellow) in the human neocortex <bold>(A)</bold>, mouse cortex <bold>(B)</bold>, and mouse hippocampus <bold>(C)</bold>, while no co-localization of OLFM3 and GFAP (red) was detected <bold>(D–F)</bold>. <bold>(G–I)</bold> Representative images show that OLFM3 expression (green) overlapped (yellow) with that of the excitatory postsynaptic marker PSD95 (red) but not the excitatory presynaptic marker vGlut1 (purple) in the human neocortex <bold>(G)</bold> and mouse cortex <bold>(H)</bold> and hippocampus <bold>(I)</bold>. DAPI (blue) indicates cell nuclei. White squares indicate positive cells, and the scale bar represents 20 or 50 μm.</p></caption><graphic xlink:href=\"fcell-08-00722-g002\"/></fig></sec><sec id=\"S3.SS3\"><title>Effect of OLFM3-LV and OLFM3-shRNA on Epileptic Seizure Activity</title><p>As shown above, OLFM3 expression was increased in epileptic tissue. Follow-up experiments were performed to detect whether OLFM3 deletion might have beneficial effects on the two epilepsy models (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Material</xref> and <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Figure S1</xref>). First, OLFM3 was overexpressed or knocked down locally in the hippocampus by intrahippocampal injection of a corresponding lentivirus bearing GFP. Next, GFP autofluorescence was detected in the mouse hippocampus by confocal microscopy after injection, confirming successful lentiviral infection (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Material</xref> and <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Figure S2a</xref>). Moreover, Western blotting revealed that OLFM3 expression was significantly higher in the OLFM3-LV-treated than the Con-LV-treated mice but lower in the OLFM3-shRNA-treated than the Con-sh-treated mice (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Material</xref> and <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Figure S2</xref>). Finally, the behavioral data for PTZ-kindled epileptic mice (<italic>n</italic> = 6–9 per group) (<italic>P</italic> < 0.05) (<xref ref-type=\"fig\" rid=\"F3\">Figures 3A,B</xref>) and mice with KA-induced epilepsy (<italic>n</italic> = 6–9 per group) (<italic>P</italic> < 0.05) (<xref ref-type=\"fig\" rid=\"F3\">Figures 3C,D</xref>) were analyzed. Seizure scores after each PTZ injection according to Racine’s standard scale were significantly increased in the OLFM3-LV-treated group (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>) but decreased in the OLFM3-shRNA-treated group compared with the scores in the corresponding control groups (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>). In the KA-induced epilepsy model, OLFM3-LV injection shortened the spontaneous seizure onset latency, while OLFM3-shRNA injection prolonged the onset latency compared with Con-sh injection (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>). Moreover, the OLFM3-LV-treated group had a significantly higher while the OLFM3-shRNA-treated group had a significantly lower SRS frequency than the corresponding control groups (<xref ref-type=\"fig\" rid=\"F3\">Figure 3D</xref>). Additionally, multichannel EEG recordings were conducted <italic>in vivo</italic> concurrently after behavior monitoring (<xref ref-type=\"fig\" rid=\"F3\">Figures 3E–H</xref>). The EEG recording data demonstrated that the total number and duration of the electrical epileptiform discharges were significantly increased in the OLFM3-LV-treated group but decreased in the OLFM3-shRNA-treated group compared with the corresponding control groups (<italic>n</italic> = 6–9 per group) (<italic>P</italic> < 0.05 or 0.01) (<xref ref-type=\"fig\" rid=\"F3\">Figures 3I,J</xref>).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Effects of OLFM3-LV and OLFM3-shRNA on epileptic seizure activity. <bold>(A,B)</bold> Daily seizure scores were significantly increased in the OLFM3-LV-treated group but were significantly decreased in the OLFM3-shRNA-treated group compared with the corresponding control groups in the PTZ kindling epileptic model (<italic>n</italic> = 6–9 in each group) (<sup>∗</sup><italic>P</italic> < 0.05). <bold>(C)</bold> The latency of SRSs in the OLFM3-LV-treated group with KA-induced epilepsy was significantly shorter than in the controls, while the latency in the OLFM3-shRNA-treated group was significantly longer than in the controls (<italic>n</italic> = 6–9 in each group) (<sup>∗</sup><italic>P</italic> < 0.05). <bold>(D)</bold> Mice injected with OLFM3-LV had more SRSs than the control group. However, mice injected with OLFM3-shRNA had less frequent SRSs than control mice (<italic>n</italic> = 6–9 in each group) (<sup>∗</sup><italic>P</italic> < 0.05). <bold>(E–H)</bold> Representative electrical epileptiform discharges in each group (Con-LV, OLFM3-LV, Con-sh, and OLFM3-shRNA). <bold>(I,J)</bold> EEG recordings showed that the mice injected with OLFM3-LV had a significantly increased frequency and total duration of electrical epileptiform discharges compared with the mice injected with Con-LV, while the frequency and total duration of the electrical epileptiform discharges were significantly decreased in the OLFM3-shRNA-treated group compared with the control group (<italic>n</italic> = 6–9 in each group) (<sup>∗</sup><italic>P</italic> < 0.05, <sup>∗∗</sup><italic>P</italic> < 0.01).</p></caption><graphic xlink:href=\"fcell-08-00722-g003\"/></fig></sec><sec id=\"S3.SS4\"><title>Effects of OLFM3-LV and OLFM3-shRNA on Electrophysiology</title><p>Whole-cell patch-clamp recording was conducted to further investigate the effect of OLFM3-LV and OLFM3-shRNA on electrophysiology in an Mg<sup>2+</sup>-free-induced brain-slice model of epileptiform activity 14 days after OLFM3-LV or OLFM3-shRNA injection. The recording data from the pyramidal neurons in the CA1 region showed that the frequency of APs was significantly increased in the OLFM3-LV-treated group compared with the Con-LV-treated group. In contrast, the frequency of APs was significantly decreased in the OLFM3-shRNA-treated group compared with the Con-sh-treated group (<italic>n</italic> = 5 per group; <italic>P</italic> < 0.05 or 0.01) (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>). To examine whether OLFM3-LV and OLFM3-shRNA altered excitatory neurotransmission, we recorded mEPSCs. The mEPSC amplitude was significantly higher in the OLFM3-LV-treated group but lower in the OLFM3-shRNA-treated group than in the corresponding control groups (<italic>n</italic> = 5 per group; <italic>P</italic> < 0.05 or 0.01) (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>). However, there was no significant difference in the frequency of mEPSCs among the groups (<italic>n</italic> = 5 per group; <italic>P</italic> > 0.05) (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>). Furthermore, PPR was assessed to determine whether OLFM3-LV and OLFM3-shRNA altered excitatory presynaptic release. There were no significant differences in PPR between the OLFM3-LV or OLFM3-shRNA group and their corresponding controls (<italic>n</italic> = 5 per group; <italic>P</italic> > 0.05) (<xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>). Finally, evoked AMPAR-mediated currents were recorded to determine whether the change in excitatory neurotransmission was mediated by AMPAR currents. The results revealed that AMPAR-mediated currents were significantly increased in the OLFM3-LV group and decreased in the OLFM3-shRNA group compared with the corresponding control groups (<italic>n</italic> = 5 per group; <italic>P</italic> < 0.05) (<xref ref-type=\"fig\" rid=\"F4\">Figure 4D</xref>).</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Effects of OLFM3-LV and OLFM3-shRNA on electrophysiology. <bold>(A)</bold> The frequency of APs was significantly higher in the OLFM3-LV group than the Con-LV group, while the frequency of APs was lower in the OLFM3-shRNA group than the Con-sh group. <bold>(B)</bold> The amplitude of mEPSCs was remarkably increased in the OLFM3-LV group but significantly decreased in the OLFM3-shRNA group. There was no significant difference in mEPSC frequency between the OLFM3-LV and OLFM3-shRNA groups and the corresponding control groups. <bold>(C)</bold> There were no significant differences in PPR between the OLFM3-LV or OLFM3-shRNA group and their corresponding control groups. <bold>(D)</bold> The AMPAR current amplitude was significantly increased in the OLFM3-LV group and significantly decreased in the OLFM3-shRNA group compared with the current amplitude in the corresponding control group (<italic>n</italic> = 5 per group; *<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01).</p></caption><graphic xlink:href=\"fcell-08-00722-g004\"/></fig></sec><sec id=\"S3.SS5\"><title>Effects of OLFM3-LV and OLFM3-shRNA on AMPARs</title><p>As demonstrated above, OLFM3 was localized to the excitatory presynaptic PSD95 and increased AMPAR-mediated currents. A series of follow-up experiments were performed to survey whether OLFM3 could interact with AMPARs and alter their expression to increase AMPA currents. Using the epileptic hippocampal tissue for co-immunoprecipitation, we demonstrated that anti-OLFM3 co-immunoprecipitation with anti-GluA1, anti-GluA2 and anti-PSD95 (<xref ref-type=\"fig\" rid=\"F5\">Figures 5A–C</xref>) but not anti-GluA3 or anti-GluA4 (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Figure S3</xref>). Furthermore, immunofluorescence showed that the expression of OLFM3 overlapped with that of GluA1 (<xref ref-type=\"fig\" rid=\"F5\">Figures 5D–F</xref>) and GluA2 (<xref ref-type=\"fig\" rid=\"F5\">Figures 5G–I</xref>) not only in the human neocortex but also in the mouse cortex and hippocampus. Taken together, these results verified that OLFM3 interacted with AMPARs (GluA1 and GluA2). Next, Western blotting was conducted to examine whether GluA1 and GluA2 protein expression levels in the hippocampus of mice with KA-induced epilepsy were altered by overexpression or knockdown of OLFM3; however, neither GluA1 nor GluA2 total protein levels were significantly different between the experimental and control groups (<italic>n</italic> = 6–10 in each group) (<italic>P</italic> > 0.05) (<xref ref-type=\"fig\" rid=\"F6\">Figures 6A–D</xref>). Next, the membrane/surface expression levels of GluA1 and GluA2 were measured. The results showed that the membrane/surface expression levels of both GluA1 and GluA2 were significantly higher in the OLFM3-LV-treated group than the Con-LV-treated group, while they were significantly lower in the OLFM3-shRNA-treated group than the Con-sh-treated group (<italic>n</italic> = 6–10 in each group) (<italic>P</italic> < 0.01) (<xref ref-type=\"fig\" rid=\"F6\">Figures 6A–D</xref>). These results revealed that OLFM3 might affect AMPA currents via interaction with AMPARs and abnormal membrane expression of the GluA1 and GluA2 proteins.</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Co-localization of OLFM3 and AMPARs. <bold>(A–C)</bold> Co-immunoprecipitation of GluA1, GluA2, and PSD95 with OLFM3 demonstrated by the reciprocal co-immunoprecipitation of anti-OLFM3 with anti-GluA1, anti-GluA2, and anti-PSD95, respectively. Immunofluorescence revealed that the distribution of OLFM3 (green) overlapped (yellow) with that of GluA1 (red) in the human neocortex <bold>(D)</bold> and mouse cortex <bold>(E)</bold> and hippocampus <bold>(F)</bold>. <bold>(G–I)</bold> Representative immunofluorescence images show that OLFM3 (green) was co-expressed (yellow) with GluA2 (red) in the human neocortex <bold>(G)</bold> and mouse cortex <bold>(H)</bold> and hippocampus <bold>(I)</bold>. Cell nuclei were counterstained with DAPI (blue). The arrows and white squares indicate positive cells, and the scale bar represents 50 μm.</p></caption><graphic xlink:href=\"fcell-08-00722-g005\"/></fig><fig id=\"F6\" position=\"float\"><label>FIGURE 6</label><caption><p>Effects of OLFM3-LV and OLFM3-shRNA on AMPARs in epileptic mice. <bold>(A–D)</bold> There was no significant difference in the expression of total GluA1 <bold>(A,B)</bold> or GluA2 <bold>(C,D)</bold> in the hippocampus of either the OLFM3-LV-treated or the OLFM3-shRNA-treated group compared with the corresponding control groups. However, the membrane expression levels of GluA1 <bold>(A,B)</bold> and GluA2 <bold>(C,D)</bold> were significantly higher in the OLFM3-LV group than in control mice, and the membrane expression levels of GluA1 <bold>(A,B)</bold> and GluA2 <bold>(C,D)</bold> membrane proteins were significantly lower in the OLFM3-shRNA group than the corresponding controls. Data are presented as the mean ± SEM (<italic>n</italic> = 6–10 in each group) (**<italic>P</italic> < 0.01).</p></caption><graphic xlink:href=\"fcell-08-00722-g006\"/></fig></sec></sec><sec id=\"S4\"><title>Discussion</title><p>In this paper, we found that OLFM3 expression was significantly increased in the brains of TLE patients and two classic models (PTZ- and KA-induced mouse models of epilepsy). We present the novel finding that lentivirus-mediated overexpression of OLFM3 in the hippocampus increased the susceptibility of mice to seizures in the models. Furthermore, OLFM3 affected AMPAR currents in a brain-slice model of epileptiform activity induced by Mg2+-free medium. Most notably, we found that OLFM3 may interact with GluA1 and GluA2 to modulate seizure activity.</p><p>As a member of the olfactomidin domain family, OLFM3 has been found to increase the activity of mucin, stimulate cells to adhere to each other, promote close connections between cells, enable cell migration, and regulate the formation of the cytoskeleton during the process of cell migration (<xref rid=\"B2\" ref-type=\"bibr\">Anholt, 2014</xref>). Differences in the gene coding for OLFM3 have been found between patients with epilepsy and controls (<xref rid=\"B13\" ref-type=\"bibr\">Heinzen et al., 2010</xref>). In the present study, most of the OLFM3-positive cells in both human and mouse brain tissues co-expressed neuronal markers but not glial cell markers. The expression of OLFM3 was significantly increased in the epileptic focus specimens of patients and in the hippocampus and cortex of the classic models of epilepsy. It is not known whether the increase of OLFM3 caused by epilepsy or epilepsy caused by the increase of OLFM3. In order to explore this problem, we need to carry out follow-up experiments. However, due to the limitations of ethics, we couldn’t find the answer in human hippocampus.</p><p>Therefore, we conducted behavioral experiments on two classic epilepsy models to further explore this phenomenon. Lentivirus-mediated overexpression of OLFM3 in the hippocampus is found increased the susceptibility of mice to PTZ-induced seizures. In order to prevent false positives of a single model, we used KA-induced chronic epilepsy model for further verification. The EEG recording data from the KA-induced epileptic mice is consistent with the results of the PTZ-induced seizures.</p><p>Furthermore, whole-cell patch-clamp recordings revealed that overexpression or knockdown of OLFM3 increased or decreased AP frequency, mEPSC amplitude and AMPAR-mediated currents, respectively, in a brain-slice model of epileptiform activity induced by Mg<sup>2+</sup>-free medium. Previous studies have demonstrated that AMPARs are tetramers composed of GluA1-4 subunits, of which GluA1-GluA2 and GluA2-GluA3 are mainly found in mature hippocampal excitatory synapses, and the role of GluA1-GluA2 is particularly important (<xref rid=\"B25\" ref-type=\"bibr\">Mayer, 2016</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Gupta, 2018</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Liu et al., 2018</xref>). AMPARs mediate the fast, transient transmission of excitatory signals in the central nervous system by combining with PSD95, which anchors AMPARs to the postsynaptic membrane, connects AMPARs to other signaling molecules, and allows for the interaction with other PSD95 molecules and AMPARs modification through phosphorylation, palm acylation and so on. Interestingly, OLFM3 co-localized with the excitatory postsynaptic marker PSD95 and with GluA1 and GluA2 in this study. By mediating excitatory synaptic transmission and enhancing the excitatory postsynaptic current, AMPARs ultimately participate in the onset of epilepsy and development (<xref rid=\"B16\" ref-type=\"bibr\">Jaremko et al., 2017</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Widagdo et al., 2017</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Joshi et al., 2018</xref>). Thus it can be seen AMPAR complexes play a major role in the pathogenesis and development of epilepsy (<xref rid=\"B25\" ref-type=\"bibr\">Mayer, 2016</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Adotevi and Leitch, 2017</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Cai et al., 2017</xref>). Proteomics and mass spectrometry analysis had been revealed that OLFM3 may be involved in the formation of AMPAR complexes (<xref rid=\"B36\" ref-type=\"bibr\">Schwenk et al., 2012</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Shanks et al., 2012</xref>). Additionally, <xref rid=\"B39\" ref-type=\"bibr\">Sultana et al. (2014)</xref> found that the formation of AMPAR complexes was inhibited by knocking out OLFM2, thereby reducing AMPAR-mediated learning, cognitive and motor functions in mice. Whether OLFM3 also plays a certain regulatory role in the formation of AMPAR complex to participate in the development of epilepsy, unfortunately, there has been no relevant report before.</p><p>Previous studies have shown that the transport of AMPARs to the membrane is a dynamic circulation process. After the receptor is internalized, a part is degraded by lysosomes, and the other part is returned to the membrane through cell phagocytosis after the reorganization of the cytoplasmic membrane network structure to restore its role in excitatory synaptic transmission. Changes in the amount of AMPAR in the membrane can cause changes in the excitability of the central nervous system (<xref rid=\"B5\" ref-type=\"bibr\">Derkach et al., 2007</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Widagdo et al., 2016</xref>). Recently, some researchers have found that the dentate gyrus granule cells of TLE and other animal models of epilepsy form abnormal mossy fiber synapses, which indicates that the recruitment of AMPAR (especially GluA1 and GluA2 subunits) is induced (<xref rid=\"B19\" ref-type=\"bibr\">Joshi et al., 2017</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Joshi and Kapur, 2018</xref>). Another study showed that GluA1 and GluA2 mediated increased excitatory transmission of hippocampal CA1 pyramidal neurons. This increase may play an important role in exacerbating menstrual seizures (<xref rid=\"B20\" ref-type=\"bibr\">Joshi et al., 2018</xref>). Other studies have further confirmed that enhanced surface expression of GluA1 in SE can induce AMPAR-mediated increase in excitatory neurotransmission in CA1 pyramidal neurons (<xref rid=\"B19\" ref-type=\"bibr\">Joshi et al., 2017</xref>). It was found that the degradation of AMPAR is regulated by the GluA2 subunit and its related proteins (<xref rid=\"B31\" ref-type=\"bibr\">Parkinson et al., 2018</xref>). The co-immunoprecipitation experiments in this study showed that OLFM3 interacted with GluA1, GluA2 and PSD95, respectively, in this study. The overexpression and knockdown of OLFM3 had no effect on the total protein of GluA1 or GluA2, but the overexpression of OLFM3 increased the membrane expression of GluA1 and GluA2 in the hippocampus, and after the decrease of OLFM3 expression, the membrane expression of GluA1 and GluA2 decreased. Therefore, OLFM3 may not participate in the synthesis or degradation of AMPAR. By regulating the translocation of GluA1 and GluA2 membrane expression, it may be related to the change in the excitability of epilepsy.</p></sec><sec id=\"S5\"><title>Conclusion</title><p>In this study, we demonstrated that OLFM3 might participate in the formation of AMPAR complexes. Upregulation of OLFM3 increased the excitatory postsynaptic currents and enhanced seizure activity by increasing the surface expression of GluA1 and GluA2 in neuronal cells. Downregulation of OLFM3 may have a therapeutic effect on epilepsy. However, the exact mechanism of these effects requires further study.</p></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>All datasets presented in this study are included in the article/<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Material</xref>.</p></sec><sec id=\"S7\"><title>Ethics Statement</title><p>This study was approved by the Ethics Committee of Zunyi Medical College and Chongqing Medical University according to the World Medical Association’s Declaration of Helsinki. Before undergoing surgery, all patients or their lineal relatives provided written informed consent for use of the resected tissue in research. No potentially identifiable human images or data are presented in this study. The methods were conducted in compliance with the ethical guidelines for medical and health research involving human subjects as established by the National Institutes of Health and the Committee on Human Research at Zunyi Medical College and Chongqing Medical University. All animal experiments were performed according to the principles outlined in the National Institutes of Health guide (NIH Publication No. 8623, revised 1987). All animal experimental protocols were reviewed and approved by the Ethics Committee of Zunyi Medical College and Chongqing Medical University (Approval No. 0002648). In addition, all efforts were made to reduce the number of animals used in the experiment and to minimize their suffering.</p></sec><sec id=\"S8\"><title>Author Contributions</title><p>ZX, TW, and ST conceived and designed the experiments. YG, DL, YZ, YL, and ST performed the experiments. PX, JZ, and ZL collected the data and carried out the statistical analysis. ST, CY, and XZ wrote the manuscript. All authors contributed to the preparation of the manuscript and approved the final contributions.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> The authors’ research was supported by grants from the National Natural Science Foundation of China (Nos. 81660227 and 81560224), the Guizhou Province Science and Technology Cooperation Project [No. (2015) 7520], and the Guizhou Province Health and Family Planning Commission Science and Technology Foundation (No: gzwjkj 2015-1-052).</p></fn></fn-group><ack><p>The authors are sincerely grateful to Beijing Tiantan Hospital and Xuanwu Hospital of the Capital University of Medical Sciences for supporting the study by supplying the brain tissues. 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Rep.</italic></source>\n<volume>6</volume>:<issue>37713</issue>.</mixed-citation></ref><ref id=\"B49\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zheng</surname><given-names>F.</given-names></name><name><surname>Yang</surname><given-names>Y.</given-names></name><name><surname>Lu</surname><given-names>S.</given-names></name><name><surname>Yang</surname><given-names>Q.</given-names></name><name><surname>Li</surname><given-names>Y.</given-names></name><name><surname>Xu</surname><given-names>X.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>CD36 deficiency suppresses epileptic seizures.</article-title>\n<source><italic>Neuroscience</italic></source>\n<volume>367</volume>\n<fpage>110</fpage>–<lpage>120</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuroscience.2017.10.024</pub-id>\n<pub-id pub-id-type=\"pmid\">29111364</pub-id></mixed-citation></ref><ref id=\"B50\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhu</surname><given-names>X.</given-names></name><name><surname>Shen</surname><given-names>K.</given-names></name><name><surname>Bai</surname><given-names>Y.</given-names></name><name><surname>Zhang</surname><given-names>A.</given-names></name><name><surname>Xia</surname><given-names>Z.</given-names></name><name><surname>Chao</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>NADPH oxidase activation is required for pentylenetetrazole kindling-induced hippocampal autophagy.</article-title>\n<source><italic>Free Rad. Biol. Med.</italic></source>\n<volume>94</volume>\n<fpage>230</fpage>–<lpage>242</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.freeradbiomed.2016.03.004</pub-id>\n<pub-id pub-id-type=\"pmid\">26969791</pub-id></mixed-citation></ref></ref-list><glossary><title>Abbreviations</title><def-list id=\"DL1\"><def-item><term>AEDs</term><def><p>antiepileptic drugs</p></def></def-item><def-item><term>AMPAR</term><def><p>α -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor</p></def></def-item><def-item><term>APs</term><def><p>action potentials</p></def></def-item><def-item><term>DAPI</term><def><p>4’,6-diamidino-2-phenylindole dihydrochloride</p></def></def-item><def-item><term>ECL</term><def><p>Enhanced chemiluminescence</p></def></def-item><def-item><term>EEGs</term><def><p>electroencephalograms</p></def></def-item><def-item><term>GFAP</term><def><p>glial fibrillary acidic protein</p></def></def-item><def-item><term>GFP</term><def><p>green fluorescent protein</p></def></def-item><def-item><term>IP</term><def><p>immunoprecipitation</p></def></def-item><def-item><term>KA</term><def><p>kainic acid</p></def></def-item><def-item><term>MAP2</term><def><p>microtubule-associated protein 2</p></def></def-item><def-item><term>mEPSCs</term><def><p>miniature excitatory postsynaptic currents</p></def></def-item><def-item><term>NMDAR</term><def><p>N-methyl -D-aspartate receptor</p></def></def-item><def-item><term>OD</term><def><p>optical density</p></def></def-item><def-item><term>OLFM3</term><def><p>Olfactomedin-3</p></def></def-item><def-item><term>PPR</term><def><p>paired pulse ratio</p></def></def-item><def-item><term>PSD-95</term><def><p>postsynaptic density 95</p></def></def-item><def-item><term>PTZ</term><def><p>pentylenetetrazol</p></def></def-item><def-item><term>PVDF</term><def><p>polyvinylidene fluoride</p></def></def-item><def-item><term>SE</term><def><p>status epilepticus</p></def></def-item><def-item><term>SPF</term><def><p>specific-pathogen-free</p></def></def-item><def-item><term>SRSs</term><def><p>spontaneous recurrent seizures</p></def></def-item><def-item><term>TLE</term><def><p>temporal lobe epilepsy</p></def></def-item><def-item><term>vGlut1</term><def><p>vesicular glutamate transporter 1.</p></def></def-item></def-list></glossary></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Microbiol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Microbiol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Microbiol.</journal-id><journal-title-group><journal-title>Frontiers in Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-302X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849425</article-id><article-id pub-id-type=\"pmc\">PMC7431668</article-id><article-id pub-id-type=\"doi\">10.3389/fmicb.2020.01821</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Microbiology</subject><subj-group><subject>Mini Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Coronavirus Interplay With Lipid Rafts and Autophagy Unveils Promising Therapeutic Targets</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Fecchi</surname><given-names>Katia</given-names></name><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/998544/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Anticoli</surname><given-names>Simona</given-names></name><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/545143/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Peruzzu</surname><given-names>Daniela</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/990474/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Iessi</surname><given-names>Elisabetta</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/917253/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Gagliardi</surname><given-names>Maria Cristina</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/545157/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Matarrese</surname><given-names>Paola</given-names></name><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/407130/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ruggieri</surname><given-names>Anna</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/549567/overview\"/></contrib></contrib-group><aff><institution>Reference Center for Gender Specific Medicine, Istituto Superiore di Sanità</institution>, <addr-line>Rome</addr-line>, <country>Italy</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Joseph Alex Duncan, The University of North Carolina at Chapel Hill, United States</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Maryam Dadar, Razi Vaccine and Serum Research Institute, Iran; Roberta Olmo Pinheiro, Oswaldo Cruz Foundation, Brazil</p></fn><corresp id=\"c001\">*Correspondence: Paola Matarrese, <email>paola.matarrese@iss.it</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>These authors have contributed equally to this work</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>11</day><month>8</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>11</volume><elocation-id>1821</elocation-id><history><date date-type=\"received\"><day>30</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>10</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Fecchi, Anticoli, Peruzzu, Iessi, Gagliardi, Matarrese and Ruggieri.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Fecchi, Anticoli, Peruzzu, Iessi, Gagliardi, Matarrese and Ruggieri</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Coronaviruses are enveloped, single-stranded, positive-sense RNA viruses that can infect animal and human hosts. The infection induces mild or sometimes severe acute respiratory diseases. Nowadays, the appearance of a new, highly pathogenic and lethal coronavirus variant, SARS-CoV-2, responsible for a pandemic (COVID-19), represents a global problem for human health. Unfortunately, only limited approaches are available to treat coronavirus infections and a vaccine against this new coronavirus variant is not yet available. The plasma membrane microdomain lipid rafts have been found by researchers to be involved in the replication cycle of numerous viruses, including coronaviruses. Indeed, some pathogen recognition receptors for coronaviruses as for other viruses cluster into lipid rafts, and it is therefore conceivable that the first contact between virus and host cells occurs into these specialized regions, representing a port of cell entry for viruses. Recent data highlighted the peculiar pro-viral or anti-viral role played by autophagy in the host immune responses to viral infections. Coronaviruses, like other viruses, were reported to be able to exploit the autophagic machinery to increase their replication or to inhibit the degradation of viral products. Agents known to disrupt lipid rafts, such as metil-β-cyclodextrins or statins, as well as autophagy inhibitor agents, were shown to have an anti-viral role. In this review, we briefly describe the involvement of lipid rafts and autophagy in coronavirus infection and replication. We also hint how lipid rafts and autophagy may represent a potential therapeutic target to be investigated for the treatment of coronavirus infections.</p></abstract><kwd-group><kwd>coronavirus</kwd><kwd>lipid rafts</kwd><kwd>autophagy</kwd><kwd>SARS-CoV-2</kwd><kwd>drugs</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Ministero della Salute<named-content content-type=\"fundref-id\">10.13039/501100003196</named-content></funding-source><award-id rid=\"cn001\">PE-2013-02354961</award-id></award-group></funding-group><counts><fig-count count=\"1\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"118\"/><page-count count=\"9\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Coronaviruses (CoVs) are a large group of enveloped animal and human viruses, with a single-stranded positive-sense RNA genome. The Coronaviridae family, to which they belong, includes four genera of CoVs, indicated as Alphacoronavirus (αCoV), Betacoronavirus (βCoV), Gammacoronavirus (γCoV), and Deltacoronavirus (δCoV) (<xref rid=\"B29\" ref-type=\"bibr\">Cui et al., 2019</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Chen et al., 2020</xref>). αCoV and βCoV have evolutionary evolved from bats and rodents, and have been responsible for human infections; whereas δCoV and γCoV derive from avian species (<xref rid=\"B103\" ref-type=\"bibr\">Woo et al., 2012</xref>). αCoV and βCoV are further divided into subgroups, 1a-1b and 2a-2d, respectively, (<xref rid=\"B47\" ref-type=\"bibr\">Graham et al., 2013</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Drexler et al., 2014</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Cui et al., 2019</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Fung and Liu, 2019</xref>) (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1</xref>). Coronaviruses have been regarded as etiologic agents of both relatively mild and severe respiratory infections/diseases in humans (<xref rid=\"B114\" ref-type=\"bibr\">Zhao et al., 2012</xref>; <xref rid=\"B78\" ref-type=\"bibr\">Paules et al., 2020</xref>). HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1 cause the common cold and mild upper respiratory diseases in immunocompetent hosts, although some of them can be potentially virulent in infants, young children, and elder individuals. Highly pathogenic human coronaviruses, responsible for more severe respiratory diseases, include SARS-CoV that caused the 2003 pandemic outbreak of severe respiratory tract infection (<xref rid=\"B54\" ref-type=\"bibr\">Ksiazek et al., 2003</xref>), MERS-CoV, responsible for the 2012 outbreak in Middle Eastern countries (<xref rid=\"B112\" ref-type=\"bibr\">Zaki et al., 2012</xref>), and the novel SARS-CoV-2, actually causing a pandemic of severe pneumonia that started in China in December 2019 (<xref rid=\"B5\" ref-type=\"bibr\">Benvenuto et al., 2019</xref>; <xref rid=\"B115\" ref-type=\"bibr\">Zhu et al., 2020</xref>).</p><p>Coronavirus and coronavirus-like infections have been described also in domestic and wild animals, such as swine [porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus], cattle (BCoV), horses, camels, cats, dogs, rodents, birds, bats, rabbits, ferrets, mink, and various wildlife species (<xref rid=\"B36\" ref-type=\"bibr\">Fehr and Perlman, 2015</xref>; <xref rid=\"B61\" ref-type=\"bibr\">Lin et al., 2016</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Banerjee et al., 2019</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Cui et al., 2019</xref>; <xref rid=\"B109\" ref-type=\"bibr\">Ye et al., 2020</xref>). Although many coronavirus infections are subclinical in animals, however, they cause a range of diseases that can have serious consequences for animal health as well as for the economic losses in livestock. In addition, domestic animals may have been important intermediate hosts for virus transmission from natural hosts to humans (<xref rid=\"B36\" ref-type=\"bibr\">Fehr and Perlman, 2015</xref>; <xref rid=\"B109\" ref-type=\"bibr\">Ye et al., 2020</xref>).</p><p>Since only limited approaches are available to treat or prevent coronavirus infections (<xref rid=\"B113\" ref-type=\"bibr\">Zhang et al., 2020</xref>), the importance of identifying novel therapeutic targets is evident.</p><p>Viruses have evolved a close interplay with the host cell and the first encounter between the virus and target cell occurs at the plasma membrane. Lipid rafts are specialized plasma membrane microdomains involved in important processes of the virus infections and of the host target cells (<xref rid=\"B86\" ref-type=\"bibr\">Rosenberger et al., 2000</xref>).</p><p>Viruses exploit lipid rafts to gain infection of the target cells; therefore, pharmacologic depletion or disruption of lipid rafts may provide a tool to reduce or inhibit viral replication (<xref rid=\"B52\" ref-type=\"bibr\">Khandia et al., 2019</xref>).</p><p>Furthermore, it has been widely demonstrated that lipid rafts play a fundamental role in autophagic machinery: they are associated with autophagosome morphogenesis, either in the initiation or in the maturation phases (<xref rid=\"B69\" ref-type=\"bibr\">Matarrese et al., 2014</xref>).</p><p>Autophagy, a cell process involved in cellular homeostasis, stress, and immune responses to viral infections, is a two-edged process in virus infections, since it may have pro- or anti-viral roles (<xref rid=\"B56\" ref-type=\"bibr\">Kudchodkar and Levine, 2009</xref>; <xref rid=\"B52\" ref-type=\"bibr\">Khandia et al., 2019</xref>). Viruses, on the other side, during co-evolution with the host cell, developed mechanisms to usurp/exploit the host autophagic system (<xref rid=\"B51\" ref-type=\"bibr\">Jheng et al., 2014</xref>).</p><p>This minireview reports on the available knowledge about the interplay between coronaviruses, including the SARS-CoV-2, with lipid rafts and autophagic pathways, in order to focus the attention to novel potential targets to inhibit coronavirus infections.</p><sec id=\"S1.SS1\"><title>Lipid Rafts and Autophagy: Focus on Coronavirus</title><p>Lipid rafts are plasma membrane microdomains (10–200 nm) enriched in cholesterol, glycosphingolipids, and phospholipids. They are also found in the endoplasmic reticulum (ER) (<xref rid=\"B7\" ref-type=\"bibr\">Browman et al., 2006</xref>), in the Golgi complex (<xref rid=\"B33\" ref-type=\"bibr\">Eberle et al., 2002</xref>), and on the membrane of endosomes (<xref rid=\"B95\" ref-type=\"bibr\">Sobo et al., 2007</xref>) and phagosomes (<xref rid=\"B30\" ref-type=\"bibr\">Dermine et al., 2001</xref>). There are two main types of lipid rafts based on their protein composition: “planar lipid rafts” and “caveolae” enriched by the proteins flotillin and caveolin, respectively (<xref rid=\"B70\" ref-type=\"bibr\">McGuinn and Mahoney, 2014</xref>). Both possess a similar lipid composition that confers resistance to solubilization by non-ionic detergents at low temperatures (<xref rid=\"B8\" ref-type=\"bibr\">Brown and Rose, 1992</xref>), and the characteristic of being able to be isolated in sucrose gradient (<xref rid=\"B89\" ref-type=\"bibr\">Sargiacomo et al., 1993</xref>). Because of their dynamic and heterogeneous structure, they can rapidly assemble and disassemble, changing their composition in response to intra- and extracellular stimuli (<xref rid=\"B94\" ref-type=\"bibr\">Simons and Toomre, 2000</xref>). A key component of lipid rafts is cholesterol that is the glue that maintains raft architecture, as demonstrated by the disorganization and disruption of lipid rafts upon depletion of plasma membrane cholesterol by the cholesterol depleting agent methyl-β-cyclodextrin (MβCD) (<xref rid=\"B93\" ref-type=\"bibr\">Simons and Ehehalt, 2002</xref>; <xref rid=\"B116\" ref-type=\"bibr\">Zidovetzki and Levitan, 2007</xref>). These membrane regions play an important role in a variety of cellular functions, but principally they recruit and concentrate several signaling molecules (<xref rid=\"B96\" ref-type=\"bibr\">Song et al., 1997</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Galbiati et al., 1999</xref>; <xref rid=\"B81\" ref-type=\"bibr\">Razani et al., 1999</xref>; <xref rid=\"B77\" ref-type=\"bibr\">Patel and Insel, 2009</xref>) that, by interacting with caveolin and flotillin, form a sort of signal transduction platform. Therefore, lipid rafts are involved in many biological functions including endocytosis, signal transduction, cell communication, and regulation of autophagy (<xref rid=\"B72\" ref-type=\"bibr\">Nabi and Le, 2003</xref>; <xref rid=\"B91\" ref-type=\"bibr\">Shi et al., 2015</xref>).</p><p>Because of their capacity to cluster into a “phagocytic synapse” several pathogen recognition receptors (Toll-like receptors, C-type lectin receptors), lipid rafts are the focus of intense research in the field of infection. In particular, they are involved in several steps along a viral infection, such as virus entry into the host cell (fusion and internalization), viral protein transport, viral assembly, and budding processes (<xref rid=\"B50\" ref-type=\"bibr\">Hogue et al., 2012</xref>). Several enveloped (HIV-1, influenza viruses, coronavirus, flavivirus) and non-enveloped (SV40 and Rrotavirus) viruses exploit the raft platform to bind to their specific receptors (<xref rid=\"B75\" ref-type=\"bibr\">Ono and Freed, 2001</xref>; <xref rid=\"B79\" ref-type=\"bibr\">Pelkmans, 2005</xref>; <xref rid=\"B60\" ref-type=\"bibr\">Li et al., 2007</xref>; <xref rid=\"B99\" ref-type=\"bibr\">Takahashi and Suzuki, 2011</xref>).</p><p>Recent studies have implicated lipid rafts in coronavirus entry and egress through a multistep endocytic process, although the detailed mechanism remains to be disclosed (<xref rid=\"B60\" ref-type=\"bibr\">Li et al., 2007</xref>). <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref> depicts a schematic view of the role of lipid rafts in two paradigmatic human coronaviruses, HCoV-229E and SARS-CoV, infections. Briefly, the receptors for the two coronaviruses, aminopeptidase N (APN/CD13) and angiotensin converting enzyme 2 (ACE2), respectively, are located into lipid rafts and play a fundamental role in the initial step of the virus infection. APN/CD13, a zinc-binding aminopeptidase, is expressed in several cell types (endothelial, granulocytic, and monocytic cells; epithelial cells of the kidney; respiratory tract and intestine) and is required also for porcine, canine, and feline coronavirus recognition. ACE2, encoded on the X chromosome, is a metalloprotease long known to be a key player in the renin–angiotensin system that co-localizes with caveolin-1 and GM1 and is expressed on type I and II pneumocytes, enterocytes, endothelial cells of the heart and kidney, epithelial cells of the kidney, and the testis (<xref rid=\"B49\" ref-type=\"bibr\">Hamming et al., 2004</xref>). SARS-CoV-2, the etiological agent of the COVID-19 pandemic, also binds ACE2 to infect host cells, by similarity to SARS-CoV, from which it differentiates for point mutations on the spike protein, essential for receptor binding (<xref rid=\"B102\" ref-type=\"bibr\">Wang et al., 2020</xref>; <xref rid=\"B107\" ref-type=\"bibr\">Yan et al., 2020</xref>). By analogy to SARS-CoV, it can be hypothesized that SARS-CoV-2 may also use lipid rafts for its entry into the host cells.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Schematic representation of the role of lipid rafts in Coronavirus infection of the host cells is a multistep endocytic process characterized by a series of complex events tightly regulated in space and time. Step 1 Entry process of coronavirus into the host cells is initiated by the binding of the spike glycoprotein with the specific receptor (ACE2, APN/CD13) located into lipid rafts/caveolae. This interaction causes conformational changes of the viral particle, which trigger specific signaling events necessary for the viral entry mechanism. Step 2 Lipid rafts/caveolae-mediated endocytosis is followed by intracellular trafficking of virus particles in transport vesicles (early and late endosomes). The low pH in late endosomes induces a conformational change in coronavirus that mediates fusion of the viral envelope with the endosomal membrane. Step 3 Viral genomes are translated in two polyproteins, pp1a and pp1ab, which encode the non-structural viral proteins that form the replication transcription complex. This complex produces genomic RNA as well as multiple subgenomic mRNAs encoding structural proteins. Translation of mRNA encoding for the nucleocapsid proteins occurs in the cytoplasm where the newly synthesized proteins interact with new genomes to form ribonucleoprotein particles. In contrast, matrix, envelope and spike proteins translation occurs into the ER. Coronavirus uses also the autophagy machinery for replication and has evolved strategies to avoid autophagy-induced lysosomal degradation. Step 4 After assembly the progeny viral particles, virus-containing vesicles (smooth-wall vesicles) are budded and released into the extracellular environment through fusion with the plasma membrane (exocytosis). Alternatively, we speculate that coronavirus might utilize multivescicular bodies (MVBs) and take advantage of the exosomal pathway for egress.</p></caption><graphic xlink:href=\"fmicb-11-01821-g001\"/></fig><p>The steps following coronavirus entry are not clearly identified. A recent study demonstrated that SARS-CoV exploits the activity of cathepsin L, an endosomal cysteine protease, to initiate proteolysis and activation of membrane fusion within endosomes (<xref rid=\"B92\" ref-type=\"bibr\">Simmons et al., 2013</xref>). In addition, coronavirus mouse hepatitis virus (MHV) infection is inhibited when early endosome-associated proteins, RAB5 and EEA1, are down-regulated by siRNA (<xref rid=\"B9\" ref-type=\"bibr\">Burkard et al., 2014</xref>) and infectious bronchitis virus (IBV) release of the nucleocapsid into the cytoplasm (<xref rid=\"B101\" ref-type=\"bibr\">Wang et al., 2018</xref>) occurs through the fusion with endosome–lysosome membranes. These data suggest that coronaviruses exploit the early and late endosome compartment after viral entry, as several other viruses (adenovirus, human papillomavirus, polyomavirus, African swine fever virus, and influenza virus) (<xref rid=\"B57\" ref-type=\"bibr\">Lakadamyali et al., 2004</xref>; <xref rid=\"B88\" ref-type=\"bibr\">Sánchez et al., 2017</xref>; <xref rid=\"B97\" ref-type=\"bibr\">Spriggs et al., 2019</xref>).</p><p>Recent evidence shows that there is a close interplay between lipid rafts and autophagy during physiological cell function, as lipid rafts can regulate autophagy by interacting with autophagosomes and autophagy-related proteins, such as ATG5 and ATG12 (<xref rid=\"B11\" ref-type=\"bibr\">Chen et al., 2014</xref>; <xref rid=\"B40\" ref-type=\"bibr\">Garofalo et al., 2016</xref>). In addition a functional and structural link between lipid rafts and autophagy comes from the evidence that chemical (e.g., MβCD, fumonisin B1) or biological (e.g., siRNA inhibition of key enzymes involved in the sphingolipids metabolism) molecules capable of altering the chemical composition or molecular organization of lipid rafts have also a strong impact on autophagy (<xref rid=\"B52\" ref-type=\"bibr\">Khandia et al., 2019</xref>).</p><p>Autophagy is an evolutionarily conserved (from yeast to mammals), selective, and finely regulated process that generates ATP and precursors for the synthesis of macromolecules (<xref rid=\"B55\" ref-type=\"bibr\">Ktistakis and Tooze, 2016</xref>) and is considered as a survival process put in place by cells under stressful conditions, such as pathogen microbial infections. It plays a key role in cellular homeostasis and is responsible for the turnover of cellular organelles (<xref rid=\"B111\" ref-type=\"bibr\">Yu et al., 2018</xref>), as its main function is to remove and recycle non-essential, damaged, or obsolete cellular components, such as whole organelles or macromolecules (<xref rid=\"B71\" ref-type=\"bibr\">Mizushima and Komatsu, 2011</xref>). During autophagy, cytoplasmic portions are enclosed into double membrane bound vesicles (autophagosomes) that then fuse with late endosomes/lysosomes, whose contents are degraded by lysosomal proteases. Autophagy has been shown to have additional function in innate immunity, by degradation of viruses, or intracellular pathogens, as well as by presenting pathogen components to the immune system (<xref rid=\"B67\" ref-type=\"bibr\">Mao et al., 2019</xref>).</p><p>Autophagy can have a pro-viral role, promoting virus infection and propagation, as well as an antiviral role, essentially depending on the virus strain, on the phase of infection, on the infected cell type, but also on the cellular microenvironment (<xref rid=\"B59\" ref-type=\"bibr\">Lennemann and Coyne, 2015</xref>). During co-evolution with their natural hosts, viruses developed the ability to hijack autophagic mechanisms to their advantage by using them for immune escaping, or using autophagosomes as a replicative niche. However, data on autophagy interplay with coronaviruses are scant so far, related on no more than 50 works published between 2004 and 2015. In general, it is suggested that CoVs can interact with some components of the autophagic pathway in an opposite way with a dual effect: utilizing autophagy components to promote viral replication and/or to inhibit degradation of viral products through the autophagic pathway (<xref rid=\"B25\" ref-type=\"bibr\">Cong et al., 2017</xref>). In some coronaviruses, infection results are still contradictory, as in the case of MERS-CoV (<xref rid=\"B26\" ref-type=\"bibr\">Corman et al., 2016</xref>), which has been reported to induce phosphorylation changes in key kinases, such as AKT1 and mTOR, that regulate the early steps of autophagic process (<xref rid=\"B53\" ref-type=\"bibr\">Kindrachuk et al., 2015</xref>), thus stimulating autophagy in infected cells. In contrast, Gassen et al. reported that MERS-CoV activates the SKP2 kinase, consequently reducing Beclin1 (BECN1) activity and inhibiting the fusion of autophagosomes with lysosomes, which results in autophagy inhibition (<xref rid=\"B41\" ref-type=\"bibr\">Gassen et al., 2019</xref>).</p><p>In general, it has been hypothesized that coronaviruses would induce accumulation of autophagic vacuoles to obtain a larger availability of the membrane structure necessary for their replication (<xref rid=\"B41\" ref-type=\"bibr\">Gassen et al., 2019</xref>), which is consistent to the observed co-localization of the rodent coronavirus, MHV, replication complex, with the autophagic proteins LC3 and Apg12 (<xref rid=\"B80\" ref-type=\"bibr\">Prentice et al., 2004</xref>). Likewise, the SARS-CoV takes advantage of autophagic pathway to replicate and transcribe its own genome, as suggested by inhibition of its replication through inactivation of GSK-3 (glycogen synthase kinase-3), a serine/threonine kinase that inhibits autophagy, through the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) (<xref rid=\"B104\" ref-type=\"bibr\">Wu et al., 2009</xref>; <xref rid=\"B118\" ref-type=\"bibr\">Zúñiga et al., 2010</xref>). A very recent study showed that SARS-CoV-2, similar to MERS-CoV, strongly reduced the autophagic flux in infected cell lines downregulating the AMPK/mTORC1 pathway, altered autophagy-relevant signaling, and also reduced autophagosome/lysosome fusion efficiency (<xref rid=\"B42\" ref-type=\"bibr\">Gassen et al., 2020</xref>). As a result, the SARS-CoV-2 virus could take advantage of the autophagy reduction, thus preventing viral product degradation and enhancing double membrane vesicles (DMV) availability, indispensable for their replication.</p><p>The cell organelle most involved in the dynamic membrane changes is the ER. It is therefore not surprising that some coronaviruses, through the insertion of their trans-membrane glycoproteins at the ER level, are able to induce ER stress. This is the case, for example, of the ORF3 protein produced by porcine epidemic diarrhea virus (PEDV), which induced ER stress-dependent autophagy in different porcine and human cell types (<xref rid=\"B117\" ref-type=\"bibr\">Zou et al., 2019</xref>). In the same vein, MHV was observed to use the host machinery to export vesicular ER to originate membranes for the genesis of DMV. As regards the non-structural protein (NSP) 6 of the avian infectious bronchitis virus (IBV), a gamma-coronavirus, in addition to providing membranes useful for the replication of the virus, it could prevent its degradation within the lysosomes, effectively escaping the autophagy-based cellular defensive mechanisms. Similar properties have also been observed for MHV and SARS NSP6 proteins and PRRSV arterivirus NSP5, NSP6, and NSP7 (<xref rid=\"B28\" ref-type=\"bibr\">Cottam et al., 2014</xref>).</p></sec></sec><sec id=\"S2\"><title>Discussion</title><p>Coronaviruses may represent a threat for human and animal health for their potential to cross the species barrier, thus acquiring human virulence, as evidenced by the previous SARS-CoV and MERS-CoV outbreaks and by the ongoing COVID-19 pandemic. Notwithstanding, treatment and prophylaxis measures to control coronavirus infections are lacking so far, either in humans or in livestock animals.</p><p>As outlined in this review, lipid rafts and autophagic pathways play a pivotal role in coronavirus infection, being critical for viral entry and replication, as well as for viral release from the host cells. Actually, lipid rafts are the focus of intense research in the field of infection, and it is conceivable to consider targeting some lipid raft components in order to inhibit virus infection at the cell level.</p><p>In particular, lipid raft disruption by cholesterol-depleting agents has been shown to inhibit infection of several microbes by blocking their entry into the host cells (<xref rid=\"B73\" ref-type=\"bibr\">Nomura et al., 2004</xref>; <xref rid=\"B100\" ref-type=\"bibr\">Thorp and Gallagher, 2004</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Choi et al., 2005</xref>; <xref rid=\"B82\" ref-type=\"bibr\">Riethmüller et al., 2006</xref>; <xref rid=\"B45\" ref-type=\"bibr\">Glende et al., 2008</xref>; <xref rid=\"B64\" ref-type=\"bibr\">Lu et al., 2008</xref>; <xref rid=\"B48\" ref-type=\"bibr\">Guo et al., 2017</xref>; <xref rid=\"B76\" ref-type=\"bibr\">Owczarek et al., 2018</xref>; <xref rid=\"B98\" ref-type=\"bibr\">Szczepanski et al., 2018</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Abu-Farha et al., 2020</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Baglivo et al., 2020</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Fantini et al., 2020</xref>; <xref rid=\"B83\" ref-type=\"bibr\">Rodrigues-Diez et al., 2020</xref>; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). In human immunodeficiency virus (<xref rid=\"B75\" ref-type=\"bibr\">Ono and Freed, 2001</xref>), herpes simplex virus, and rotavirus infections, MβCD, a widely used raft-disrupting agent, has been shown to affect virus entry, thereby reducing their infectivity (<xref rid=\"B31\" ref-type=\"bibr\">Dou et al., 2018</xref>; <xref rid=\"B105\" ref-type=\"bibr\">Wudiri et al., 2017</xref>). In addition, in Japanese encephalitis virus and dengue virus infection, disruption of lipid rafts by MβCD has been shown to decrease viral infection acting at both viral entry and intracellular replication step (<xref rid=\"B58\" ref-type=\"bibr\">Lee et al., 2008</xref>). β-cyclodextrins are extensively utilized in pharmaceutical formulations as excipients to enhance solubility, bioavailability, and stability of many drugs (<xref rid=\"B63\" ref-type=\"bibr\">Loftsson and Brewster, 2012</xref>). In the light of the above-described antiviral activity, further studies on pharmacokinetic and safety of MβCD could foster its clinical application as an antimicrobial agent in humans. Statins, used in human therapy for their ability to inhibit cellular synthesis of cholesterol, have also been reported to have an anti-viral effect, inhibiting infection of flavivirus, such as dengue virus (DENV), hepatitis C virus (HCV), West Nile virus (WNV), and Zika virus (<xref rid=\"B65\" ref-type=\"bibr\">Mackenzie et al., 2007</xref>; <xref rid=\"B2\" ref-type=\"bibr\">Andrus and East, 2010</xref>; <xref rid=\"B68\" ref-type=\"bibr\">Martínez-Gutierrez et al., 2011</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Españo et al., 2019</xref>), and their application to inhibition of the coronavirus infection would be worth considering.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Function of lipid rafts and autophagy in coronavirus lifecycles: overview on drugs.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Drugs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mechanism of action</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Target virus</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Antiviral effects</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">References</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MβCD, Nystatin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Disrupt lipid raft architecture by depleting cell membrane cholesterol</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CRCoV HCoV-229E HCoV-NL63 HCoV-OC43 IBV MERS-CoV MHV SARS-CoV-2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibit viral entry/endocytosis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B73\" ref-type=\"bibr\">Nomura et al., 2004</xref>; <xref rid=\"B100\" ref-type=\"bibr\">Thorp and Gallagher, 2004</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Choi et al., 2005</xref>; <xref rid=\"B45\" ref-type=\"bibr\">Glende et al., 2008</xref>; <xref rid=\"B64\" ref-type=\"bibr\">Lu et al., 2008</xref>; <xref rid=\"B48\" ref-type=\"bibr\">Guo et al., 2017</xref>; <xref rid=\"B76\" ref-type=\"bibr\">Owczarek et al., 2018</xref>; <xref rid=\"B98\" ref-type=\"bibr\">Szczepanski et al., 2018</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Baglivo et al., 2020</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Fantini et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Statins (mevastatin)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibit lipid raft formation by lowering cholesterol biosynthesis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IBV SARS-CoV-2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibit viral entry/endocytosis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B48\" ref-type=\"bibr\">Guo et al., 2017</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Abu-Farha et al., 2020</xref>; <xref rid=\"B83\" ref-type=\"bibr\">Rodrigues-Diez et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chloroquine and Hydroxychloroquine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Interfere with lysosome-mediated autophagy function by increasing the endosomal/lysosomal pH</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MERS-CoV HCoV-229E HCoV-OC43 SARS-CoV SARS-CoV-2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Prevent viral fusion and inhibit the viral entry by endocytosis, uncoating and exit (exocytosis) process</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B14\" ref-type=\"bibr\">ClinicalTrials.gov, 2020a</xref>,<xref rid=\"B15\" ref-type=\"bibr\">b</xref>,<xref rid=\"B16\" ref-type=\"bibr\">c</xref>,<xref rid=\"B17\" ref-type=\"bibr\">d</xref>,<xref rid=\"B18\" ref-type=\"bibr\">e</xref>,<xref rid=\"B1\" ref-type=\"bibr\">f</xref>,<xref rid=\"B20\" ref-type=\"bibr\">g</xref>,<xref rid=\"B21\" ref-type=\"bibr\">h</xref>,<xref rid=\"B22\" ref-type=\"bibr\">i</xref>,<xref rid=\"B23\" ref-type=\"bibr\">j</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rapamycin (Sirolimus)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits mammalian target of rapamycin (mTOR) kinase</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MERS-CoV PEDV SARS-CoV-2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Downregulates virus infection</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B24\" ref-type=\"bibr\">ClinicalTrials.gov, 2020k</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nitazoxanide</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits Akt/mTOR/ULK1 signaling pathway</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HCoVOC43 MERS-CoV SARS-CoV-2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits virus replication</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B90\" ref-type=\"bibr\">Shakya et al., 2018</xref>; <xref rid=\"B108\" ref-type=\"bibr\">Yang et al., 2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Niclosamide</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">mTORC1 inhibitor</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MERS-CoV SARS-CoV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits viral antigen synthesis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B62\" ref-type=\"bibr\">Liu et al., 2019</xref>; <xref rid=\"B106\" ref-type=\"bibr\">Xu et al., 2020</xref></td></tr></tbody></table></table-wrap><p>Likewise, the pharmacological modulation of autophagic processes can represent an attractive therapeutic strategy for elimination of the viral pathogen or containment of the infection (<xref rid=\"B87\" ref-type=\"bibr\">Rubinsztein et al., 2009</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Clark et al., 2018</xref>).</p><p>In fact, different drugs described as inhibitors or inducers of the autophagy that control host cell pathways process involved in coronavirus infection, have sparked interest for their potential antiviral activity (<xref rid=\"B90\" ref-type=\"bibr\">Shakya et al., 2018</xref>; <xref rid=\"B62\" ref-type=\"bibr\">Liu et al., 2019</xref>; <xref rid=\"B106\" ref-type=\"bibr\">Xu et al., 2020</xref>; <xref rid=\"B108\" ref-type=\"bibr\">Yang et al., 2020</xref>; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). One of this, chloroquine (CQ), and its derivative hydroxychloroquine (HCQ), known as anti-malarial drugs (<xref rid=\"B74\" ref-type=\"bibr\">O’Neill et al., 1998</xref>), which are able to inhibit autophagy by raising the lysosomal pH (<xref rid=\"B46\" ref-type=\"bibr\">Golden et al., 2015</xref>), have also been evaluated for HIV infection (<xref rid=\"B84\" ref-type=\"bibr\">Romanelli et al., 2004</xref>). Very recently, clinicians have paid attention to CQ and HCQ as a possible treatment of patients infected by the novel emerged SARS-CoV-2 (<xref rid=\"B14\" ref-type=\"bibr\">ClinicalTrials.gov, 2020a</xref>,<xref rid=\"B15\" ref-type=\"bibr\">b</xref>,<xref rid=\"B16\" ref-type=\"bibr\">c</xref>,<xref rid=\"B17\" ref-type=\"bibr\">d</xref>,<xref rid=\"B18\" ref-type=\"bibr\">e</xref>,<xref rid=\"B19\" ref-type=\"bibr\">f</xref>,<xref rid=\"B20\" ref-type=\"bibr\">g</xref>,<xref rid=\"B21\" ref-type=\"bibr\">h</xref>,<xref rid=\"B22\" ref-type=\"bibr\">i</xref>,<xref rid=\"B23\" ref-type=\"bibr\">j</xref>). This insight is also supported by some recent works, including a recent publication by Gao et al. that indicates some positive effects of CQ on the course of pneumonia associated with the infection and on the reduction in healing (<xref rid=\"B27\" ref-type=\"bibr\">Cortegiani et al., 2020</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Gao et al., 2020</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Geleris et al., 2020</xref>; <xref rid=\"B110\" ref-type=\"bibr\">Yu et al., 2020</xref>).</p><p>On the contrary, other studies have highlighted the ineffectiveness of these drugs both in the viremic phase and in respiratory complications (<xref rid=\"B6\" ref-type=\"bibr\">Boulware et al., 2020</xref>; <xref rid=\"B66\" ref-type=\"bibr\">Mahévas et al., 2020</xref>; <xref rid=\"B85\" ref-type=\"bibr\">Rosenberg et al., 2020</xref>). Although some data, both referring to COVID-19 and SARS, have highlighted some positive effects of CQ and HCQ in the evolution of the disease, thus showing their therapeutic potential, at the moment, there are no data that can support the use of these drugs to control the entire pathological process related to SARS-CoV-2 (<xref rid=\"B44\" ref-type=\"bibr\">Gies et al., 2020</xref>).</p><p>In the same vein, rapamycin, also known as sirolimus, an autophagy inducer already used as an immunosuppressant, has been tested, with some success, in the treatment of COVID-19 (NCT04341675) (<xref rid=\"B24\" ref-type=\"bibr\">ClinicalTrials.gov, 2020k</xref>). These cases may represent a repositioning of the drugs with clinical success in treatment areas beyond their original approved use. According to this, the antiviral <italic>in vitro</italic> activity of spermidine, niclosamide, and nitazoxanide (known autophagy inducers) vs. SARS-CoV-2 was recently reported (<xref rid=\"B90\" ref-type=\"bibr\">Shakya et al., 2018</xref>; <xref rid=\"B62\" ref-type=\"bibr\">Liu et al., 2019</xref>; <xref rid=\"B106\" ref-type=\"bibr\">Xu et al., 2020</xref>; <xref rid=\"B108\" ref-type=\"bibr\">Yang et al., 2020</xref>). Thus, a prophylactic approach to COVID-19 using these drugs, which are well tolerated, clinically applied, or FDA-approved compounds, would be rational (<xref rid=\"B42\" ref-type=\"bibr\">Gassen et al., 2020</xref>). Importantly, treatments for emerging infections by targeting host cell pathways, rather than the infectious agent directly, or to complement antivirals with drugs that enhance host cell resistance mechanisms have thus become an active and promising therapeutic strategy. This strategy is even more important and urgent to be explored in the case of such potentially and suddenly pandemic virus family, as coronaviruses are.</p></sec><sec id=\"S3\"><title>Author Contributions</title><p>KF contributed to the design of this study, drafted the manuscript, and drew figures relative to lipid rafts. SA contributed to the conception of the idea and drafted the manuscript and figure relative to coronavirus description. DP conducted a wide literature search and drafted the manuscript on the lipid rafts section. EI drafted the manuscript on the autophagy section. PM provided autophagy expertise, contributed to draft and revision of the manuscript, and concurred with the final version of the review. MG provided lipid rafts expertise, and contributed to draft and review the manuscript and to the organization of the review. AR conceived the idea, drafted and revised the manuscript, coordinated the activity, and concurred with the final version of the review. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> The authors gratefully acknowledge the financial support obtained from the Italian Ministry of Health (to MG), grant PE-2013-02354961, and Peretti Foundation (to PM), grant 3603.</p></fn></fn-group><ack><p>We thank Massimo Delle Femmine for his support with the figures.</p></ack><sec id=\"S6\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fmicb.2020.01821/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fmicb.2020.01821/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"FS1\"><label>FIGURE S1</label><caption><p>Taxonomy of CoVs Classification scheme of some representative human (in red) and animal (in black) coronaviruses.Abbreviations: FCoV, feline coronavirus; TGEV, transmissible gastroenteritis virus; PRCV, porcine respiratory coronavirus; HCoV, human coronavirus; PEDV, porcine epidemic diarrhea virus; Ro-BatCoV, Rousettus bat CoV; PHEV, porcine hemagglutinating encephalomyelitis virus; MHV, mouse hepatitis virus; BCV, bovine coronavirus; SARS-CoV, severe acute respiratory syndrome coronavirus; Bat SARSr-CoV, Bat SARS-like coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; BuCoV, bulbul coronavirus; PDCoV, porcine deltacoronavirus; BWCoV, beluga whale coronavirus; IBV, avian infectious bronchitis virus; TCoV, Turkey coronavirus.</p></caption><media xlink:href=\"Image_1.TIF\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abu-Farha</surname><given-names>M.</given-names></name><name><surname>Thanaraj</surname><given-names>T. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Cell Dev. Biol.</journal-id><journal-title-group><journal-title>Frontiers in Cell and Developmental Biology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-634X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850851</article-id><article-id pub-id-type=\"pmc\">PMC7431669</article-id><article-id pub-id-type=\"doi\">10.3389/fcell.2020.00741</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cell and Developmental Biology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>m<sup>6</sup>A Reader: Epitranscriptome Target Prediction and Functional Characterization of <italic>N</italic><sup>6</sup>-Methyladenosine (m<sup>6</sup>A) Readers</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Zhen</surname><given-names>Di</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1004973/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wu</surname><given-names>Yuxuan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1004960/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Yuxin</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1008258/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Kunqi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/846473/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Song</surname><given-names>Bowen</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/846406/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Xu</surname><given-names>Haiqi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Tang</surname><given-names>Yujiao</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/643158/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wei</surname><given-names>Zhen</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Meng</surname><given-names>Jia</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/643203/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Biological Sciences, Xi’an Jiaotong-Liverpool University</institution>, <addr-line>Suzhou</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Institute of Ageing and Chronic Disease, University of Liverpool</institution>, <addr-line>Liverpool</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Mathematical Sciences, Xi’an Jiaotong-Liverpool University</institution>, <addr-line>Suzhou</addr-line>, <country>China</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Institute of Integrative Biology, University of Liverpool</institution>, <addr-line>Liverpool</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff5\"><sup>5</sup><institution>AI University Research Centre, Xi’an Jiaotong-Liverpool University</institution>, <addr-line>Suzhou</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Yu Xue, Huazhong University of Science and Technology, China</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Fuyi Li, Monash University, Australia; Nicolas Reynoird, INSERM U1209 Institut pour l’Avancée des Biosciences (IAB), France; Lin Zhang, China University of Mining and Technology, China</p></fn><corresp id=\"c001\">*Correspondence: Kunqi Chen, <email>kunqi.chen@liverpool.ac.uk</email>; <email>kunqi.chen@xjtlu.edu.cn</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>These authors have contributed equally to this work</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Cellular Biochemistry, a section of the journal Frontiers in Cell and Developmental Biology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>741</elocation-id><history><date date-type=\"received\"><day>19</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>16</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Zhen, Wu, Zhang, Chen, Song, Xu, Tang, Wei and Meng.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Zhen, Wu, Zhang, Chen, Song, Xu, Tang, Wei and Meng</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p><italic>N</italic><sup>6</sup>-methyladenosine (m<sup>6</sup>A) is the most abundant post-transcriptional modification in mRNA, and regulates critical biological functions via m<sup>6</sup>A reader proteins that bind to m<sup>6</sup>A-containing transcripts. There exist multiple m<sup>6</sup>A reader proteins in the human genome, but their respective binding specificity and functional relevance under different biological contexts are not yet fully understood due to the limitation of experimental approaches. An <italic>in silico</italic> study was devised to unveil the target specificity and regulatory functions of different m<sup>6</sup>A readers. We established a support vector machine-based computational framework to predict the epitranscriptome-wide targets of six m<sup>6</sup>A reader proteins (YTHDF1-3, YTHDC1-2, and EIF3A) based on 58 genomic features as well as the conventional sequence-derived features. Our model achieved an average AUC of 0.981 and 0.893 under the full-transcript and mature mRNA model, respectively, marking a substantial improvement in accuracy compared to the sequence encoding schemes tested. Additionally, the distinct biological characteristics of each individual m<sup>6</sup>A reader were explored via the distribution, conservation, Gene Ontology enrichment, cellular components and molecular functions of their target m<sup>6</sup>A sites. A web server was constructed for predicting the putative binding readers of m<sup>6</sup>A sites to serve the research community, and is freely accessible at: <ext-link ext-link-type=\"uri\" xlink:href=\"http://m6areader.rnamd.com\">http://m6areader.rnamd.com</ext-link>.</p></abstract><kwd-group><kwd><italic>N6</italic>-methyladenosine</kwd><kwd>m<sup>6</sup>A reader</kwd><kwd>machine learning (ML)</kwd><kwd>YTH domain</kwd><kwd>eIF3a</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Natural Science Foundation of China<named-content content-type=\"fundref-id\">10.13039/501100001809</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"7\"/><table-count count=\"3\"/><equation-count count=\"2\"/><ref-count count=\"95\"/><page-count count=\"14\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>In the exploration of RNA epigenetics, more than 150 types of RNA modification have been identified (<xref rid=\"B5\" ref-type=\"bibr\">Boccaletto et al., 2018</xref>). The methylation of adenosine at the N6 position (m<sup>6</sup>A) is the most prevalent post-transcriptional modification in the mRNA (<xref rid=\"B63\" ref-type=\"bibr\">Meyer and Jaffrey, 2017</xref>), which was discovered in a wide range of eukaryotic RNAs (<xref rid=\"B1\" ref-type=\"bibr\">Adams and Cory, 1975</xref>) as well as viral RNAs (<xref rid=\"B31\" ref-type=\"bibr\">Gokhale et al., 2016</xref>). m<sup>6</sup>A was considered as a potential mRNA processing regulator in 1970s (<xref rid=\"B25\" ref-type=\"bibr\">Desrosiers et al., 1974</xref>), and subsequent studies noticed intensive functions of it (<xref rid=\"B67\" ref-type=\"bibr\">Patil et al., 2018</xref>), including cardiac gene expression (<xref rid=\"B45\" ref-type=\"bibr\">Kmietczyk et al., 2019</xref>), cell growth, neuronal development (<xref rid=\"B9\" ref-type=\"bibr\">Chen J. et al., 2019</xref>), stress response (<xref rid=\"B29\" ref-type=\"bibr\">Engel et al., 2018</xref>), translation initiation, and stabilizing junctional RNA (<xref rid=\"B57\" ref-type=\"bibr\">Liu B. et al., 2018</xref>).</p><p>Similar to other epigenetic modifications, m<sup>6</sup>A is thought to be dynamic and reversible (<xref rid=\"B73\" ref-type=\"bibr\">Song et al., 2019</xref>). It can be installed by methyltransferase (writers) or removed by demethylase (erasers). This internal modification also attracts specific binding proteins, namely readers, which bind selectively to m<sup>6</sup>A-containing transcripts (<xref rid=\"B51\" ref-type=\"bibr\">Liao et al., 2018</xref>). Additionally, m<sup>6</sup>A performs many functions through interacting with “reader” proteins (<xref rid=\"B33\" ref-type=\"bibr\">Hazra et al., 2019</xref>). The most widely studied readers are YT521-B homology (YTH) family of proteins, which possess the evolutionarily conserved YTH domain that recognizes m<sup>6</sup>A mark. The YTH domain consists of 100–150 residues and adopts alpha/beta fold, with 4–5 alpha helices surrounding a curved six-stranded beta sheet (<xref rid=\"B91\" ref-type=\"bibr\">Zhang et al., 2010</xref>). In human, five m<sup>6</sup>A readers were reported to have the YTH domain, namely YTHDF1,2,3 and YTHDC1,2. However, the YTH domain is not indispensable for m<sup>6</sup>A readers, a subunit of translation initiation complex factor EIF3 complex, called EIF3A, was reported as an m<sup>6</sup>A reader lacking YTH domain (<xref rid=\"B64\" ref-type=\"bibr\">Meyer et al., 2015</xref>).</p><p>The m<sup>6</sup>A reader YTHDC1 is predominantly found in the nucleus, while YTHDC2 and YTHDF1,2,3 are cytoplasmic (<xref rid=\"B66\" ref-type=\"bibr\">Patil et al., 2016</xref>). YTHDC1 and YTHDC2 are unrelated to other members of the YTH family based on amino acid sequence, size or overall YTH domain organization (<xref rid=\"B67\" ref-type=\"bibr\">Patil et al., 2018</xref>). By contrast, YTHDF family comprises three paralogs, YTHDF1-3, that share high sequence identity with about 85% of sequence similarity (<xref rid=\"B33\" ref-type=\"bibr\">Hazra et al., 2019</xref>). YTHDC1 and three YTHDF proteins contain a single C-terminal YTH domain that binds to m<sup>6</sup>A marker by a segment rich of proline, glutamate and aspartate. Compared to other YTH domain-containing proteins, whose YTH domains are embedded in low complexity regions, YTHDC2 has a unique multidomain structure (<xref rid=\"B33\" ref-type=\"bibr\">Hazra et al., 2019</xref>). N-terminal R3H domain, central DEAH-box helicase domain and helicase associated 2 domain are also found in YTHDC2 apart from the C-terminal YTH domain. Different from the structures of five YTH domain-containing proteins, EIF3 is a large multiprotein complex comprising 13 subunits (<xref rid=\"B64\" ref-type=\"bibr\">Meyer et al., 2015</xref>). The EIF3 binding sites are predominantly mapped at the 5′ untranslated region (5′ UTR) (<xref rid=\"B48\" ref-type=\"bibr\">Lee et al., 2015</xref>), whereas the binding sites of YTH domain-containing proteins are usually located near the stop codon.</p><p>In addition to different cellular locations and structures, m<sup>6</sup>A readers appear to function through various post-transcriptional control mechanisms to regulate RNAs dynamically. Human YTHDC1 has been demonstrated to participate in RNA splicing by interacting with serine/arginine splicing factor SRSF3, which is involved in exon inclusion and exclusion splicing (<xref rid=\"B85\" ref-type=\"bibr\">Ye et al., 2017</xref>). As a putative RNA helicase, YTHDC2 enhances the translation of target RNAs and reduces the abundance of target RNAs (<xref rid=\"B36\" ref-type=\"bibr\">Hsu et al., 2017</xref>). YTHDF2 is verified to decrease the stability and control the lifetime of its targeted methylated mRNA transcripts (<xref rid=\"B27\" ref-type=\"bibr\">Du et al., 2016</xref>), while YTHDF1 ensures efficient protein expression from their shared regions (<xref rid=\"B78\" ref-type=\"bibr\">Wang et al., 2015</xref>). YTHDF3, the third member of YTHDF family, has been proposed to share common targets (about 60%) with both YTHDF1 and YTHDF2 (<xref rid=\"B71\" ref-type=\"bibr\">Shi et al., 2017</xref>). This suggests potential coordination in regulating gene expression by YTHDF family proteins. YTHDF3 can promote the function of YTHDF1 by interacting with some ribosomal proteins to facilitate mRNA translation. When associating with YTHDF2, YTHDF3 could participate in mRNA decay. In addition to the five members of YTH family, EIF3A plays an important role in biological processes as well. It can act as both repressor and activator of cap-dependent transcript-specific translation through directly binding to m<sup>6</sup>A marked mRNA sequence (<xref rid=\"B48\" ref-type=\"bibr\">Lee et al., 2015</xref>).</p><p>Since the five YTH family proteins (YTHDC1-2 and YTHDF1-3) and EIF3A present distinctive structures and properties, it is worth studying the preferential binding sites in the m<sup>6</sup>A marked transcripts for each m<sup>6</sup>A reader.</p><p>Single base resolution techniques such as miCLIP (<xref rid=\"B53\" ref-type=\"bibr\">Linder et al., 2015</xref>) are developed and are fairly effective on screening m<sup>6</sup>A sites, and it is usually based on the iCLIP or Par-CLIP approach (<xref rid=\"B64\" ref-type=\"bibr\">Meyer et al., 2015</xref>) to identify the binding sites of each m<sup>6</sup>A reader. As these wet-lab experiments are costly and laborious, computational methods may provide a viable avenue. To date, a large number of RNA methylation sites have been reported, providing sufficient information for effective computational prediction. A huge amount of data extracted from experiments encouraged the establishment of a number of effective m<sup>6</sup>A site predictors, including WHISTLE (<xref rid=\"B15\" ref-type=\"bibr\">Chen K. et al., 2019</xref>), SRAMP (<xref rid=\"B94\" ref-type=\"bibr\">Zhou et al., 2016</xref>), BERMP (<xref rid=\"B38\" ref-type=\"bibr\">Huang et al., 2018</xref>), and Gene2vec (<xref rid=\"B95\" ref-type=\"bibr\">Zou et al., 2019</xref>). However, to our knowledge, the prediction dedicated to the target specificity of the readers is absent. In this project, we constructed a predictor, m<sup>6</sup>A reader, to distinguish the substrate of each m<sup>6</sup>A reader. A comprehensive analysis of these readers was then performed, including the analysis of distribution, conservation, GO enrichment, cellular components and molecular functions of their respective epitranscriptome target sites.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Collection of m<sup>6</sup>A Sites and the Target Sites of m<sup>6</sup>A Readers</title><p>The transcriptome-wide m<sup>6</sup>A sites were collected from 17 different conditions generated from 6 different epitranscriptome profiling approaches of base-resolution or high resolution (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Base-resolution or high resolution datasets of m<sup>6</sup>A sites.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dataset</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Technique</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Cell line</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GEO</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">References</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">miCLIP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MOLM13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE98623</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B76\" ref-type=\"bibr\">Vu et al., 2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S2</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HEK293</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE63753</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B53\" ref-type=\"bibr\">Linder et al., 2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S3</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HepG2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE73405</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B64\" ref-type=\"bibr\">Meyer et al., 2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S4</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HEK293T</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE122948</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B6\" ref-type=\"bibr\">Boulias et al., 2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S5</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HepG2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE121942</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B37\" ref-type=\"bibr\">Huang et al., 2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S6</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HCT116</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE128699</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B75\" ref-type=\"bibr\">van Tran et al., 2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">m<sup>6</sup>A-CLIP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HeLa</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE86336</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B43\" ref-type=\"bibr\">Ke et al., 2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S8</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">CD8T</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE71154</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B42\" ref-type=\"bibr\">Ke et al., 2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S9</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A549</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MAZTER-seq</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HEK293T</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE122961</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B30\" ref-type=\"bibr\">Garcia-Campos et al., 2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S11</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ESC</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">m<sup>6</sup>A-REF-seq</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HEK293</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE125240</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B89\" ref-type=\"bibr\">Zhang et al., 2019c</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S13</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Brain</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S14</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Kidney</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S15</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Liver</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PA-m<sup>6</sup>A-seq</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HeLa</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE54921</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B10\" ref-type=\"bibr\">Chen K. et al., 2015a</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">S17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">m<sup>6</sup>A-seq (improved protocol)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A549</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE54365</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B70\" ref-type=\"bibr\">Schwartz et al., 2014</xref></td></tr></tbody></table></table-wrap><p>In this study, we consider the binding sites of six m<sup>6</sup>A readers identified by Par-CLIP or iCLIP approaches. Specifically, a total of 16,664 m<sup>6</sup>A sites located on 4,722 different genes reported by four experiments were considered as the target sites of YTHDC1, and 1,234 sites on 275 genes identified by two experiments were considered as the target sites for YTHDC2. For the three proteins from YTHDF family, three experiments for each reader proposed 25,597, 28,970, and 7,253 target sites located on 6,714, 6,677, and 3,495 genes for YTHDF1, YTHDF2, and YTHDF3, respectively. Two CLIP experiments conducted on HEK2937T cell line discovered 756 sites located in 470 genes on marked RNA transcripts, which are targeted by EIF3A. The testing datasets and training datasets are strictly segregated under all conditions. Detailed information of the target sites of m<sup>6</sup>A readers analyzed in this study was summarized in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>.</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Target sites of m<sup>6</sup>A readers identified by Par-CLIP or iCLIP.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dataset</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Reader</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Source</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Site #</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Total #</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Gene #</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Cell line</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">YTHDC1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE74397 (<xref rid=\"B69\" ref-type=\"bibr\">Roundtree et al., 2017</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">482</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16,664</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,722</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HeLa</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D2</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE58352 (<xref rid=\"B83\" ref-type=\"bibr\">Xu et al., 2014</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,633</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D3</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE71096 (<xref rid=\"B82\" ref-type=\"bibr\">Xiao et al., 2016</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,430</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D4</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE78030 (<xref rid=\"B66\" ref-type=\"bibr\">Patil et al., 2016</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12,309</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HEK293T</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">YTHDC2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE98085 (<xref rid=\"B36\" ref-type=\"bibr\">Hsu et al., 2017</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,183</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,234</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">275</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HeLa</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D6</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE78030 (<xref rid=\"B66\" ref-type=\"bibr\">Patil et al., 2016</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">131</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HEK293T</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">YTHDF1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE63591 (<xref rid=\"B78\" ref-type=\"bibr\">Wang et al., 2015</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,541</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25,597</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6,714</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HeLa</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D8</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE83438 (<xref rid=\"B31\" ref-type=\"bibr\">Gokhale et al., 2016</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,527</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Huh7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D9</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE78030 (<xref rid=\"B66\" ref-type=\"bibr\">Patil et al., 2016</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20,694</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HEK293T</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">YTHDF2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE49339 (<xref rid=\"B77\" ref-type=\"bibr\">Wang et al., 2014</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22,688</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28,970</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6,677</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HeLa</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D11</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE83438 (<xref rid=\"B31\" ref-type=\"bibr\">Gokhale et al., 2016</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5,147</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Huh7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D12</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE78030 (<xref rid=\"B66\" ref-type=\"bibr\">Patil et al., 2016</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6,280</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HEK293T</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">YTHDF3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE86214 (<xref rid=\"B71\" ref-type=\"bibr\">Shi et al., 2017</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,608</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,253</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3,495</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HeLa</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D14</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE83438 (<xref rid=\"B31\" ref-type=\"bibr\">Gokhale et al., 2016</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">177</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Huh7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D15</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE78030 (<xref rid=\"B66\" ref-type=\"bibr\">Patil et al., 2016</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5,082</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HEK293T</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">EIF3A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE65004 (<xref rid=\"B48\" ref-type=\"bibr\">Lee et al., 2015</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">45</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">756</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">470</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HEK293T</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D17</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSE73405 (<xref rid=\"B64\" ref-type=\"bibr\">Meyer et al., 2015</xref>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">731</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></table-wrap></sec><sec id=\"S2.SS2\"><title>Feature Encoding Scheme and Selection</title><p>We considered both the conventional sequence-derived features and the genome-derived features.</p><p>The sequence-derived features were summarized in the iLearn (<xref rid=\"B21\" ref-type=\"bibr\">Chen Z. et al., 2019</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Chen et al., 2020</xref>) and BioSeq-Analysis (<xref rid=\"B54\" ref-type=\"bibr\">Liu, 2019</xref>; <xref rid=\"B55\" ref-type=\"bibr\">Liu et al., 2019</xref>), which can be divided into six different classes. Based on their classification, we chose one method from each class including nucleic acid composition (<xref rid=\"B49\" ref-type=\"bibr\">Lee et al., 2011</xref>), binary encoding method (<xref rid=\"B80\" ref-type=\"bibr\">Wu et al., 2015</xref>), position-specific tendencies of trinucleotide (<xref rid=\"B34\" ref-type=\"bibr\">He et al., 2018</xref>), electron-ion interaction pseudopotentials (<xref rid=\"B35\" ref-type=\"bibr\">He et al., 2019</xref>), Autocorrelation and pseudo k-tupler composition (<xref rid=\"B56\" ref-type=\"bibr\">Liu et al., 2015</xref>). Also, the chemical property combined with nucleic frequency, which is a popular encoding method in recent years (<xref rid=\"B4\" ref-type=\"bibr\">Bari et al., 2013</xref>; <xref rid=\"B16\" ref-type=\"bibr\">Chen et al., 2016a</xref>, <xref rid=\"B17\" ref-type=\"bibr\">b</xref>, <xref rid=\"B18\" ref-type=\"bibr\">2017a</xref>; <xref rid=\"B50\" ref-type=\"bibr\">Li et al., 2018</xref>), was also used in performance testing for m<sup>6</sup>A reader target prediction.</p><p>The genomic features shown in previous projects (<xref rid=\"B15\" ref-type=\"bibr\">Chen K. et al., 2019</xref>; <xref rid=\"B73\" ref-type=\"bibr\">Song et al., 2019</xref>) are effective in RNA modification prediction. In order to improve the performance of the predictor, 58 mammalian genome features belonging to 9 classes were applied. All the features used were generated by the “GenomicFeatures R/Bioconducter” package using the transcript annotations hg19 TxDb package (<xref rid=\"B47\" ref-type=\"bibr\">Lawrence et al., 2013</xref>). The first class involves dummy variables indicating whether the adenosine site overlaps the topological region within the RNA transcript. The second class specifies the relative position of the adenosine site on the region, while the third class tells the length of the target mRNA transcript. Features belonging to the fourth class measure the nucleotide distances to the splicing junction and the nearest neighboring site. The fifth and sixth classes are based on clustering information of modification sites and scores related to conservation (<xref rid=\"B72\" ref-type=\"bibr\">Siepel et al., 2005</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Gulko et al., 2015</xref>), respectively. The last three feature groups describe RNA secondary structures (<xref rid=\"B60\" ref-type=\"bibr\">Lorenz et al., 2011</xref>), genomic properties and attributes of the genes or transcripts, respectively. More details of the genomic features considered in our analysis were presented in <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Table S1</xref>.</p></sec><sec id=\"S2.SS3\"><title>Feature Selection Technique</title><p>With multiple features, the dimension of dataset increases, leading to overfitting, information redundancy or increased computational time. To solve this problem, feature selection is effective in optimizing relevant modeling variables and improving the accuracy of the constructed models. In this study, we performed feature selection using F-score technique (<xref rid=\"B52\" ref-type=\"bibr\">Lin et al., 2014</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Dao et al., 2019</xref>). Technically, F-score is a wrapper-type feature selection algorithm, used to measure the degree of difference between two real-number data sets. For a given training sample <italic>x</italic><sub><italic>d</italic></sub>, there are <italic>n</italic><sup>+</sup> positive samples and <italic>n</italic><sup>−</sup> negative samples. The <italic>F</italic>-score for the i-th feature can be calculated as:</p><p>\n<disp-formula id=\"S2.Ex1\"><mml:math id=\"M1\"><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msup><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:msubsup><mml:mover accent=\"true\"><mml:mi>x</mml:mi><mml:mo stretchy=\"false\">¯</mml:mo></mml:mover><mml:mi>i</mml:mi><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mo>+</mml:mo><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:msubsup><mml:mo>-</mml:mo><mml:msub><mml:mover accent=\"true\"><mml:mi>x</mml:mi><mml:mo stretchy=\"false\">¯</mml:mo></mml:mover><mml:mi>i</mml:mi></mml:msub></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mn>2</mml:mn></mml:msup><mml:mo>+</mml:mo><mml:msup><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:msubsup><mml:mover accent=\"true\"><mml:mi>x</mml:mi><mml:mo stretchy=\"false\">¯</mml:mo></mml:mover><mml:mi>i</mml:mi><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mo>-</mml:mo><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:msubsup><mml:mo>-</mml:mo><mml:msub><mml:mover accent=\"true\"><mml:mi>x</mml:mi><mml:mo stretchy=\"false\">¯</mml:mo></mml:mover><mml:mi>i</mml:mi></mml:msub></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mrow><mml:mrow><mml:mrow><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:msup><mml:mi>n</mml:mi><mml:mo>+</mml:mo></mml:msup><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfrac><mml:mo>⁢</mml:mo><mml:mrow><mml:msubsup><mml:mo largeop=\"true\" symmetric=\"true\">∑</mml:mo><mml:mrow><mml:mi>k</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:msup><mml:mi>n</mml:mi><mml:mo>+</mml:mo></mml:msup></mml:msubsup><mml:msup><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:msubsup><mml:mover accent=\"true\"><mml:mi>x</mml:mi><mml:mo stretchy=\"false\">¯</mml:mo></mml:mover><mml:mrow><mml:mi>d</mml:mi><mml:mo rspace=\"4.2pt\">,</mml:mo><mml:mi>i</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mo>+</mml:mo><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:msubsup><mml:mo>-</mml:mo><mml:msubsup><mml:mover accent=\"true\"><mml:mi>x</mml:mi><mml:mo stretchy=\"false\">¯</mml:mo></mml:mover><mml:mi>i</mml:mi><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mo>+</mml:mo><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:msubsup></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:mrow><mml:mo>+</mml:mo><mml:mrow><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:msup><mml:mi>n</mml:mi><mml:mo>-</mml:mo></mml:msup><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfrac><mml:mo>⁢</mml:mo><mml:mrow><mml:msubsup><mml:mo largeop=\"true\" symmetric=\"true\">∑</mml:mo><mml:mrow><mml:mi>d</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:msup><mml:mi>n</mml:mi><mml:mo>+</mml:mo></mml:msup></mml:msubsup><mml:msup><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:msubsup><mml:mover accent=\"true\"><mml:mi>x</mml:mi><mml:mo stretchy=\"false\">¯</mml:mo></mml:mover><mml:mrow><mml:mi>d</mml:mi><mml:mo rspace=\"4.2pt\">,</mml:mo><mml:mi>i</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mo>-</mml:mo><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:msubsup><mml:mo>-</mml:mo><mml:msubsup><mml:mover accent=\"true\"><mml:mi>x</mml:mi><mml:mo stretchy=\"false\">¯</mml:mo></mml:mover><mml:mi>i</mml:mi><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mo>-</mml:mo><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:msubsup></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:mrow></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula></p><p>where <inline-formula><mml:math id=\"INEQ7\"><mml:msubsup><mml:mover accent=\"true\"><mml:mi>x</mml:mi><mml:mo stretchy=\"false\">¯</mml:mo></mml:mover><mml:mi>i</mml:mi><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mo>+</mml:mo><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula>, <inline-formula><mml:math id=\"INEQ8\"><mml:msubsup><mml:mover accent=\"true\"><mml:mi>x</mml:mi><mml:mo stretchy=\"false\">¯</mml:mo></mml:mover><mml:mi>i</mml:mi><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mo>-</mml:mo><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula> and <inline-formula><mml:math id=\"INEQ9\"><mml:msub><mml:mover accent=\"true\"><mml:mi>x</mml:mi><mml:mo stretchy=\"false\">¯</mml:mo></mml:mover><mml:mi>i</mml:mi></mml:msub></mml:math></inline-formula> denote the average frequency of the i-th feature in the positive, negative and the whole samples, respectively; <inline-formula><mml:math id=\"INEQ10\"><mml:msubsup><mml:mover accent=\"true\"><mml:mi>x</mml:mi><mml:mo stretchy=\"false\">¯</mml:mo></mml:mover><mml:mrow><mml:mi>d</mml:mi><mml:mo rspace=\"4.2pt\">,</mml:mo><mml:mi>i</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mo>+</mml:mo><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula> and <inline-formula><mml:math id=\"INEQ11\"><mml:msubsup><mml:mover accent=\"true\"><mml:mi>x</mml:mi><mml:mo stretchy=\"false\">¯</mml:mo></mml:mover><mml:mrow><mml:mi>d</mml:mi><mml:mo rspace=\"4.2pt\">,</mml:mo><mml:mi>i</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mo>-</mml:mo><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula> represent the value of the i-th feature of the d-th sequence in the positive and negative samples, respectively. A larger F-score value means better predictive ability of a feature. To demonstrate this relative distinguishing ability of every genomic feature, the computed <italic>F</italic>-score values were rescaled between 0 and 1, and ranked in the descending order. Referring to this ranking, we used incremental feature selection (IFS) and SVM method to complete the selection process (<xref rid=\"B20\" ref-type=\"bibr\">Chen and Lin, 2006</xref>; <xref rid=\"B52\" ref-type=\"bibr\">Lin et al., 2014</xref>). Specifically, the feature subset begins with the feature with the highest <italic>F</italic>-score, and the next feature subset contains the last feature subset and one next feature. AUC values of 5-fold cross-validation were obtained for each feature subset.</p></sec><sec id=\"S2.SS4\"><title>Machine Learning Approach and Performance Evaluation</title><p>To reduce the bias in the experiment, especially when selecting the polyA RNAs during library preparation, we built separate prediction models using full transcript data and mature mRNA data, respectively. In the mature mRNA predictor, only m<sup>6</sup>A sites located in exon regions are considered.</p><p>Since the positive-to-negative ratio of our datasets was highly unbalanced (1:10), we randomly split the negative data into ten parts and combined with the positive dataset with 1:1 positive-to-negative ratio to avoid the unfavorable choice of machine learning classifiers. Subsequently, 10 models were trained and the average outcome score was reported as the performance of the classifier. For each m<sup>6</sup>A reader, the target sites identified in different experiments were mixed, and then the predictor was trained with 80% of the total sites before being evaluated by the remaining 20% of sites for independent testing. Specifically, the mature mRNA datasets for YTHDF1-3, YTHDC1-2, EIF3a have 39577, 44025, 11065, 24312, 1245, and 1200 training data, and 9895, 11007, 2767, 6078, 311, and 300 testing data. The full transcript datasets for those m<sup>6</sup>A readers have 40955, 46352, 11605, 26662, 1970, and 1210 training data, and 10239, 11588, 2901, 6666, 492, and 302 testing data.</p><p>Machine learning algorithms have been widely applied in many fields of biological research such as predicting structural and functional properties of biological sequences. We applied Support Vector Machine (SVM) (<xref rid=\"B8\" ref-type=\"bibr\">Chang and Lin, 2011</xref>) to compare encoding schemes and approaches. To identify a better algorithm for model construction, we compared multiple machine learning algorithms including SVM, Logistic Regression (LR), Random Forest (RF), and XGBoost.</p><p>To validate the model performance, besides 5-fold cross-validation, we also applied the cross-sample test, in which the sites reported from one sample (or condition) were reserved for testing purpose and the sites reported in all other samples (or conditions) were used for training. This testing mode directly evaluates the capability of the prediction approach to detect reader-specific target sites under a single biological condition not profiled previously. Besides, four commonly used performance metrics are used for performance evaluation, including Area under the ROC Curve (AUC) (<xref rid=\"B7\" ref-type=\"bibr\">Bradley, 1997</xref>), Precision-Recall Curve (PR AUC) (<xref rid=\"B44\" ref-type=\"bibr\">Keilwagen et al., 2014</xref>), accuracy (Acc) (<xref rid=\"B41\" ref-type=\"bibr\">Jin and Ling, 2005</xref>) and Mathew’s correlation coefficient (MCC) (<xref rid=\"B68\" ref-type=\"bibr\">Powers, 2008</xref>). The formula of Acc and MCC are as follows:</p><p>\n<disp-formula id=\"S2.Ex2\"><mml:math id=\"M2\"><mml:mtable><mml:mtr><mml:mtd><mml:mi>A</mml:mi><mml:mi>c</mml:mi><mml:mi>c</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>T</mml:mi><mml:mi>P</mml:mi><mml:mo>+</mml:mo><mml:mi>T</mml:mi><mml:mi>N</mml:mi></mml:mrow><mml:mrow><mml:mi>T</mml:mi><mml:mi>P</mml:mi><mml:mo>+</mml:mo><mml:mi>F</mml:mi><mml:mi>N</mml:mi><mml:mo>+</mml:mo><mml:mi>T</mml:mi><mml:mi>N</mml:mi><mml:mo>+</mml:mo><mml:mi>F</mml:mi><mml:mi>P</mml:mi></mml:mrow></mml:mfrac></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mi>M</mml:mi><mml:mi>C</mml:mi><mml:mi>C</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>T</mml:mi><mml:mi>P</mml:mi><mml:mo>×</mml:mo><mml:mi>T</mml:mi><mml:mi>N</mml:mi><mml:mo>−</mml:mo><mml:mi>F</mml:mi><mml:mi>P</mml:mi><mml:mo>×</mml:mo><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:mrow><mml:mrow><mml:msqrt><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>T</mml:mi><mml:mi>P</mml:mi><mml:mo>+</mml:mo><mml:mi>F</mml:mi><mml:mi>P</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>×</mml:mo><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>T</mml:mi><mml:mi>P</mml:mi><mml:mo>+</mml:mo><mml:mi>F</mml:mi><mml:mi>N</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>×</mml:mo><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>T</mml:mi><mml:mi>N</mml:mi><mml:mo>+</mml:mo><mml:mi>F</mml:mi><mml:mi>P</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>×</mml:mo><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>T</mml:mi><mml:mi>N</mml:mi><mml:mo>+</mml:mo><mml:mi>F</mml:mi><mml:mi>N</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:msqrt></mml:mrow></mml:mfrac></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula></p><p>where TP is the number of true positives, TN the number of true negatives, FP the number of false positives and FN the number of false negatives.</p><p>Model construction and performance evaluation were conducted in R (Version 3.6.3). Machine learning algorithms were supported by caret package (<xref rid=\"B46\" ref-type=\"bibr\">Kuhn, 2020</xref>).</p></sec></sec><sec id=\"S3\"><title>Results and Discussion</title><sec id=\"S3.SS1\"><title>Feature Selection</title><p>Due to the high reliability and effectiveness in reflecting intrinsic relation to the targets, sequence-derived features have been widely used and achieved high accuracy in extensive researches focusing on the m<sup>6</sup>A site prediction. However, genome-derived features have been discovering and currently showing a new perspective in feature extraction (<xref rid=\"B94\" ref-type=\"bibr\">Zhou et al., 2016</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Chen et al., 2017a</xref>). Here, we extracted genome features from 41 bp sequence data. We employed WHISTLE approach to combine both sequence-derived features and genome-derived features to predict the target specificity of m<sup>6</sup>A readers. To increase robustness and reduce overfitting of the predicter, feature selection was performed, where those most relevant features to the targets were identified.</p><p>Initially, all the genomic features were normalized to ensure the equal contribution of each feature. Then the <italic>F</italic>-score method was applied to allow all features to be ranked accordingly. Combining IFS and SVM, AUC value of 5-fold cross-validation were obtained for each feature subset. By examining AUC scores, the best performance was achieved by the optimal feature subset. The detailed feature selection results were summarized in <xref ref-type=\"supplementary-material\" rid=\"SM3\">Supplementary Figures S1</xref>–<xref ref-type=\"supplementary-material\" rid=\"SM3\">S6</xref> for YTHDF1-3, YTHDC1-2 and EIF3A under both the full transcript and mature mRNA transcript, respectively. For example, it can be observed in <xref ref-type=\"supplementary-material\" rid=\"SM3\">Supplementary Figure S6A</xref> that, the best performance of EIF3A target prediction was achieved with the top 44 features for the mature mRNA model. Therefore, only the top 44 features were used ultimately to build the mature mRNA prediction models for EIF3A target prediction. Likewise, feature selection in target prediction was conducted for every other reader, and the predictors were constructed in the same way.</p></sec><sec id=\"S3.SS2\"><title>Performance Based on Different Features</title><p>With the nucleotide encoding methods based on chemical properties, extensive studies have achieved high accuracy in the m<sup>6</sup>A site prediction. However, for the first time, we explored and compared different sequence encoding schemes for predicting the target specificity of m<sup>6</sup>A-binding proteins.</p><p>For each m<sup>6</sup>A reader, the target sites identified in different experiments were mixed, and then the predictor was trained with 80% of the total sites before being evaluated by the remaining 20% of sites for independent testing. As a comparison, the performance of 5-fold cross-validation on the training data was also reported. As shown in <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Table S7</xref>, m<sup>6</sup>A reader achieved AUC scores of 0.981 and 0.893 in independent testing under the full transcript and mature mRNA models, respectively. This performance is substantially better than other approaches that did not take advantage of genome-derived features.</p><p>Subsequently, we evaluated the capability of the proposed method in identifying the reader-specific target m<sup>6</sup>A sites under different biological contexts. In this test, the sites generated from each sample were used for independent testing, while all other samples were used for training, so the training sites and the test sites were not reported from the same condition. This is often the real scenario of interest where models are constructed to predict target sites in a new biological context. Besides this cross-condition test, the results of 5-fold cross-validation on the training data were also presented. The detailed evaluation results on every individual sample for every reader are shown in <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Tables S2</xref>–<xref ref-type=\"supplementary-material\" rid=\"SM2\">S6</xref>, with a summary of the cross-condition tests presented in <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>. It can be seen that our approach achieved a high accuracy with AUC scores of 0.975 and 0.873 under full transcript and mature mRNA models in the cross-condition test. The performance is again substantially better than the competing methods.</p><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>Target prediction performance under cross-condition test.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mode</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Method</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">YTHDC1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">YTHDC2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">YTHDF1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">YTHDF2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">YTHDF3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">EIF3A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Average</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Full transcript model</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">m<sup>6</sup>A reader</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.974</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.920</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.983</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.983</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.992</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.000</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.975</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Composition</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.769</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.713</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.773</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.778</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.782</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.893</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.785</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MethyRNA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.763</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.611</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.795</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.794</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.787</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.849</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.767</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">EIIP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.770</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.713</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.768</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.778</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.782</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.894</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.784</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PseKNC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.733</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.643</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.743</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.755</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.753</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.852</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.747</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">AutoCo</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.651</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.586</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.673</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.684</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.737</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.835</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.694</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PSNP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.777</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.654</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.816</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.816</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.894</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.869</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.804</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">onehot</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.750</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.603</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.796</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.795</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.791</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.858</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.766</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mature mRNA model</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">m<sup>6</sup>A reader</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.815</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.730</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.983</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.839</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.883</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.987</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.873</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Composition</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.660</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.503</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.773</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.667</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.707</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.872</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.697</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">MethyRNA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.659</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.631</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.795</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.695</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.733</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.833</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.724</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">EIIP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.670</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.504</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.768</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.667</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.727</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.871</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.701</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PseKNC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.635</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.593</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.743</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.630</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.706</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.837</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.691</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">AutoCo</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.527</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.556</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.673</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.559</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.688</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.820</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.637</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PSNP</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.703</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.675</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.816</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.754</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.858</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.870</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.779</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">onehot</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.662</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.622</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.796</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.696</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.757</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.836</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.728</td></tr></tbody></table><table-wrap-foot><attrib><italic>In this test, the sites generated from each sample were used for independent testing, while all other samples were used for training, so the training sites and the test sites were not reported from the same condition. This is often the real scenario of interest where models are constructed to predict target sites under a new biological context. See <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Tables S2</xref>–<xref ref-type=\"supplementary-material\" rid=\"SM2\">S6</xref> for more detailed results.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS3\"><title>Detect Potential Substrate of m<sup>6</sup>A Readers</title><p>In order to further confirm the reliability and efficiency of our predictors, we used our predictors to detect m<sup>6</sup>A reader binding sites on the unidentified regions. As expected, all m<sup>6</sup>A readers bind to more than 20% m<sup>6</sup>A sites, while they bind to less than 10% unmethylated motifs as shown in <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>. The binding preference is significant and reasonable, which demonstrated the high discrimination ability of our predictors. Moreover, we compared the previous binding sites of YTHDF family (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>) and the prediction result of them on unidentified regions (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). The wet-lab and prediction result shows that readers in YTHDF family have both common and distinct binding sites, suggesting that the binding sites of YTHDF proteins are not exactly identical. This is not consistent with the conclusion in the previous study that YTHDF proteins bind to identical sites on all m<sup>6</sup>A mRNAs (<xref rid=\"B86\" ref-type=\"bibr\">Zaccara and Jaffrey, 2020</xref>). Our result suggests that YTHDF family proteins have similar functions of mediating degradation of m<sup>6</sup>A mRNAs, and they also have different functions in mRNA regulation simultaneously. This result is consistent with our GO enrichment analysis, and also partially supports that m<sup>6</sup>A readers’ effect on downstream processes are much more heterogeneous and context-dependent across transcripts (<xref rid=\"B92\" ref-type=\"bibr\">Zhang et al., 2020</xref>). The predicted probabilities for the targeting of each m<sup>6</sup>A reader are provided on the download page of the website<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup>.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Potential substrate of m<sup>6</sup>A readers.</p></caption><graphic xlink:href=\"fcell-08-00741-g001\"/></fig><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Substrate overlap between YTHDF family.</p></caption><graphic xlink:href=\"fcell-08-00741-g002\"/></fig></sec><sec id=\"S3.SS4\"><title>Model Comparison</title><p>To discover a better machine learning algorithm for our proposed models, we compared the performance of SVM, LR, RF, and XGBoost on mature mRNA and full transcript data for the prediction of target specificity of six m<sup>6</sup>A readers. In general, the performances of different machine learning algorithms are all very high (>0.8 for mature mRNA models and >0.9 for full transcript models) and have little difference among them as shown in <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Table S8</xref>. Therefore, we decided to use SVM classifier for the predictors.</p></sec><sec id=\"S3.SS5\"><title>Characterizing the Target Specificity of m<sup>6</sup>A Readers</title><p>Our result suggests that the substrates of m<sup>6</sup>A readers can be classified, reflecting the distinct biological characteristics of each m<sup>6</sup>A reader. We thus explored the distribution, conservation, and functional relevance of the substrates of each m<sup>6</sup>A reader.</p><p>Here, we firstly examined the distribution of binding sites for each reader (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). High enrichment of YTHDC1 is observed around stop codons and CDSs. However, it can be noticed that the binding abundance of YTHDC1 is relatively lower than members of YTHDF family in stop codons, while it is highly enriched in CDSs. This is consistent with the fact that YTHDC1 is not only targeting to m<sup>6</sup>A sites at its C terminus but also directly interacting with pre-mRNA splicing factor SRSF3 or SRSF10, which prefers to reside on the upper stream of m<sup>6</sup>A sites (<xref rid=\"B69\" ref-type=\"bibr\">Roundtree et al., 2017</xref>). The spatial association among those proteins implicates the process of recruiting pre-mRNA splicing factors and inducing mRNA splicing outcomes. Surprisingly, YTHDC2 targets are more enriched in CDSs near stop codons than in 3′ UTR, suggesting that YTHDC2 is distinct from other m<sup>6</sup>A readers. As YTHDC2 is reported to be the largest protein (∼160 kDa) among all YTH family members and with numerous RNA binding domains (e.g., helicase domain and two Ankyrin repeats, <xref rid=\"B36\" ref-type=\"bibr\">Hsu et al., 2017</xref>) apart from YTH domain, besides its acknowledged functions of accelerating translation and degradation of mRNAs as an m<sup>6</sup>A reader, it is possible that there are potential underlying functions independent from m<sup>6</sup>A-binding remained to be discovered. For instance, the recent study indicated that YTHDC2 as an RNA induced ATPase moves along the RNA from 3′ to 5′ with helicase activity, and interacts with 5′ to 3′ exoribonuclease XRN1 mediated by two Ankyrin repeats (ANK) on YTHDC2 (<xref rid=\"B79\" ref-type=\"bibr\">Wojtas et al., 2017</xref>). Remarkably, YTHDF family shows a similar binding distribution in CDSs and 3′ UTRs with peaks at around stop codons of mRNAs. A similar pattern of results was obtained in previous studies suggesting that YTHDFs directly interplay among one another to collaboratively regulate translation and decay of targeted mRNAs in the cytoplasm (<xref rid=\"B71\" ref-type=\"bibr\">Shi et al., 2017</xref>). The binding sites of EIF3A are uniquely enriched at 5′UTRs. This is directly in line with previous findings that the HLH motif of EIF3A interacts predominantly with the m<sup>6</sup>A residues on the 5′UTR, and EIF3A specifically functions to promote cap-independent translation under diverse cellular stresses.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Distribution of m<sup>6</sup>A readers binding sites on mRNAs. <bold>(A)</bold> Distribution of the binding sites of YTHDC1, YTHDC2, YTHDF1, YTHDF2, and YTHDF3 on mRNAs. <bold>(B)</bold> Distribution of the binding sites of EIF3A on mRNAs. The figures were plotted using the Guitar R/Bioconductor package (<xref rid=\"B23\" ref-type=\"bibr\">Cui et al., 2016</xref>).</p></caption><graphic xlink:href=\"fcell-08-00741-g003\"/></fig><p>We then compared the conservation of all m<sup>6</sup>A readers by phastCons score and high conservation ratio (>0.5). As seen in <xref ref-type=\"fig\" rid=\"F4\">Figures 4A,B</xref>, the m<sup>6</sup>A sites (targeted or not targeted by the studied six readers) are more conservative than unmethylated m<sup>6</sup>A motifs (DRACH). This suggests that m<sup>6</sup>A sites and the m<sup>6</sup>A reader binding sites are more evolutionarily conserved at the gene level, and the occurrence of m<sup>6</sup>A should be considered of functional importance and maintained under selection pressure. Moreover, the YTH family is more conserved compared with other regulation components, which is similar to the finding that YT521-B homology (YTH) RNA-binding domain in eukaryotes is known to be highly conserved with essential Lys-364, Trp-380, and Arg-478 (<xref rid=\"B91\" ref-type=\"bibr\">Zhang et al., 2010</xref>). Additionally, as shown in <xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>, compared with EIF3a binding sites and unmethylated sites which are mostly not in 3′ UTR, targets of other m<sup>6</sup>A readers and other untargeted m<sup>6</sup>A sites are more correlated with the miRNA binding sites. This result agrees well with existing studies investigated that miRNA targets are more enriched in 3′ UTR and m<sup>6</sup>A peaks prior to the present of miRNA binding for a majority of the time, suggesting that m<sup>6</sup>A modification functions to enhance initiation of miRNA biogenesis (<xref rid=\"B65\" ref-type=\"bibr\">Meyer et al., 2012</xref>; <xref rid=\"B2\" ref-type=\"bibr\">Alarcón et al., 2015</xref>). And the relative low overlapping rate between YTHDC2 binding sites and miRNA binding sites could be explained by multiple RNA-binding domains of YTHDC2. Furthermore, the proportions of overlapping of RNA-binding proteins (RBPs) and each m<sup>6</sup>A reader’s binding site are calculated. <xref ref-type=\"fig\" rid=\"F4\">Figure 4D</xref> shows that RBPs binding regions overlap with m<sup>6</sup>A reader binding sites in mRNA more than the other m<sup>6</sup>A sites, while there are even fewer overlapping regions with unmethylated sites. This is consistent with our knowledge that some RBPs are essential in post-transcriptional control of RNAs including splicing, stabilization, localization and translation of mRNA. In the process of regulating transcription and translation, m<sup>6</sup>A readers may recruit large numbers of regulators or factors to their targeted RNAs so as to functionally regulate biological processes (<xref rid=\"B71\" ref-type=\"bibr\">Shi et al., 2017</xref>).</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Conservation analysis of the m<sup>6</sup>A sites targeted by different readers. <bold>(A)</bold> Average phastCons; <bold>(B)</bold> High conservation ratio; <bold>(C)</bold> Frequency of miRNA binding site among the targets of six m<sup>6</sup>A readers; <bold>(D)</bold> Frequency of RBP binding site among the targets of six m<sup>6</sup>A readers.</p></caption><graphic xlink:href=\"fcell-08-00741-g004\"/></fig><p>To explore the association among m<sup>6</sup>A modification, readers and biological functions, the gene ontology (GO) enrichment analysis was conducted to measure the biological functions of substrates of each reader using DAVID websites (<xref rid=\"B39\" ref-type=\"bibr\">Huang da et al., 2009</xref>). The resulting top 10 GO functions related to each m<sup>6</sup>A readers were illustrated in <xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>. Interestingly, YTHDC1 is involved in mRNA splicing, mRNA processing and nuclear-transcribed mRNA catabolic process, which is consistent with our understanding of its role of mediating nuclear to cytoplasmic export of nascent m<sup>6</sup>A-containing mRNAs (<xref rid=\"B69\" ref-type=\"bibr\">Roundtree et al., 2017</xref>). The targeting of YTHDC2, shown to accelerate the degradation of mRNA and enhance translation efficiency (<xref rid=\"B36\" ref-type=\"bibr\">Hsu et al., 2017</xref>), are more related to nonsense-mediated decay, protein stabilization and translational initiation. YTHDF1 targets are enriched under the GO terms of nuclear-transcribed mRNA catabolic process and translation initiation (<xref rid=\"B78\" ref-type=\"bibr\">Wang et al., 2015</xref>), suggesting its function in selectively recruiting of ribosomes and facilitating translation. YTHDF2 and YTHDF3 targets are both associated with proteasome-mediated ubiquitin-dependent protein catabolic process, which corresponds to our knowledge of their regulation in the metabolism of cytosolic m<sup>6</sup>A-modified mRNAs (<xref rid=\"B77\" ref-type=\"bibr\">Wang et al., 2014</xref>; <xref rid=\"B71\" ref-type=\"bibr\">Shi et al., 2017</xref>). EIF3A, reported to serve as a driver of specialized translation (<xref rid=\"B48\" ref-type=\"bibr\">Lee et al., 2015</xref>), is enriched with gene expression, translation and SRP-dependent co-translational protein targeting to the membrane. Moreover, as summarized in <xref ref-type=\"supplementary-material\" rid=\"SM3\">Supplementary Figure S7</xref>, six m<sup>6</sup>A readers show high enrichment in cytosol, cytoplasm, and membrane. Five of them (YTHDC1, YTHDF1-3, and EIF3a) are enriched in nucleus and nucleoplasm. While YTHDC2 is more enriched in extracellular exosome, extracellular matrix and myelin sheath instead of nucleus or nucleoplasm. All six proteins are enriched in the function of protein binding and poly(A) RNA binding, while they each have other specialized functions. This is consistent with analysis above on the enrichment of biological process and previous relevant literature. All gene ontology enrichment results were shown in <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Table S9</xref>.</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Gene ontology (GO) enrichment analysis for each reader’s substrates. The top 10 GO functions related to each m<sup>6</sup>A readers are presented.</p></caption><graphic xlink:href=\"fcell-08-00741-g005\"/></fig><p>Additionally, we further confirmed the biological meanings of the substrates of all m<sup>6</sup>A readers. Based on the results of previous GO enrichment analysis (<xref rid=\"B13\" ref-type=\"bibr\">Chen K. et al., 2018</xref>), the most significant <italic>p</italic>-values of top 10 terms treated with the negative logarithm were firstly added up, and then those computed results of identified targets were compared with those of randomly selected substrates. With the bootstrap sampling approach, substrates were randomly selected and analyzed for 100 times before the results were summarized as proportions and displayed in pie charts. Conceivably, if our results achieved on real data are more biologically meaningful than random permutation, it is possible that our analysis reliably unveiled the true biological functions. Specifically, there are 88, 100, 73, 68, 80, and 100% chances for each reader to be more enriched in biological functions than random permutation as illustrated in <xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>, suggesting high possibility that our functional prediction for each individual reader is statistically meaningful.</p><fig id=\"F6\" position=\"float\"><label>FIGURE 6</label><caption><p>Comparing detection of m<sup>6</sup>A readers’ targets based on biological significance. The most significant <italic>p</italic>-values of top 10 GO terms treated with negative logarithm were added up, and those results of identified targets were compared with those of randomly selected substrates. With the bootstrap sampling approach, substrates were randomly selected and analyzed for 100 times before the results were summarized as proportions and displayed in pie charts.</p></caption><graphic xlink:href=\"fcell-08-00741-g006\"/></fig></sec><sec id=\"S3.SS6\"><title>Web Sever for m<sup>6</sup>A Reader</title><p>A web server with a friendly graphical user interface (<xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref>) was constructed to properly share the predictive models we constructed for predicting target specificity of the m<sup>6</sup>A readers. Users may upload the genome ranges in BED format to the website, and a notification email will be sent to the given email address once the job is finished.</p><fig id=\"F7\" position=\"float\"><label>FIGURE 7</label><caption><p>m<sup>6</sup>A reader web server. The web server takes genome ranges in BED format as the input, and supports prediction for the target sites of six m<sup>6</sup>A readers (YTHDC1, YTHDC2, YTHDF1, YTHDF2 and YTHDF3 and EIF3A). All the materials used in the project, including the training data and codes, are also available on the website.</p></caption><graphic xlink:href=\"fcell-08-00741-g007\"/></fig></sec></sec><sec id=\"S4\"><title>Conclusion</title><p>With the great breakthroughs made in RNA modification-mediated regulation of gene expression, studies of emerging transcriptome modifications have driven rapid development of the high-throughput sequencing technologies. With the aid of the invention of m<sup>6</sup>A-seq (<xref rid=\"B26\" ref-type=\"bibr\">Dominissini et al., 2012</xref>) and MeRIP-seq (<xref rid=\"B65\" ref-type=\"bibr\">Meyer et al., 2012</xref>), transcriptome-wide profiling of m<sup>6</sup>A is now possible. Based on comprehensive high-throughput sequencing data, MeT-DB (<xref rid=\"B58\" ref-type=\"bibr\">Liu H. et al., 2018</xref>) and RMBase (<xref rid=\"B84\" ref-type=\"bibr\">Xuan et al., 2018</xref>) were established, providing the site information of RNA modifications. Subsequently, single-based technologies such as m<sup>6</sup>A-CLIP (<xref rid=\"B42\" ref-type=\"bibr\">Ke et al., 2015</xref>) and miCLIP (<xref rid=\"B53\" ref-type=\"bibr\">Linder et al., 2015</xref>) were also developed to precisely identify the positions of m<sup>6</sup>A. Complementary to experimental methods, well-established computational models facilitate the analysis of sequencing data and address the challenges presented in the bioinformatics community by predicting potential RNA methylation sites. The exomePeak R/Bioconductor package (<xref rid=\"B61\" ref-type=\"bibr\">Meng et al., 2013</xref>, <xref rid=\"B62\" ref-type=\"bibr\">2014</xref>), MACS algorithm (<xref rid=\"B90\" ref-type=\"bibr\">Zhang et al., 2008</xref>) and DRME software (<xref rid=\"B59\" ref-type=\"bibr\">Liu et al., 2016</xref>) were introduced to analyze epitranscriptome profiling data, which improved our understanding of RNA methylation. Sequence-based site prediction models such as iRNA(m<sup>6</sup>A)-PseDNC (<xref rid=\"B14\" ref-type=\"bibr\">Chen W. et al., 2018</xref>) and iRNAMethyl (<xref rid=\"B11\" ref-type=\"bibr\">Chen et al., 2015b</xref>) applied statistical methods, whereas m<sup>6</sup>Apred (<xref rid=\"B12\" ref-type=\"bibr\">Chen et al., 2015c</xref>), RAM-ESVM (<xref rid=\"B19\" ref-type=\"bibr\">Chen et al., 2017b</xref>), and RNAMethPre (<xref rid=\"B81\" ref-type=\"bibr\">Xiang et al., 2016</xref>) integrated machine learning approaches, predicting m<sup>6</sup>A sites in different species’ transcriptome. Furthermore, potential RNA methylation-disease associations have been revealed by m<sup>6</sup>Avar (<xref rid=\"B93\" ref-type=\"bibr\">Zheng et al., 2018</xref>) and m<sup>6</sup>ASNP (<xref rid=\"B40\" ref-type=\"bibr\">Jiang et al., 2018</xref>). With a similar purpose, heterogeneous networks have been used in DRUM (<xref rid=\"B74\" ref-type=\"bibr\">Tang et al., 2019</xref>), FunDMDeep-m<sup>6</sup>A (<xref rid=\"B88\" ref-type=\"bibr\">Zhang et al., 2019b</xref>) and Deepm<sup>6</sup>A (<xref rid=\"B87\" ref-type=\"bibr\">Zhang et al., 2019a</xref>), showing a new perspective in studying disease-associated RNA methylation.</p><p>In this study, we constructed SVM-based models for the prediction of target specificity of m<sup>6</sup>A readers (YTHDC1, YTHDC2, YTHDF1, YTHDF2, YTHDF3, and EIF3A). The proposed models rely on 58 genomic features integrated with the sequence features related to chemical properties. After feature selection using the <italic>F</italic>-score method, those models achieved high prediction performance in 5-fold cross-validation and independent testing. Additionally, we compared the performance of different sequence encoding schemes on each reader’s substrate prediction. As existing m<sup>6</sup>A base-resolution data suffer from the bias of polyA selection, mature mRNA model was also considered besides the full transcript model. Moreover, we compared different machine learning algorithms and showed that four algorithms all demonstrate high performance with little difference in the prediction of target specificity of m<sup>6</sup>A readers. We eventually decided to use SVM classifier for our predictors.</p><p>It is also worth mentioning that our comprehensive analysis of m<sup>6</sup>A readers revealed potential regulatory patterns and biological relationships. We showed that m<sup>6</sup>A reader binding sites on mRNAs were concentrated in CDSs and 3′ UTR near stop codons, which is in line with m<sup>6</sup>A localization. Although distribution analysis of m<sup>6</sup>A readers has been conducted in previous studies and suggested similar binding patterns (<xref rid=\"B83\" ref-type=\"bibr\">Xu et al., 2014</xref>; <xref rid=\"B78\" ref-type=\"bibr\">Wang et al., 2015</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Hsu et al., 2017</xref>), the results we presented were substantially enhanced with the incorporation of multiple datasets. Our result shed lights on the post-transcriptional and translational functions of m<sup>6</sup>A readers on m<sup>6</sup>A-containing mRNAs with more reliable evidence. Moreover, computed phastCons score and conservation ratio revealed a high conservation of the target sites of m<sup>6</sup>A readers, suggesting that they are possibly playing necessary or essential roles in regulating m<sup>6</sup>A-containing mRNAs. This is remarkable since we focused on the conservation of binding sites of m<sup>6</sup>A readers on mRNAs, rather than the conservation of m<sup>6</sup>A motifs itself as widely studied currently (<xref rid=\"B65\" ref-type=\"bibr\">Meyer et al., 2012</xref>), thus the biologically meaningful relationship between m<sup>6</sup>A readers and m<sup>6</sup>A modifications was confirmed. Besides, different from enrichment analysis alone in previous studies (<xref rid=\"B36\" ref-type=\"bibr\">Hsu et al., 2017</xref>), we not only unveiled functional relevance through the enrichment of the targets of m<sup>6</sup>A readers in biological process, cellular components and molecular functions by GO analysis, but also confirmed that reader-regulated sites are more likely to be biologically significant than randomly selected sites. The combination of statistical analysis and GO analysis ensures the robust detection and critical evaluation of the biological functions with a higher degree of confidence. Furthermore, our GO enrichment analysis result is also consistent with the wet-lab experiment and our prediction on unidentified regions that YTHDF proteins have both similar functions and different functions in the m<sup>6</sup>A mRNA regulation. This supports the conclusion made in previous study that m<sup>6</sup>A readers’ effect on downstream processes are much more heterogeneous and context-dependent across transcripts (<xref rid=\"B92\" ref-type=\"bibr\">Zhang et al., 2020</xref>).</p><p>However, this study has a number of limitations that could be improved in the future. Firstly, it has been argued that 4SU PAR-CLIP suffers from U-bias in contrast with UV-254 crosslinking or 6SG crosslinking (<xref rid=\"B3\" ref-type=\"bibr\">Ascano et al., 2012</xref>), thus other CLIP techniques are recommended to ensure crosslinking efficiency. Secondly, although data from different experiments were combined to build the predictors and 5-fold cross-validation was used to balance the bias-variance tradeoff, data of YTHDC2 and EIF3A substrates are still limited, which may make overfitting of the models possible. Thus, the analysis and prediction will benefit from other data from wet experiments in the future. Thirdly, as genome-derived features improved the performance of predictors dramatically, this suggests that genomic features carry important characteristics of biological data. Considering only 58 of them were involved in the feature selection procedure, it is worth exploring more genomic features so as to allow more effective features to be selected and reduce the bias as much as possible. In the future, it is expected to see the expanded studies of the enzyme target specificity and functional associations of other RNA modifications, such as m<sup>1</sup>A and Pseudouridine, on other types of RNAs, such as lncRNA and snRNAs, and in other species, such as mouse and yeast. Additional studies are clearly needed to investigate RNA-sequence-dependent m<sup>6</sup>A readers other than YTH domain-containing proteins such as FMR1 (<xref rid=\"B28\" ref-type=\"bibr\">Edupuganti et al., 2017</xref>). And it could be quite interesting to explore disease-associated RNA modification based on cellular binding patterns of regulatory proteins on modified RNAs.</p></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ <xref ref-type=\"supplementary-material\" rid=\"SM3\">Supplementary Material</xref>.</p></sec><sec id=\"S6\"><title>Author Contributions</title><p>KC conceived the idea, initialized the project, collected and processed the training and benchmark datasets. ZW generated the genomic features. DZ, YW, YZ, and HX built machine learning models. YT and BS designed and built the web server. DZ, YW, and YZ drafted the manuscript. All authors read, critically revised, and approved the final manuscript.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work has been supported by the National Natural Science Foundation of China (31671373) and XJTLU Key Program Special Fund (KSF-T-01). This work was partially supported by the AI University Research Centre through XJTLU Key Programme Special Fund (KSF-P-02).</p></fn></fn-group><fn-group><fn id=\"footnote1\"><label>1</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://m6Areader.rnamd.com\">http://m6Areader.rnamd.com</ext-link></p></fn></fn-group><sec id=\"S8\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fcell.2020.00741/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fcell.2020.00741/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Table_1.XLSX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM2\"><media xlink:href=\"Table_2.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM3\"><media xlink:href=\"Image_1.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Adams</surname><given-names>J. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Bioeng Biotechnol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Bioeng Biotechnol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Bioeng. Biotechnol.</journal-id><journal-title-group><journal-title>Frontiers in Bioengineering and Biotechnology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-4185</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850765</article-id><article-id pub-id-type=\"pmc\">PMC7431670</article-id><article-id pub-id-type=\"doi\">10.3389/fbioe.2020.00943</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Bioengineering and Biotechnology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Melanoma Peptide MHC Specific TCR Expressing T-Cell Membrane Camouflaged PLGA Nanoparticles for Treatment of Melanoma Skin Cancer</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Yaman</surname><given-names>Serkan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/972906/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ramachandramoorthy</surname><given-names>Harish</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/972356/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Oter</surname><given-names>Gizem</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhukova</surname><given-names>Daria</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Nguyen</surname><given-names>Tam</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1032065/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Sabnani</surname><given-names>Manoj K.</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1007839/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Weidanz</surname><given-names>Jon A.</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Nguyen</surname><given-names>Kytai T.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/147572/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Bioengineering, The University of Texas at Arlington</institution>, <addr-line>Arlington, TX</addr-line>, <country>United States</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Joint Bioengineering Program, The University of Texas Southwestern Medical Center</institution>,<addr-line> Dallas, TX</addr-line>, <country>United States</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Biology, University of Texas at Arlington</institution>, <addr-line>Arlington, TX</addr-line>, <country>United States</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Stefano Leporatti, Institute of Nanotechnology (CNR), Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Paolo Bigini, Mario Negri Institute for Pharmacological Research (IRCCS), Italy; Pradipta Ranjan Rauta, The University of Texas MD Anderson Cancer Center, United States</p></fn><corresp id=\"c001\">*Correspondence: Jon A. Weidanz, <email>weidanz@uta.edu</email></corresp><corresp id=\"c002\">Kytai T. Nguyen, <email>knguyen@uta.edu</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>These authors have contributed equally to this work</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>943</elocation-id><history><date date-type=\"received\"><day>07</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>21</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Yaman, Ramachandramoorthy, Oter, Zhukova, Nguyen, Sabnani, Weidanz and Nguyen.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Yaman, Ramachandramoorthy, Oter, Zhukova, Nguyen, Sabnani, Weidanz and Nguyen</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Melanoma is one of the most aggressive skin cancers, and the American Cancer Society reports that every hour, one person dies from melanoma. While there are a number of treatments currently available for melanoma (e.g., surgery, chemotherapy, immunotherapy, and radiation therapy), they face several problems including inadequate response rates, high toxicity, severe side effects due to non-specific targeting of anti-cancer drugs, and the development of multidrug resistance during prolonged treatment. To improve chemo-drug therapeutic efficiency and overcome these mentioned limitations, a multifunctional nanoparticle has been developed to effectively target and treat melanoma. Specifically, poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) were coated with a cellular membrane derived from the T cell hybridoma, 19LF6 endowed with a melanoma-specific anti-gp100/HLA-A2 T-cell receptor (TCR) and loaded with an FDA-approved melanoma chemotherapeutic drug Trametinib. T-cell membrane camouflaged Trametinib loaded PLGA NPs displayed high stability, hemo- and cyto-compatibility. They also demonstrated membrane coating dependent drug release profiles with the most sustained release from the NPs proportional with the highest amount of membrane used. 19LF6 membrane-coated NPs produced a threefold increase in cellular uptake toward the melanoma cell line <italic>in vitro</italic> compared to that of the bare nanoparticle. Moreover, the binding kinetics and cellular uptake of these particles were shown to be membrane/TCR concentration-dependent. The <italic>in vitro</italic> cancer killing efficiencies of these NPs were significantly higher compared to other NP groups and aligned with binding and uptake characteristics. Particles with the higher membrane content (greater anti-gp100 TCR content) were shown to be more effective when compared to the free drug and negative controls. <italic>In vivo</italic> biodistribution studies displayed the theragnostic capabilities of these NPs with more than a twofold increase in the tumor retention compared to the uncoated and non-specific membrane coated groups. Based on these studies, these T-cell membrane coated NPs emerge as a potential theragnostic carrier for imaging and therapy applications associated with melanoma.</p></abstract><abstract abstract-type=\"graphical\" id=\"G1\"><title>Graphical Abstract</title><p><graphic xlink:href=\"fbioe-08-00943-g001a.jpg\" position=\"float\"/></p></abstract><kwd-group><kwd>membrane-based drug delivery</kwd><kwd>T-cell membrane-coated nanoparticles</kwd><kwd>theragnostic nanoparticles</kwd><kwd>melanoma</kwd><kwd>cancer therapy</kwd></kwd-group><counts><fig-count count=\"6\"/><table-count count=\"0\"/><equation-count count=\"1\"/><ref-count count=\"32\"/><page-count count=\"14\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Melanoma is one of the most common cancer types in the United States. It is the most aggressive type of skin cancer and influences people of all ages. Heavy exposure to ultraviolet (UV) radiation, from sunlight or the use of indoor tanning devices (classified as carcinogenic by the International Agency for Research on Cancer) are some of the major causes of melanoma. Risk is also increased for people who are sun-sensitive and those with a weakened immune system (<xref rid=\"B8\" ref-type=\"bibr\">Hayes et al., 2016</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Wheatley et al., 2016</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Street, 2019</xref>). Although many advances have been made for the treatment of aggressive melanoma stages, including improved chemotherapy, targeted therapy, immunotherapy, radiotherapy, and the combinations of the two (<xref rid=\"B7\" ref-type=\"bibr\">Hauschild et al., 2003</xref>), most advanced melanoma cases are incurable. Chemotherapy and targeted therapy involve the administration of drugs that often stop DNA transcription or block intracellular signaling pathways in the hope of inhibiting cell growth and replication. In chemotherapy, drugs such as dacarbazine, cisplatin, vinblastine, and temozolomide are widely used. A novel mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor, Trametinib, was approved by the United States Food and Drug Administration (FDA) in May 2013 as a single agent for the treatment of BRAF V600E/K mutant metastatic melanoma (<xref rid=\"B14\" ref-type=\"bibr\">Lugowska et al., 2015</xref>). While there are a wide number of therapies and drug combinations available for the treatment of melanoma, a key limitation in the efficacy of chemotherapy is the severe side effects and the development of multidrug resistance during prolonged treatment. Chemotherapy and the listed traditional treatments, however, are often accompanied with insufficient response rates and numerous severe side effects owing to the low efficacy and non-specific targeting mechanisms of drug delivery (<xref rid=\"B14\" ref-type=\"bibr\">Lugowska et al., 2015</xref>).</p><p>One particular focus of interest in immunotherapy encompasses technologies such as cell therapy. Immune cells are isolated from immunosuppressed cancer patients and reprogrammed <italic>in vitro</italic> against cancer antigens (specific to the cancer present in the patient). Current research has mostly focused on the modification of dendritic cells and T-cells, with T-cell based cancer immunotherapy having been used effectively in cancer treatment (<xref rid=\"B27\" ref-type=\"bibr\">Tran et al., 2014</xref>). T-cells are collected from patient’s own body and engineered to generate specific receptors on their surface. These receptors are referred to as chimeric antigen receptors (CARs), which are a specific type of protein located on tumor cells and provide the T-cells with a way to recognize and kill cancer cells (<xref rid=\"B31\" ref-type=\"bibr\">Zhao and Cao, 2019</xref>). Although these methods have been proven to be effective, they are accompanied by several limitations. Along the high costs and complexities of isolation, the long-term presence of re-programmed cells in the body poses some major concerns. The possible risks of long-term survival of re-programmed cells have included the development of autoimmune disease and can subsequently lead to death (<xref rid=\"B20\" ref-type=\"bibr\">Sharpe and Mount, 2015</xref>; <xref rid=\"B2\" ref-type=\"bibr\">Charrot and Hallam, 2019</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Stoiber et al., 2019</xref>).</p><p>Nanoparticle-based drug delivery systems have recently gained extensive attention for cancer detection and therapy. Nanoparticles possess several advantages, including increased stability, enhanced carrier capacity, varied feasible methods of administration and the ability to incorporate both hydrophilic and hydrophobic types of drugs (<xref rid=\"B18\" ref-type=\"bibr\">Pinto-Alphandary et al., 2000</xref>). Nano-vehicles often serve to protect the drug and can be customized to release the drug in a sustained fashion. Sustained kinetics could lead to enhanced drug bioavailability at the cancer site and reduced toxicity to healthy tissues. Several currently studied, nano-based drug delivery platforms for melanoma include polymeric carriers, liposomes, polymersomes, carbon-based nanoparticles, and protein-based nanoparticles (<xref rid=\"B1\" ref-type=\"bibr\">Cai et al., 2014</xref>). Polymer based nanocarriers are widely studied due to their long-term stability and ability to provide a sustained drug release over prolonged periods of time. Current FDA approved examples of polymers include PLGA (polylactic-co-glycolic acid) and PLA (polylactic acid). Even though such polymers are biocompatible, they are still far from complete immune evasion (<xref rid=\"B4\" ref-type=\"bibr\">Danhier et al., 2012</xref>). To overcome the limitations of current drug delivery and immunotherapy methods, numerous novel modifications have been studied over the last few decades in the hopes of giving polymeric nano-carriers the ability to escape the immune surveillance. Several of these modifications include PEGylation, plasma-surface modification and lipid-based or cell membrane masking. In addition, with the emergence of nanotechnology, the use of bio/nano-carriers is widely expected to alter the landscape of cancer treatment. For example, cell membrane-based drug delivery applications have raised an interest due to the ability of providing better “stealth” properties for foreign biomaterial-based vehicles to remain unnoticed in the face of immune cells. Several of the stealth functionalities of synthetic and biopolymers are used to enable prolonged pharmacokinetics and improve bio-distribution of the particles.</p><p>In this study, we have hypothesized that membrane-coated nanoparticles present a comprehensive evasion strategy against the multi-faceted nature of immune clearance and a cancer cell specificity approach. Cell membrane coated biomimetic nanoparticles have made an impressive contribution to the improvement of cancer therapy (<xref rid=\"B5\" ref-type=\"bibr\">Fang et al., 2014</xref>) due to the cell membrane structure and retained cellular antigens. Biomimetic NPs carry special advantages, such as long blood circulation, ligand recognition, immune escape, homotypic targeting, and the ability for a sustained drug delivery (<xref rid=\"B25\" ref-type=\"bibr\">Tan et al., 2015</xref>). Recently, cell membrane coated nano-systems with unique features and functions have been tested and proved to have higher circulation time and immune evasion (<xref rid=\"B17\" ref-type=\"bibr\">Parodi et al., 2013</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Hu et al., 2015</xref>). In this study, we have used a hybridoma T-cell with specific targeting against the melanoma cancer. The hybridoma 19LF6 expressed gp100 antigen-MHC molecule recognizing moiety. This cell membrane would be extracted and coated on PLGA nanoparticles encapsulating dyes (coumarin-6 for <italic>in vitro</italic> studies/lipophilic DiD for <italic>in vivo</italic> studies)/Drug (Trametinib) wherein serving as a highly melanoma specific carrier for diagnosis/treatment, respectively. The presence of anti-gp100 TCR on T-MNPs would specifically target gp100 presenting melanoma cells and enhance cellular uptake of T-MNPs. The chemo-drug, Trametinib, was selected due to its effectiveness against cell lines carrying a V600E BRAF oncogenic mutation acquired by DM-6 and 1520 cancer cell lines. Trametinib is an FDA approved MEK (MEK 1 and 2) that can also be used in combination with other approved anti-melanoma drugs. PLGA polymer was chosen for its excellent properties, including biocompatibility, biodegradability, FDA and European Medicine Agency approval, adaptability to both hydrophobic and hydrophilic drugs, and sustained drug release kinetics. Properties of T-MNPs, including binding affinity with skin cancer cells, cellular uptake, therapeutic efficacy, particle retention, and biodistribution, were evaluated using cell cultures and animal models. Their results were also compared to non-specific membrane coated (A-MNPs and D-MNPs) and naked PLGA NPs (NNPs).</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Materials</title><p>Poly (<sc>D</sc>, <sc>L</sc> lactide-co-glycolide) (PLGA) (inherent viscosity 50:50 with carboxyl end groups) was purchased from Akina (PolySciTech; West Lafayette, IN, United States). Poly (vinyl alcohol) (PVA, MW 30,000–70,000) and DCM (Dichloromethane) were obtained from Sigma Aldrich (St. Louis, MO, United States). Trametinib, coumarin-6, Tris-HCL, <sc>D</sc>-glucose, <italic>B</italic>-Mercaptoethanol, Phenylmethylsulfonyl fluoride (PMSF), Protease inhibitor cocktail, Triton<sup>®</sup> X-100, Dimethyl sulfoxide (DMSO) and RPMI-1640 were also received from Sigma-Aldrich. The Avanti Polar Lipids Mini Extruder Kit was ordered from Avanti Polar Lipids (Alabaster, AL, United States). RIPA buffer was purchased from Alfa Aesar (Haverhill, MA, United States). SDS-PAGE gel, and Mini PVDF transfer pack were obtained from Bio-Rad. TCR β chain (Armenia Hamster IgG) – antibody and its isotype antibody were bought from BD Biosciences. Nuetravidin biotin binding protein, Superblock solution, BCA assay kit and DNAse were obtained from Thermofisher Scientific. Formvar coated copper TEM grids were purchased from Electron Microscopy Sciences. Fetal bovine serum (FBS), 1X trypsin EDTA, Dulbecco’s Modified Eagle’s Medium (DMEM) and penicillin-streptomycin were ordered from Invitrogen. Biotinylated gp100-HLA-A2/B2M (gp100 refold) and biotinylated GIGL peptide-HLA-A<sup>∗</sup>201/B2M complexes were synthesized from Dr. Weidanz’s laboratory. gp100 refold heavy chain and Beta-2-Microglobulin chain were expressed in and harvested from <italic>Escherichia coli</italic> in the form of inclusion bodies. The complex was biotinylated using Avidin Biotinylation kit [Bulk BirA: BirA biotin-protein ligase bulk reaction kit]. gp100-2M (209–217) peptide was received from Genscript. bGIGL complex was synthesized in a similar fashion. DM-6, and 1520 cell lines were obtained from Dr. Weidanz’s lab. T-cell hybridomas 19LF6 and DO11.10 were kind gifts from Dr. Devin Lowes (Texas Tech University Health Sciences Center) and Dr. Philippa Marrack (National Jewish Health, Denver, CO, United States), respectively.</p></sec><sec id=\"S2.SS2\"><title>Synthesis of PLGA Nanoparticles</title><p>Poly (lactic-co-glycolic acid) nanoparticles (naked NPs; NNPs) were synthesized via a modified single emulsion (O/W) technique as described previously by <xref rid=\"B15\" ref-type=\"bibr\">Menon et al. (2014)</xref>. Briefly, 90 mg of PLGA 50:50 and 4.5 mg of Trametinib were dissolved in 3 mL of DCM and sonicated at 30 W for 2 min to allow dispersion of PLGA and Trametinib in the solvent. The resulting solution was added in dropwise to 15 ml of filtered 5% (w/v) PVA solution under stirring conditions. The suspension was then sonicated at 30 W for 10 min and then allowed to stir overnight to evaporate the organic solvent. The obtained nanoparticle suspension was centrifuged at 15,000 rpm for 30 min. The supernatant was saved for the drug loading evaluation, and the PLGA NP pellet was re-suspended in 3 ml of DI water followed by freeze-drying for 24 h. Nanoparticles for imaging/diagnostic techniques were synthesized by a similar procedure with coumarin-6 instead of the Trametinib.</p></sec><sec id=\"S2.SS3\"><title>Cell Lines and Culture Conditions</title><p>The cell lines used for the experiments, DM-6, 1520, A549 and 19LF6, were maintained in RPMI-1640, supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin streptomycin. DO11.10 and HDF cell lines were maintained in high glucose DMEM, supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin streptomycin. All cells were incubated at 37°C, 5% CO<sub>2</sub>.</p></sec><sec id=\"S2.SS4\"><title>Synthesis of Cell Membrane-Coated NPs (MNPs)</title><p>To harvest the cell membrane, cells were grown to confluency in T-225 cell culture flasks. Cells were isolated by trypsinization and centrifuged at 1,000 rpm for 5 min. To remove any remaining media contents, the cells were washed with cold 1X PBS and centrifuged at 1,000 g for 10 min. The resulting pellet was re-suspended in hypotonic lysis buffer (10 mm Tris-HCL, pH = 7.5) and supplemented with the ready-to-use 1× protease inhibitor cocktail. The solution was kept on ice for 20 min and then centrifuged at 1,000 <italic>g</italic> for 10 min. The pellet was re-suspended in cold 0.25 × PBS and kept on ice for 20 min followed by centrifugation at 800 <italic>g</italic> for 5 min. The final pellet was collected in cold 1× PBS. The cell membrane mix was analyzed for the DNA and protein content using Nano-Drop 1000 Spectrophotometer (Thermo Fisher Scientific)/PicoGreen DNA assays and BCA assay kits, respectively. To eliminate any remaining DNA contents, DNAse reaction was performed on the cell extract by incubation with DNAase (the amount of DNAse added varied with the DNA content in the cell extract sample) for least 1 h at 37°C.</p><p>Poly (lactic-co-glycolic acid) (PLGA) nanoparticles (naked NPs; NNPs) were coated with different cell membranes (MNPs) including 19LF6, DO11.10 and A549 by adapting a previously used technique by <xref rid=\"B9\" ref-type=\"bibr\">Hu et al. (2011)</xref>. These particles were loaded with an FDA-approved chemotherapeutic drug, Trametinib, suitable for treatment of melanoma cell lines containing V600E BRAF mutation. Briefly, NNPs were re-suspended in a cell membrane solution at different NNP weight to membrane protein weight ratios (w/w): 1:0.5, 1:1, 1:2 and 1:3. The mixture was then extruded 15 times using a pre-heated Avanti Polar Lipids Mini Extruder (37°C). The extrusion was performed using a 200-nm polycarbonate membrane. The resulting membrane coated NPs (MNPs) were dialyzed with a 100 kDa MWCO dialysis membrane for 2 h. Prior to freeze-drying, dialyzed MNP solution was supplemented with <sc>D</sc>-glucose at a final concentration of 1 mg/ml. Three types of membrane-coated NPs were created namely: (1) 19LF6 cell line (T-cells specific to melanoma) coated NPs (T-MNPs) – our treatment, (2) DO11.10 cell line (non-specific T-cells) coated NPs (D-MNPs) – control for T-cells, and (3) A549 cell line (lung cancer) coated NPs (A-MNPs) – control for other cell types.</p></sec><sec id=\"S2.SS5\"><title>Physiochemical Characterizations</title><p>Nanoparticle size, polydispersity index, and zeta potential were investigated using Dynamic Light Scattering (DLS) technique. To measure the size of the nanoparticles, NP suspension (10 μl of 500 μg/ml) was added to 3 ml of deionized water and inserted into the DLS in a compatible cuvette. To generate TEM images of MNPs, 10 μl of 250 μg/ml MNP suspension was added to plasma treated Formvar Square Mesh Copper Grids and airdried. An H-7500 TEM (Hitachi) transmission electron microscope was used to visualize the particle morphologies. Confirmation of T-cell receptor membrane protein (TCR) on the 19LF6 cell membranes coated nanoparticles was performed via flow cytometry. The particles were also stained with TCR- β chain to evaluate the presence of any TCR or TCR components on synthesized T-MNPs. Briefly, 1mg/ml of T-MNP solution was prepared in a staining buffer (0.5% BSA, 1mm EDTA in 1× PBS). 200 μl of the solutions was added with antibodies Armenian Hamster anti-TCR β and Armenian Hamster IgG antibodies at 1:100 dilution, for test group and isotype control, respectively. The solutions were then incubated for 30 min and then washed 3× times with the staining solution. The cells were collected by centrifuging at 14,000 rpm for 20 min and resuspending in 200 μl of staining buffer. Each of the groups was then analyzed using a BD Biosciences LSR II Flow cytometer without an FSC threshold to be able to detect nanoparticles.</p><p>Stability of T-MNPs with varying NP weight to membrane protein weight ratios (w/w) was evaluated by monitoring the particle size at pre-determined time-points using DLS. To observe the stability of T-MNPs, particles of different membrane ratios were incubated in 0.9% saline over 48 h. T-MNP suspensions were prepared as described above, and the size of particles was measured at different time points (0, 0.5, 1, 3, 6, 12, 24, and 48 h).</p></sec><sec id=\"S2.SS6\"><title>Binding Kinetics Assay</title><p>The binding characteristics of T-MNPs were studied using ResoSens label-free optical detection system. Biotinylated pMHC complexes HLA-A<sup>∗</sup>02:01–IMDQVPFSV (gp100<sub>209</sub><sub>–</sub><sub>217</sub>) and HLA-A<sup>∗</sup>02:01–GILGFVFTL (Influenza-M, negative control) were synthesized by our group previously. In this study, T-MNPs with the highest and lowest NP weight to membrane weight ratios were tested at varying concentrations. D-MNPs were tested at the highest NP to membrane protein ratio (w/w) (1:2), and at a concentration 2× higher than the highest concentration used for T-MNPs. All samples were tested against a specific gp100-b monomer and non-specific b monomer GILG.</p></sec><sec id=\"S2.SS7\"><title>Drug Loading and Drug Release Kinetics of T-MNPs</title><p>The drug/dye loading efficiency was calculated by an indirect method where the drug/dye present in the supernatant collected from the nanoparticle synthesis process was measured, and the following formula was used for loading efficiency calculation:</p><disp-formula id=\"S2.Ex1\"><mml:math id=\"M1\"><mml:mrow><mml:mo rspace=\"5.3pt\">%</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>loading</mml:mi></mml:mpadded><mml:mi>efficiency</mml:mi><mml:mo>=</mml:mo><mml:mpadded width=\"+3.3pt\"><mml:mfrac><mml:mtable rowspacing=\"0pt\"><mml:mtr><mml:mtd columnalign=\"center\"><mml:mrow><mml:mrow><mml:mpadded width=\"+2.8pt\"><mml:mi>Amount</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>of</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>drug</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>used</mml:mi></mml:mrow><mml:mo>-</mml:mo></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd columnalign=\"center\"><mml:mrow><mml:mpadded width=\"+2.8pt\"><mml:mi>Amount</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>of</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>drug</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>in</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>supernatant</mml:mi></mml:mrow></mml:mtd></mml:mtr></mml:mtable><mml:mrow><mml:mpadded width=\"+2.8pt\"><mml:mi>Amount</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>of</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mpadded width=\"+2.8pt\"><mml:mi>drug</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>used</mml:mi></mml:mrow></mml:mfrac></mml:mpadded><mml:mo rspace=\"5.8pt\">×</mml:mo><mml:mn>100</mml:mn></mml:mrow></mml:math></disp-formula><p>The drug (Trametinib) release study was carried out for a period of 28 days. Briefly, 1 mg of NNPs and T-MNPs [NP: membrane protein weight (w/w) ratios of 1:0.5, 1:1 and 1:2] were taken at a concentration of 1 mg/ml and were incubated at 37°C. At each pre-determined time-point, the samples were centrifuged at 14,000 rpm for 30 min, and supernatants were collected and stored at −20°C for later analysis. The pellets were re-suspended in fresh 1× PBS and incubated for further time points. Each of the drug release aliquots was analyzed using a UV-vis Spectrophotometer at 330 nm. The amount of drug released was determined against a standard curve for Trametinib.</p></sec><sec id=\"S2.SS8\"><title><italic>In vitro</italic> Studies of T-MNPs</title><sec id=\"S2.SS8.SSS1\"><title>Assessment of Glycoprotein Expression (gp100) in Tumor Cells by Western Blot and RT PCR Analysis</title><p>The expression of glycoprotein (gp100) expression in melanoma cell lines, DM6 and 1520, and lung carcinoma cells A549 were evaluated by western blotting and RT (reverse transcriptase)-PCR. Cellular proteins were extracted from cancer cells using RIPA lysis buffer containing protease cocktail, and protein concentration was measured using the Pierce BCA protein assay kit (#23227, Thermo Fisher, United States). Cellular proteins (12 and 6 μg from each cell line) were separated by 10% SDS-PAGE and electrophoretically transferred onto PVDF membranes. The membranes were blocked with 5% BSA in TBST for 1 h at room temperature, followed by incubation with primary antibodies overnight at 4°C. The membranes were washed with TBST 3× for 5 min and then incubated with HRP conjugated secondary antibodies for 1 h at room temperature. Protein bands were developed using an ECL detection system and imaged using the Chemidoc TM Imaging system.</p><p>Total RNA was extracted from DM6, 1520, and A549 cells by using RNeasy plus Mini kits (Qiagen) according to the manufacturer’s instructions. Approximately 3 μg of total RNA was reverse transcribed into cDNA (High-Capacity cDNA RT Kit) and subjected to PCR in Bio-Rad Thermocycler (T100TM) with the following conditions, pre-denaturation at 95°C for 1 min and 30 cycles of denaturation at 95°C for 15 s, primer annealing at 57°C for 30 s, and extension at 72°C for 60 s. The gp100 gene was amplified using the following primers: 5′ GCTTGGTGTCTCAAGGCAACT 3′ (gp100 for) and 5′ CTCCAGGTAAGTATGAGTGAC 3′ (gp100 rev). β-actin gene was amplified using 5′ GGCACCACACCTTCTACAAT 3′ and 5′ GCCTGGATAGCAACGTACAT 3′. The relative amount of each gene was normalized to the amount of β-actin and the RT-PCR result for each gene was expressed as fold change over the basal level.</p></sec><sec id=\"S2.SS8.SSS2\"><title>Cellular Uptake and Therapeutic <italic>in vitro</italic> Studies</title><p>To determine the cell specific targeting function of the nanoparticle <italic>in vitro</italic>, cellular uptake studies were conducted. Briefly, 1520 (gp100 positive), DM-6 (gp100 positive) and A549 (gp100 negative) cell lines were seeded in a 96 well plate at a density of 20,000 cells/well. Coumarin-6 (C-6; fluorescent dye) loaded T-MNPs (gp100 refold specific) and D-MNPs (non-specific to gp100 refold) at varying NP to membrane weight ratios: 1:0.5, 1:1 and 1:2, were tested. Serially diluted MNP concentrations (100, 250, 500, and 1000 μg/ml) at different NP to membrane ratios were prepared. The cells were exposed to different NP groups for approximately 2 h, subsequently washed with 1× PBS and lysed with 250 μl/well of 1% Triton<sup>®</sup> X-100. Cell lysis extracts were then analyzed for the protein content using the Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, United States) and C-6 fluorescent intensity using a UV-vis Spectrophotometer (458/540 ex/em). Total protein concentration in each lysate was determined using a BSA standard curve. The uptake of the nanoparticles was calculated by normalizing the particle concentration (determined from fluorescence intensity in a lysate) in each sample with total cell protein, which correlated to the number of cells in the sample. Untreated cells were used as a negative control.</p><p>For the therapeutic efficiency of Trametinib loaded T-MNPs, melanoma cell lines, DM-6 and 1520, were used. Firstly, the effective concentration (IC50) of the Trametinib on the cell lines were determined by exposing cells seeded in a 96 well plate at density of 5,000 cells/well to a free drug of known concentrations (1.2, 2.5, 5, 10, 15, 20, 25, 50, 75, and 100 μg/mL) for a period of 72 h. After 72 h of incubation, the cell death was analyzed using MTS assays (CellTiter 96<sup>®</sup> AQueous Non-Radioactive Cell Proliferation Assay, Promega) according to manufacturer instructions. Therapeutic efficiency of different nanoparticle groups including 1:2 NP weight to membrane weight ratio T-MNPs (melanoma-specific), A-MNPs (non-specific), and D-MNPs (non-specific) as well as free drug and NNPs (bare/naked NPs) was determined. Three different NP concentrations (0.83, 1.66, and 2.5 μg/ml for DM-6; 15, 29, and 44 μg/ml for 1520) were used based on the calculation of the IC50 studies. Cells seeded in a 96 well plate at a density of 20,000 cells/well were exposed to the described groups for 72 h, and the cell viability was determined using MTS assays.</p></sec></sec><sec id=\"S2.SS9\"><title>Cyto-Compatibility and Hemo-Compatibility</title><p>A cyto-compatibility study was performed on human dermal fibroblast cells (HDFs). T-MNPs and NNPs were re-suspended in media and added to cells at various concentrations (50, 100, 250, 500, and 1,000 μg/ml) followed by 24 h of incubation at 37°C. After incubation, cell viability was evaluated using MTS assays. Cells without treatment and cells treated with 1% Triton<sup>®</sup> X-100 served as positive and negative controls, respectively.</p><p>Blood clotting and hemolysis property of T-MNPs were performed on fresh human blood. Briefly, for hemolysis, 200 μl of blood was added with 10 μl of the T-MNPs prepared in 0.9% saline at varying concentrations (50, 100, 250, 500, and 1,000 μg/ml). 10 μl of 0.9% saline and 10 μl of DI water was used as positive and negative controls. The tubes were incubated at 37°C for 2 h under gentle agitation and then were centrifuged at 1,000 <italic>g</italic> for 10 min. The absorbance of sample supernatants was monitored at 545 nm using a UV-vis Spectrophotometer. For hemo-compatibility, 10 μl of T-MNPs at the various concentrations (50, 100, 250, 500, and 1,000 μg/ml) in 0.9% saline was mixed in 50 μl activated blood (0.1 M CaCI<sub>2</sub> added blood). At pre-determined time-points (10, 20, 30, and 60 min), 1.5 ml of DI water was added to all samples to inactivate blood clotting, and the samples were incubated for 5 min at room temperature. Supernatants were collected and monitored at 540 nm using a UV-vis Spectrophotometer. 0.9% saline and DI water were used as positive and negative controls.</p></sec><sec id=\"S2.SS10\"><title><italic>In vivo</italic> Biodistribution Studies of T-MNPs via Near-Infrared Fluorescence Imaging</title><p>The tumor specific targeting and accumulation of T-MNPs were assessed using a subcutaneous xenograft model of melanoma. It was established by injecting 0.5 × 10<sup>6</sup> DM-6 cells in 150 μl of Matrigel (Corning, United States) subcutaneously in athymic nude mice. Tumor volumes were checked at regular intervals and treatment started when the volume reached ∼150 mm<sup>3</sup>. For imaging of NP biodistribution <italic>in vivo</italic>, lipophilic DiD (Thermo, United States) dye was incubated with 19LF6 T-MNPs to stain membranes for 30 min and unbound dye was washed away. All nanoparticle groups were injected intravenously through the tail vein. Following, anesthetized mice were scanned at different time points using a Kodak<sup>TM</sup>\n<italic>in vivo</italic> imaging system. After 24 h time point, the mice were euthanized under anesthesia. Tumors and organs were excised and prepared for <italic>ex vivo</italic> imaging and fluorescent analysis. Excised organs and tumors then were analyzed by their weights and fluorescent responses.</p></sec><sec id=\"S2.SS11\"><title>Statistical Analysis</title><p>All results were expressed as mean ± SD performed with <italic>n</italic> = 3 for most of the experiments if not specified. Results were analyzed using either one-way or two-way ANOVA depending on experiments with <italic>p</italic> < 0.05, and the student’s <italic>t</italic>-test was used to identify differences between groups. <italic>P</italic> < 0.05 was considered to be statistically significant.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Particle Size, Zeta Potential, and Morphology</title><p>The DLS results showed that the T-MNPs with 1:2 NP to membrane ratio had a particle size of about 193 ± 56 nm with a polydispersity of 0.265. The particle size of naked NPs (NNPs) was around 171.7 ± 76 nm with a polydispersity of 0.109. The particle sizes were confirmed with Transmission Electron Microscopy (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). Furthermore, TEM images of T-MNPs showed that the particles were smooth and spherical with a clear core-shell structure. The T-MNPs also had a stable ZETA potential value of −36 mV compared to a −20 mV ZETA potential of the NNPs. The stability studies also showed that the T-MNPs were stable for the whole incubation period, which was denoted by no significant change in the size of the T-MNPs during the course of experiment and, therefore, there was no sign of significant aggregation (<xref ref-type=\"fig\" rid=\"F1\">Figures 1B,C</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Characterization of T-MNPs. <bold>(A)</bold> TEM images of T-MNPs at 1:2 NP to membrane protein weight ratio. <bold>(B)</bold> Size, polydispersity and zeta potential of PLGA NPs and T-MNPs analyzed using Dynamic Light Scattering (DLS). <bold>(C)</bold> Stability of T-MNPs in 0.9% saline (at varying NP to membrane protein weight ratios: 1:0.5, 1:1, 1:2, and 1:3) evaluated by change in NPs size over a 2-day period using DLS. <bold>(D)</bold> Cumulative% drug release from T-MNPs (varying NP to membrane protein ratios of 1:0.5, 1:1, 1:2 versus PLGA NPs) performed in phosphate buffered saline over 28 days. Samples were analyzed using UV-vis spectrophotometer.</p></caption><graphic xlink:href=\"fbioe-08-00943-g001\"/></fig><p>Drug loading efficiency of T-MNPs was found to be 61%. To analyze how different membrane coating ratios would affect Trametinib release from T-MNPs, the drug release kinetics of T-MNPs with different NP to membrane weight ratios (1:0.5, 1:1, and 1:2) and naked NPs were evaluated over 28 days. The rate of release kinetics differed across the three ratios of NP to membrane protein (w/w) T-MNPs. The Trametinib release rate was significantly slower and sustained for all ratios of T-MNPs when compared to MNPs, with the lowest rate of release for 1:2 T-MNPs and the highest one for 1:0.5 (<xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>). The presence of TCR β chain on 19LF6 cells and T-MNPs were confirmed by flow cytometry analysis as seen by a shift in the absorbance curve (<xref ref-type=\"fig\" rid=\"F2\">Figures 2A,B</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Validation of TCR on T-MNPs. <bold>(A)</bold> Histogram curves demonstrate 19LF6 cells expressing the TCR β chain. 19LF6 cells were stained with either mouse IgG (isotype) or anti-TCR antibody. Unstained, isotype, and stained groups are represented as black, gray, and red curves, respectively. <bold>(B)</bold> T-MNPs also expressed the TCR β chain [19LF6 MNP’s were stained with either mouse IgG (isotype) or anti-TCR antibody]. Unstained, isotype, and stained groups are represented as black, gray, and red curves, respectively. Histograms were analyzed using BD LSR II with no threshold on the forward scatter to detect the nanoparticles. <bold>(C)</bold> Binding kinetics of D-MNPs (non-specific control), T-MNPs (our treatment group) and NNPs (negative control) at varying concentrations and NP to membrane protein (w/w) ratios; kinetics were measured in terms of resonance shift over a period of time using ResoSens label-free optical detection.</p></caption><graphic xlink:href=\"fbioe-08-00943-g002\"/></fig></sec><sec id=\"S3.SS2\"><title>Binding Kinetics of T-MNPs</title><p>As shown in <xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>, T-MNPs bound onto immobilized gp100b in a dose-dependent manner with a higher binding strength observed in samples with higher concentration and higher ratio. In addition, T-MNPs at concentrations 500 and 250 μg/ml were significantly higher when compared to the binding kinetics of 1:2 D-MNPs at 1,000 μg/ml concentration.</p></sec><sec id=\"S3.SS3\"><title>Western Blot and Reverse Transcription PCR</title><p>DM6 and 1520 cell lines exhibited significant expression of glycoprotein gp100. The expression level in the 1520 cell line was found to be approximately 2× higher than DM-6, whereas the lung cancer cells A549 showed no apparent gp100 protein expression (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>). RT-PCR identified gp100 gene amplicon (751 bp) in all 3 cell lines (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>). The melanoma cancer cell 1520 has demonstrated its higher expression of gp100/HLA-A2 complex protein compared to that of the DM-6 cell line previously (<xref rid=\"B28\" ref-type=\"bibr\">Weidanz et al., 2006</xref>).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>gp100 expression and Cellular uptake of T-MNPs in different cell lines. <bold>(A)</bold> Western blot for assessing of gp100 protein content in 3 cancer cell lines: DM-6, 1520, and A549. β-actin was served as a control (42 kDa). Proteins are represented at two concentrations: 6 μg (right) and 12 μg (left) for each cell line. <bold>(B)</bold> RT-PCR: gp100 (751 bp) expression presented in 1520, DM-6, and A549 cell lines, β-actin served as a control (157 bp). <bold>(C)</bold> DM-6, <bold>(D)</bold> 1520, and <bold>(E)</bold> A549 cell lines were treated with MNPs for 2 h. Cell lysates were analyzed for MNP content using a UV-vis Spectrophotometer and cellular uptake was normalized with total cellular protein. Uptake of D-MNPs, T-MNPs and PLGA NPs (PLGA-C6) are presented at varying NP to membrane protein weight ratios. MNPs were loaded with a fluorescent dye, Coumarin 6 (<italic>n</italic> = 3).</p></caption><graphic xlink:href=\"fbioe-08-00943-g003\"/></fig></sec><sec id=\"S3.SS4\"><title><italic>In vitro</italic> Properties of T-MNPs</title><p>The cellular uptake of NPs was evaluated on DM-6 (gp100-containing melanoma), 1520 (gp100-containing melanoma), A549 (lung cancer, no gp100 antigen) cell lines. For uptake studies, fluorescent dye (Coumarin 6) loaded NPs were used. To analyze how different NP to membrane weight ratios would affect NP cellular uptake, NPs with 1:0.5, 1:1, and 1:2 ratios were utilized. Such ratios were prepared for D-MNPs and T-MNPs. The results demonstrated that T-MNPs displayed significantly higher uptake compared to the negative control, D-MNPs (<xref ref-type=\"fig\" rid=\"F3\">Figures 3C–E</xref>) in gp100-presenting melanoma cell lines. As expected, A549 did not show any selectivity/cellular uptake toward T-MNPs or D-MNPs. Such result was expected due to the absence of the gp100 antigen on the surface. The IC50 values of Trametinib were calculated to be approximately 29.3 and 1.66 μg/ml for 1520 and DM-6 cell lines, respectively. The therapeutic potential of the Trametinib-loaded T-MNPs was evaluated on both 1520 and DM-6 cell lines. It was shown that T-MNPs had a significantly higher therapeutic efficiency on both melanoma cell lines when compared to the negative controls, specifically at IC50 and IC75 drug concentrations (<xref ref-type=\"fig\" rid=\"F4\">Figures 4A,B</xref>).</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Therapeutic efficiency of T-MNPs. Therapeutic capabilities of T-MNPs, D-MNPs, A-MNPs, NNPs and free Trametinib on <bold>(A)</bold> 1520 melanoma cell line and <bold>(B)</bold> DM-6 melanoma cell lines. Cell were exposed to the nanoparticle suspensions for 72 h (ranging concentrations from IC25 to IC75), and cell viability was evaluated using MTS assays. *Statistically significant with <italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"fbioe-08-00943-g004\"/></fig></sec><sec id=\"S3.SS5\"><title>Cyto- and Hemo-Compatibility Studies</title><p>The viability of HDF cells was evaluated after interaction with the T-MNPs and PLGA NPs (naked NPs, NNPs) at various concentrations for 24 h. Since PLGA is an FDA approved and biocompatible polymer, the cytocompatibility of both NNPs and T-MNPs were also observed in our studies. The result illustrated that T-MNPs did not show any toxicity to the cell line up to a concentration of 1,000 μg/ml, similar to PLGA NPs (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>). Blood clotting assay was also performed to elucidate potential blood clotting interferences by T-MNPs. The coagulation time of blood in the presence of T-MNPs was examined at different time-points: 10, 20, 30, and 60 min. Blood coagulation initiates an activation of a cascade of coagulation factors and surface mediated reactions (<xref rid=\"B21\" ref-type=\"bibr\">Smith et al., 2015</xref>). At all the tested time points, T-MNPs did not display a significantly different blood clotting pattern when compared to that of the saline control (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>). Furthermore, hemolysis study was performed to test T-MNPs against potential negative effects on red blood cells. T-MNPs showed hemolysis properties lower than 5% up to 1,000 μg/ml concentration.</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Cyto-/Hemo-compatibility of T-MNPs. <bold>(A)</bold> Cyto-compatibility of T-MNPs was analyzed on human dermal fibroblasts (HDFs) at varying NPs concentrations (50—1,000 μg/ml) for 24 h, and cell viability was quantified using MTS assays (<italic>n</italic> = 3). <bold>(B)</bold> Blood clotting kinetics of T-MNPs. Clotting efficiency was measured in absorbance units (OD) of the supernatants collected from T-MNPs treated blood samples at pre-determined time-points: 10, 20, 30, and 60 min. The absorbances were quantified using a UV-vis Spectrophotometer (<italic>n</italic> = 9).</p></caption><graphic xlink:href=\"fbioe-08-00943-g005\"/></fig></sec><sec id=\"S3.SS6\"><title><italic>In vivo</italic> Tumor Targeting and Imaging</title><p>The <italic>in vivo</italic> tumor targeting and imaging abilities of the T-MNPs were examined to demonstrate whether injected T-MNPs accumulated at the tumor site during the treatment period. <italic>In vivo</italic> imaging results showed that the T-MNPs (melanoma-specific) had better tumor accumulation than those of the control D-MNPs (non-specific) and NNPs or bare PLGA NPs (<xref ref-type=\"fig\" rid=\"F6\">Figure 6A</xref>). The T-MNPs mainly accumulated in the tumor within 6 h. Furthermore, the tumor signal from the T-MNPs remained relatively constant throughout 24 h, while the overall signal of the NNP and D-MNP groups was decreased with time. In addition, <italic>ex vivo</italic> organ images revealed that T-MNPs could efficiently accumulate at tumor tissues and avoid liver accumulation compared to those of D-MNPs and NNPs in a xenograft mouse model as shown in <xref ref-type=\"fig\" rid=\"F6\">Figure 6B</xref>. The accumulation of T-MNPs was more than twice the amount of nanoparticle accumulation of D-MNPs or NNPs. This result is also confirmed with fluorescent intensity measurements from homogenized tumor tissues (<xref ref-type=\"fig\" rid=\"F6\">Figure 6C</xref>). Therefore, <italic>in vivo</italic> biodistribution results showed that the T-MNP’s targeting ability was confirmed via more efficient accumulation than the NNPs and D-MNPs at the tumor sites.</p><fig id=\"F6\" position=\"float\"><label>FIGURE 6</label><caption><p><italic>In vivo</italic> and <italic>ex vivo</italic> analysis of T-MNP biodistribution. <bold>(A)</bold> Real-time tumor targeting characteristics of IV injected NPs on melanoma tumor models. <bold>(B)</bold>\n<italic>Ex vivo</italic> organ images of biodistribution in different study groups. <bold>(C)</bold> Measured fluorescent intensity of <italic>in vivo</italic> biodistribution study groups in tissue homogenates (<italic>n</italic> = 6 per group). PLGA, poly-lactic-<italic>co</italic>-glycolic acid; NP, nanoparticle; T-MNPs, T-cell membrane-coated PLGA NPs. D-MNP’s, DO10.11 membrane coated PLGA NPs; C-6, coumarin-6; A-MNP’s, A549 membrane coated PLGA NPs; NNP, naked nanoparticle; OD, optical density; IC, inhibitory concentration; RT-PCR, reverse transcriptase polymerase chain reaction; UV-Vis, ultraviolet visible; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium. *Statistically significant with <italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"fbioe-08-00943-g006\"/></fig></sec></sec><sec id=\"S4\"><title>Discussion</title><p>We have thus developed a theragnostic system for effective diagnosis and treatment of melanoma. The developed nanoparticle system offers great advantages compared to the conventional drug delivery systems including enhanced retention in the cancer cells and tumor sites compared to non-targeted systems. These NPs also provide controlled/sustained drug release for up to 28 days and the least/null cytotoxic behavior. The NP drug delivery system helps to reduce the cytotoxic effects of chemotherapy drugs and is predicted to minimize immune reactions as the nanoparticles are coated in endogenous T-cell membranes. The dye loaded nanoparticles will serve as an effective tool to accurately visualize the tumor site, whereas the drug loaded nanoparticles will provide the therapeutic effectiveness to treat melanoma. Thus, our results indicate the potential of our designed T-MNPs for theragnostic application to detect and treat melanoma.</p><p>In the last decade, it has been proven that cells can be used as effective drug carriers via either themselves or their membranes that can facilitate payload delivery to desired regions (<xref rid=\"B22\" ref-type=\"bibr\">Stephan et al., 2010</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Tan et al., 2015</xref>). Previous works have also shown that healthy immune cells (i.e., leukocytes) are able to circulate around the body, migrate into tissues, transport through inflamed tissues and adhere to inflamed vessel walls (<xref rid=\"B16\" ref-type=\"bibr\">Muller, 2013</xref>). In this manner, cell membrane surface proteins such as ICAM’s, CCLs, CXCLs, TCR’s, and/or specific CD macromolecules become important to provide an effective drug delivery platform for biomimetic delivery (<xref rid=\"B26\" ref-type=\"bibr\">Thanuja et al., 2018</xref>). Recently, studies have shown that cytotoxic T lymphocyte membranes can deliver their payloads to tumor regions; for example, it has been shown that payload delivery can be facilitated through the natural lymphocyte surface proteins such as LFA-1 (<xref rid=\"B30\" ref-type=\"bibr\">Zhang et al., 2017</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Huang et al., 2018</xref>). Therefore, this study explored the role of target specific cytotoxic T lymphocyte membranes as a drug delivery platform.</p><p>The observed shortcomings of several drug/dye-loaded polymeric nanoparticles are their poor stability/increased aggregation, low control over the payload release rate, and rapid clearance from the body. Our results of T-MNPs showed that coating the nanoparticle with cell membranes could overcome these limitations. Physiochemical characterization of synthesized T-MNPs showed excellent colloidal stability in physiological conditions. The increase in stability due to the membrane coating onto the nanoparticles was evident by the stable Zeta potential of the T-MNPs compared to an unstable potential of the NNPs as described by the −30 mV Zeta rule (<xref rid=\"B13\" ref-type=\"bibr\">Kumar and Dixit, 2017</xref>). The negative surface charge (−36 mV) of the T-MNPs repel negative charged albumin molecules in the serum, preventing potential aggregations. The stability of T-MNPs in 0.90% saline solution was found to be similar across different NP weight to membrane protein weight (w/w) ratios for up to 2 days (<xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref>). <xref rid=\"B5\" ref-type=\"bibr\">Fang et al. (2014)</xref> reported a similar observation where they showed a higher stability of the coated nanoparticle for up to 15 days across a wide range of NP to membrane ratios (1:0.5 to 1:4). The loading efficiency of T-MNPs was observed to be approximately 61%, and the drug release kinetics displayed an initial burst release followed by a sustained release up to 28 days. When comparing T-MNPs and NNPs, it can be clearly seen that cloaking of drug loaded PLGA nanoparticles with T-cell membranes reduced the drug release rates, especially those coated with higher amounts of the cell membrane. This reduction has also been observed in nanoparticles cloaked with membranes from erythrocytes (<xref rid=\"B9\" ref-type=\"bibr\">Hu et al., 2011</xref>) where erythrocyte membrane-coated nanoparticles showed slow drug release compared to uncoated nanoparticles. <xref rid=\"B9\" ref-type=\"bibr\">Hu et al. (2011)</xref> attributed this effect to the membrane’s ability to act as a diffusion barrier to provide better sustained drug release, as compared with PEG-based nanoparticles, thereby enhancing the therapeutic efficacy of the drug in acute myeloid leukemia cells. Furthermore, T-MNPs hold the unique characteristics of natural 19LF6 cell membranes thus avoiding clearance and enhancing circulation. <xref rid=\"B32\" ref-type=\"bibr\">Zhong et al. (2013)</xref> previously defined the 19LF6 cell line to contain a metastatic melanoma antigen, gp100 (209–217) – specific T-cell receptor. Our flow cytometric analysis and the binding kinetic results not only confirmed the higher presence of the specific TCR on the nanoparticle coating, but also its ability to bind to its specific target.</p><p>As part of the toxicological analysis, the T-MNPs, cyto- and hemo-compatibility were analyzed. The T-MNPs were found to be cyto-compatible up to 1,000 μg/ml concentration, which was shown to be at least as cyto-compatible as NNPs or bare PLGA NPs. This was similar to the results observed by <xref rid=\"B6\" ref-type=\"bibr\">Guo et al. (2015)</xref>, who demonstrated that erythrocyte-membrane coated PLGA nanoparticles possessed a similar cyto-compatibility compared to bare PLGA nanoparticles. This attributes to the cell mimicking characteristic and the slow drug release characteristics of T-MNPs. Blood clotting characteristics of T-MNPs were compared to the saline control and found to have no significant effect on the blood clotting cascade up to 1,000 μg/ml. According to the criterion in the ASTM E2524-08 standard, percent hemolysis >5% is considered toxic to red blood cells (<xref rid=\"B3\" ref-type=\"bibr\">ASTM E2524-08, 2013</xref>). T-MNPs-induced blood hemolysis was observed to be <5% up to 500 μg/ml. Although T-MNPs were found to be toxic to red blood cells at a concentration of 1,000 μg/ml, such high concentrations of the particles might not be used for later studies.</p><p>We also continued to investigate the <italic>in vitro</italic> characteristics of these membrane coated nanoparticles and their ability as a highly specific vehicle for cancer targeted delivery. In light of past studies that showed bare PLGA NPs have limitations owing to non-specific targeting and result in uncontrolled tissue distribution of the drug (<xref rid=\"B4\" ref-type=\"bibr\">Danhier et al., 2012</xref>) and that cell membrane coating improves immune evasion, target specificity and drug efficacy (<xref rid=\"B9\" ref-type=\"bibr\">Hu et al., 2011</xref>), we have devised the proposed design of T-MNPs. <xref rid=\"B5\" ref-type=\"bibr\">Fang et al. (2014)</xref>, devised surface-engineered PLGA NPs with platelet-membrane-derived vesicles since platelet cells have a natural ability to adhere to injured blood vessels as well as circulating pathogens. Such membrane coating provided the particles with natural platelet-like targeting functions. However, there is no data found in the literature for natural specific targeting (i.e., via TCR receptors) abilities of membrane coated drug carriers so far. The data obtained from our <italic>in vitro</italic> cell studies sheds light to the undiscovered potential of cytotoxic T-cell membrane coated nanoparticles as a biomimicking drug delivery vehicle. In our study, anti-gp100 TCR influence on T-MNP cellular uptake was observed in DM-6 and 1520 melanoma cell lines. Since A549 lung cell lines did not show any apparent gp100 presentation, A549 was used as a negative control cell line. Data collected from binding and cellular uptake studies showed that T-MNPs illustrated superior binding and uptake kinetics attributed to their anti-gp100 TCR. The uptake of T-MNPs at all ratios was significantly higher than that of D-MNPs, the negative control nanoparticles (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). These particles showed selective and effective binding to gp100 carrying melanoma cells when compared to that of D-MNPs made from the negative T-cell control. The uptake of T-MNPs increased with increasing concentrations and was enhanced in the 1520 cell line even more than that of DM-6 cell line. The enhanced 1520 uptake of the T-MNPs might have been due to its ability to better present gp100/HLA-A2 complex protein compared to that of the DM-6 cell line (<xref rid=\"B28\" ref-type=\"bibr\">Weidanz et al., 2006</xref>).</p><p>Furthermore, therapeutic efficiency of T-MNPs was evaluated. The IC50 values of Trametinib for DM-6 and 1520 were found to be approximately 29 and 1.6 μg/ml, respectively, which were very much similar to the values observed in a previous study by <xref rid=\"B19\" ref-type=\"bibr\">Roller et al. (2016)</xref>. However, the IC values often depend on numerous human and in-house factors, therefore, they must be performed for each study. T-MNPs were found to be significantly more efficient in killing melanoma cancer cells than any of other groups compared, even much more than free Trametinib at the same concentration, which would attribute to the binding and uptake characteristics of T-MNPs. Particles with a higher membrane content (greater anti-gp100 TCR content) showed to be more effective when compared to that of the lower NP to membrane ratio. These results indicate that proposed membrane coated natural targeting nanoparticles could potentially be used to improve chemotherapeutic efficacy to effectively treat melanoma.</p><p>Following the <italic>in vitro</italic> characterizations, the preliminary <italic>in vivo</italic> investigation by the biodistribution study examined whether intravenously injected T-MNPs targeted and recruited/retained at the subcutaneous tumors in the tumor implanted mice. In biodistribution studies, comparing between T-MNPs and other study groups, expected tumor accumulation difference was observed. NPs from almost all groups accumulated mainly at the liver and spleen in 2 h. The T-MNP group showed distinctive accumulation in the tumor region at almost a threefold higher accumulation on the tumor site than D-MNPs and about a twofold increase in accumulation than NNPs. D-MNPs and NNPs, on the other hand, were captured more in the liver. Similarly, <xref rid=\"B30\" ref-type=\"bibr\">Zhang et al. (2017)</xref> used non-specific hCTL membranes to target gastric cancer combined with low dose irradiation and observed very similar results in <italic>in vivo</italic> targeting capabilities of hCTL membrane coated nanoparticles. In their study, membrane coated nanoparticles showed a gradual increase in the tumor sites after low dose irradiation exposure. However, they reported that low dose irradiation was helped for these NPs to accumulate on target tissues at later time points of the study. Surprisingly, they were still able to confirm that non-specific hCTL membrane coated nanoparticles accumulated on tumor tissues without LDI and targeting molecule (<xref rid=\"B30\" ref-type=\"bibr\">Zhang et al., 2017</xref>). This effect might be due to the natural adhesion molecules like LFA-1 or integrin of the T-cell membrane surface. In our <italic>in vivo</italic> biodistribution study, anti-gp100 TCR decorated T-cell membranes were able to accumulate on target tissues. What is more to the above-mentioned literature results is tumor tissue accumulation of TCR decorated T-MNPs was superior compared to all control groups from the very early time points. All in all, our proposed T-cell membrane coated drug carrier system showed superior <italic>in vitro</italic> and <italic>in vivo</italic> targeting and uptake capabilities. Therefore, such natural biomaterials as engineered cells, bacteria membranes, biocompatible proteins, viral capsids and others in our drug carrier design can take the theragnostic field further than its current capabilities without compromising the effective drug delivery requirements.</p><p>Although cell membrane coated nanocarriers have great potential to deliver drugs to the desired location and could be a promising carrier to improve the theragnostic outcomes in treating melanoma, there are a few associated limitations and challenges. For instance, the cell membrane is comprised of a lot of different protein or peptide types, some of them are required for targeting, evading immune response while the other abundant proteins have unknown interactions in the host environment (<xref rid=\"B12\" ref-type=\"bibr\">Klammt and Lillemeier, 2012</xref>). Thus, further in-depth immune response and toxicity profiles must be performed for individual membrane proteins. Cell membrane isolation procedures are not robust and are limited to particular laboratory settings which can be a challenge in clinical translation such as isolation and culture of abundant T-cells in a short duration. Therefore, quality control such as maintaining the functional and structural aspects of cell membranes for longer periods needs to be investigated. Future work should include the required studies to address the above-mentioned limitations of the proposed research. Yet a new approach, utilization of T-MNPs that have been formulated using donor cells and investigating their drug delivery potential as a translational medicine application might be beneficial toward personalized cancer therapies in the future.</p></sec><sec id=\"S5\"><title>Conclusion</title><p>Overall, we successfully developed T-cell coated nanoparticle carriers that displayed superior targeting capabilities toward skin cancer cells and that could serve as a potential tool as a theragnostic system to image and treat melanoma. T-MNPs maintained excellent <italic>in vitro</italic> targeting ability and had a biomimicking shell that minimizes toxicity and systemic clearance concerns in the conventional drug carrier designs. The natural targeting molecule TCR receptor on the surface of the T-MNPs was preserved after membrane isolation and synthesis of T-MNPs. <italic>In vitro</italic> assessments of T-MNPs also showed their therapeutic ability as a drug carrier platform. Finally, biodistribution studies showed the <italic>in vivo</italic> targeting abilities of T-MNPs. The cyto-compatibility and natural targeting T-MNPs recruited to the tumor regions and displayed a distinctive accumulation signal. We showed the therapeutic capabilities of T-MNPs including intrinsic targeting, prolonged drug release and therapeutic potential making T-MNPs an excellent biomimicking theragnostic carrier platform for future cancer therapy.</p></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.</p></sec><sec id=\"S7\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by Julia Kissling, IACUC/IBC Specialist, Office of Regulatory Services, The University of Texas at Arlington, Texas-76010.</p></sec><sec id=\"S8\"><title>Author Contributions</title><p>SY conceived the original idea whereas JW and KN guided in developing the final nanoparticle design and the planned experiments. GO and DZ carried out the synthesis and characterization of designed nanoparticles and performed some <italic>in vitro</italic> experiments. HR cultured and isolated the cell membranes required for coating of nanoparticles used for some <italic>in vitro</italic> experiments and animal studies. SY, HR, TN, and MS designed and performed the <italic>in vivo</italic> (animal) experiments. SY and HR wrote the manuscript. All authors discussed the results and contributed to the final manuscript. JW and KN supervised the work and helped with the editing of the manuscript.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Chem</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Chem</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Chem.</journal-id><journal-title-group><journal-title>Frontiers in Chemistry</journal-title></journal-title-group><issn pub-type=\"epub\">2296-2646</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850656</article-id><article-id pub-id-type=\"pmc\">PMC7431671</article-id><article-id pub-id-type=\"doi\">10.3389/fchem.2020.00640</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Chemistry</subject><subj-group><subject>Mini Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Polyethylene Oxide-Based Composites as Solid-State Polymer Electrolytes for Lithium Metal Batteries: A Mini Review</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Zhao</surname><given-names>Shuangshuang</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/987781/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wu</surname><given-names>Qinxia</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1045954/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ma</surname><given-names>Wenqing</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/968573/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Yang</surname><given-names>Lishan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/830629/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education of China), National and Local Joint Engineering Laboratory for New Petrochemical Materials and Fine Utilization of Resources, Key Laboratory of the Assembly and Application of Organic Functional Molecules of Hunan Province, Hunan Normal University</institution>, <addr-line>Changsha</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>School of Materials Science and Engineering, Qilu University of Technology (Shandong Academy of Sciences)</institution>, <addr-line>Jinan</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Du Yuan, Nanyang Technological University, Singapore</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Liqiang Xu, Shandong University, China; Xifei Li, Xi'an University of Technology, China; Zhicheng Ju, China University of Mining and Technology, China</p></fn><corresp id=\"c001\">*Correspondence: Lishan Yang <email>lsyang.chemistry@gmail.com</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Electrochemistry, a section of the journal Frontiers in Chemistry</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>640</elocation-id><history><date date-type=\"received\"><day>02</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>22</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Zhao, Wu, Ma and Yang.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Zhao, Wu, Ma and Yang</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Solid-state polymer electrolytes (SPEs) have great processing flexibility and electrode–electrolyte contact and have been employed as the promising electrolytes for lithium metal batteries. Among them, poly(ethylene oxide) (PEO)-based SPEs have attracted widespread attention because of easy synthesis, low mass density, good mechanical stability, low binding energy with lithium salts, and excellent mobility of charge carriers. In order to overcome the low room-temperature ionic conductivity and the poor thermodynamic stability in high-voltage devices (>4.2 V) of the PEO materials, composition modulations by incorporating PEO with inorganic and/or organic components have been designed, which could effectively enable the applications of PEO-based SPEs with widened electro-stable voltage ranges. In this mini review, we describe recent progresses of several kinds of PEO composite structures for SPEs, and we compare the synthesis strategies and properties of these SPEs in lithium batteries. Further developments and improvements of the PEO-based materials for building better rechargeable batteries are also discussed.</p></abstract><kwd-group><kwd>solid-state polymer electrolytes</kwd><kwd>polyethylene oxide</kwd><kwd>lithium metal batteries</kwd><kwd>ionic conductivity</kwd><kwd>electrochemical stability</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Natural Science Foundation of China<named-content content-type=\"fundref-id\">10.13039/501100001809</named-content></funding-source></award-group><award-group><funding-source id=\"cn002\">Natural Science Foundation of Hunan Province<named-content content-type=\"fundref-id\">10.13039/501100004735</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"2\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"48\"/><page-count count=\"7\"/><word-count count=\"5407\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Lithium ion batteries (LIBs) are critical for large-scale applications, including power sources for 3C electronics, portable devices, and electric vehicle (Liu et al., <xref rid=\"B21\" ref-type=\"bibr\">2019b</xref>). The innovation of advanced batteries with higher energy/power densities, longer cycling life, and, especially, guaranteed levels of safety at a satisfactory cost is crucially needed (Manthiram et al., <xref rid=\"B24\" ref-type=\"bibr\">2017</xref>; Liu et al., <xref rid=\"B19\" ref-type=\"bibr\">2018</xref>). As the most potential anode material, Li-metal possesses advantages such as high theoretical capacity (3,860 mA h g<sup>−1</sup>), negative potential [−3.04 V vs. standard hydrogen electrode (SHE)], and low density (~0.59 g cm<sup>−3</sup>) (He et al., <xref rid=\"B14\" ref-type=\"bibr\">2018</xref>; Zhao C. Z. et al., <xref rid=\"B45\" ref-type=\"bibr\">2019</xref>). However, Li-metal batteries (LMBs) with liquid electrolytes cannot coordinate a high energy density with excellent electro-stability for real applications (Choudhury et al., <xref rid=\"B9\" ref-type=\"bibr\">2019b</xref>). The robustness and integrity of the electrode–electrolyte interface through cycling ensure the efficacy of the whole cell (Maleki Kheimeh Sari and Li, <xref rid=\"B23\" ref-type=\"bibr\">2019</xref>). To address these issues, solid-state polymer electrolytes (SPEs) in the replacement electrolytes have already demonstrated feasibility and superiority in lithium secondary batteries (e.g., solid-state LMBs, lithium-sulfur batteries, and lithium-gas batteries) (Xiao et al., <xref rid=\"B36\" ref-type=\"bibr\">2019</xref>; Zhou et al., <xref rid=\"B48\" ref-type=\"bibr\">2019</xref>; Zhao et al., <xref rid=\"B47\" ref-type=\"bibr\">2020</xref>).</p><p>SPEs are generally composed of polymeric solid host and lithium salt without the use of liquid solvents. The construct of SPEs was derived from the discovery by Fenton et al. (<xref rid=\"B12\" ref-type=\"bibr\">1973</xref>) that alkali metal salts dissolved in polyethylene oxide (PEO) could form conductive complexes (Fenton et al., <xref rid=\"B12\" ref-type=\"bibr\">1973</xref>). In general, various lithium-ion conductive polymer materials, such as PEO (Zhao Q. et al., <xref rid=\"B46\" ref-type=\"bibr\">2019</xref>), poly(acrylonitrile) (PAN) (Wang et al., <xref rid=\"B35\" ref-type=\"bibr\">1996</xref>), poly(methyl methacrylate) (PMMA) (Appetecchi et al., <xref rid=\"B2\" ref-type=\"bibr\">1995</xref>), and poly(vinylidene fluoride) (PVDF) (Choe et al., <xref rid=\"B7\" ref-type=\"bibr\">1995</xref>), have been employed as SPEs for the development of solid-state LMB batteries. Generally, a preferred SPE, used to replace the electrolyte in lithium batteries, should contain a high dielectric host polymer and a lithium salt with a low lattice energy. In this regard, the polymers with polar functional groups are required for consideration from the dissociation and transportation of the lithium ions (Liang et al., <xref rid=\"B17\" ref-type=\"bibr\">2019</xref>). Among all polymers, PEO is widely studied because of its excellent salt-solvating ability and electrode interfacial compatibility. From the above considerations, we selected and reviewed PEO-based SPEs (Xu et al., <xref rid=\"B37\" ref-type=\"bibr\">2019</xref>; Nie et al., <xref rid=\"B27\" ref-type=\"bibr\">2020</xref>; Qiu et al., <xref rid=\"B29\" ref-type=\"bibr\">2020</xref>).</p><p>However, the room-temperature (RT) ionic conductivity of PEO-based SPEs is lower than that of the liquid-based counterpart. Moreover, the batteries constructed with PEO-based SPEs usually suffer from Li dendrite because of the poor mechanical strength of the polymer matrix. Therefore, future studies of PEO-based SPEs should focus on the improvement of the mechanical properties for preventing Li dendrite formation and increasing electrochemical stability of PEO-based SPEs. By compositing with inorganic solid oxides/organic molecules, as-obtained composite PEO-based polymer electrolytes show a high ionic conductivity and are stable under relative high voltage (Liang et al., <xref rid=\"B17\" ref-type=\"bibr\">2019</xref>). For inorganic-PEO SPEs, the hybrid electrolyte consists of a soft polymer PEO electrolyte and a rigid inorganic solid-state electrolyte (SSE), which has a relatively high ionic conductivity at RT (Choudhury et al., <xref rid=\"B8\" ref-type=\"bibr\">2019a</xref>; Qiu et al., <xref rid=\"B29\" ref-type=\"bibr\">2020</xref>). As for organic material-modified PEO SPEs, chemical strategies (copolymerization and crosslinking) frustrate crystallization to enable acceptable ionic conductivities at temperatures near ambient or within the thermal window of applications, including in transport applications (Liu et al., <xref rid=\"B20\" ref-type=\"bibr\">2019a</xref>; Zhao et al., <xref rid=\"B47\" ref-type=\"bibr\">2020</xref>). Moreover, complex synergistic optimization is the primary efficient method of improving the antioxidant stability of systems with PEO-based electrolytes because of its high mechanical strength and high ionic conductivity (Feng et al., <xref rid=\"B11\" ref-type=\"bibr\">2018</xref>; Zhang et al., <xref rid=\"B44\" ref-type=\"bibr\">2019</xref>).</p><p>The amorphous PEO domain benefits Li<sup>+</sup> diffusion and is preferred as compared with the crystalline one. With the addition of filler particles, the PEO SPEs show increased mechanical strength and thermal stability and can thus suppress crystallization and facilitate the dissociation of the salt with an improved ionic conductivity (Zhao et al., <xref rid=\"B47\" ref-type=\"bibr\">2020</xref>). With PEO-based electrolyte, the all-solid-state Li|LiFePO<sub>4</sub> (LFP) cell, Li|LiCoO<sub>2</sub> cell, and Li|LiNi<sub>1−x−<italic>y</italic></sub>Mn<sub><italic>x</italic></sub>Co<sub><italic>y</italic></sub>O<sub>2</sub> (NMC) cell all show excellent cycling stability and rate capability. The PEO-based SPEs are of great significance to improve the practical application of hot ternary cathode materials (Wang C. et al., <xref rid=\"B33\" ref-type=\"bibr\">2019</xref>; Wang X. et al., <xref rid=\"B34\" ref-type=\"bibr\">2019</xref>; Xu et al., <xref rid=\"B37\" ref-type=\"bibr\">2019</xref>).</p><p>In this work, we review the recent progress in the synthesis strategies and polymer engineering of PEO-based electrolytes. We specifically compare the ion conductivity and electro-stability of different compound electrolytes and thus provide a certain reference for structural optimization of the PEO-based SPEs.</p></sec><sec id=\"s2\"><title>PEO Polymer Solid Electrolytes Based on Different Compositions</title><p>PEO-based composite SPEs show a high probability in all-solid-state lithium batteries, including inorganic-PEO composite, organic-PEO composite, and other complex composite electrolytes. The relevant synthesis/modification avenues and their performance/characteristics are summarized in the following sections.</p><sec><title>Inorganic-PEO Composite SPEs</title><p>In initial studies, PEO electrolyte is employed in solid-state LMBs but shows a low conductivity and inferior thermal stability. It is found that the increment of lithium salts within the polymeric matrix can lead to an improved ionic conductivity, accompanied by a degradation of mechanical strength and electro-stability of the PEO-based SPEs (Lopez et al., <xref rid=\"B22\" ref-type=\"bibr\">2019</xref>). Integration of nanostructured inorganic fillers (including metal oxide, neotype lithium salts, and some oxide solid-solutions) into polymeric solid host, named as “inorganic-PEO composite,” has emerged as an effective approach to design SPEs with the most needed improvements.</p><p>Basically, composite casting strategies can be divided into two-step solution casting and <italic>in situ</italic> casting combined with fillers. To date, the <italic>in situ</italic> approach, which possesses good distribution of nanofillers and is scalable for practical application, is commonly used for the syntheses of composite SPEs (Yap et al., <xref rid=\"B39\" ref-type=\"bibr\">2013</xref>; Xu et al., <xref rid=\"B38\" ref-type=\"bibr\">2020</xref>). Two-step reactions usually include the formation of the fillers (step I) and the incorporation operation (step II). For example, in a two-step synthesis process, self-synthesized MgAl<sub>2</sub>O<sub>4</sub> nanoparticles were incorporated into PEO polymers through a rapid hot press procedure (Angulakshmi et al., <xref rid=\"B1\" ref-type=\"bibr\">2013</xref>). For example, Al<sub>2</sub>O<sub>3</sub>-PEO SPEs synthesized via the <italic>in situ</italic> method could get a conductivity of 2.970 × 10<sup>−5</sup> S cm<sup>−1</sup> with microsized Al<sub>2</sub>O<sub>3</sub> fillers at RT; however, composite SPEs with Al<sub>2</sub>O<sub>3</sub> (<50 nm) displayed rougher surface structures and a reduced conductivity of 4.843 × 10<sup>−6</sup> S cm<sup>−1</sup> (Yap et al., <xref rid=\"B39\" ref-type=\"bibr\">2013</xref>). Generally, the conductivity of the Al<sub>2</sub>O<sub>3</sub>-PEO SPEs can be improved with a small Al<sub>2</sub>O<sub>3</sub> filler particle, which can probably have a more powerful impact on the immobilization of the long polymer chains. However, the fine (nanosized) filler grains would be too close to each other, inducing the blocking effect of the filler grains with enhanced immobilization and leading to the decrease in the ionic conductivity. Recently, through a rigid–flexible coupling technology, nano-SiO<sub>2</sub> particles were incorporated into 3D PEO networks to <italic>in situ</italic> construct SiO<sub>2</sub>-PEO SPEs (shown in <xref ref-type=\"fig\" rid=\"F1\">Figures 1A–C</xref>). SiO<sub>2</sub>-PEO SPEs show an outstanding RT ionic conductivity (σ ≈ 1.1 × 10<sup>−4</sup> S cm<sup>−1</sup>) along with dramatically improved solid–solid interface stabilization and excellent high-temperature capability (~90 mA h g<sup>−1</sup> after 100 cycles under 2 C at 90°C) (Xu et al., <xref rid=\"B38\" ref-type=\"bibr\">2020</xref>).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p><bold>(A–C)</bold> Synthetic routes of the SiO<sub>2</sub>-PEO–LiClO<sub>4</sub> solid-state polymer electrolytes (SPEs). <bold>(D)</bold> The schematic preparation process of the ice-templated Li<sub>1.5</sub>Al<sub>0.5</sub>Ge<sub>1.5</sub>(PO<sub>4</sub>)<sub>3</sub> (LAGP)-PEO SPEs. Reproduced with permission from Wang X. et al. (<xref rid=\"B34\" ref-type=\"bibr\">2019</xref>) and Xu et al. (<xref rid=\"B38\" ref-type=\"bibr\">2020</xref>). <bold>(E)</bold> Schematic structure of the lamellar PS–PEO–LiTFSI SPEs. Reproduced with permission from Chen et al. (<xref rid=\"B6\" ref-type=\"bibr\">2019</xref>). <bold>(F)</bold> Schematic of the PEO|PEO–perovskite|PEO composite solid electrolyte. <bold>(G)</bold> Schematic of the multilayered solid polymer electrolyte (DSM-SPE). The bilateral layers are <italic>in situ</italic> formed on the surfaces of electrodes. Reproduced with permission from Wang C. et al. (<xref rid=\"B33\" ref-type=\"bibr\">2019</xref>) and Liu K. et al. (<xref rid=\"B18\" ref-type=\"bibr\">2019</xref>).</p></caption><graphic xlink:href=\"fchem-08-00640-g0001\"/></fig><p>In recent years, neotype lithium salts (inorganic ion conductors) are regarded as the most promising filler materials for inorganic-PEO SPEs, which can effectively enhance the battery properties in the electrochemical aspect. Phosphate ion conductor nanomaterials such as Li<sub>1.5</sub>Al<sub>0.5</sub>Ge<sub>1.5</sub>(PO<sub>4</sub>)<sub>3</sub> (LAGP) (Wang X. et al., <xref rid=\"B34\" ref-type=\"bibr\">2019</xref>) and Li<sub>1+x</sub>Al<sub><italic>x</italic></sub>Ti<sub>2−x</sub>(PO<sub>4</sub>)<sub>3</sub> (LATP) (Zhai et al., <xref rid=\"B43\" ref-type=\"bibr\">2017</xref>) are reported to present optimal ionic conductivities and superior flexibilities than do the SPEs before compounding. The inorganic particles were first dispersed into water and cast onto a substrate. Thereafter, the bottom of the dispersion is slowly cooled down and forms a vertical temperature gradient with ice nucleated from the bottom of the suspension, during which the ceramic particles are compelled to form vertically aligned structures. After ice sublimation, inorganic particles are sintered together to form vertically aligned walls (Wang X. et al., <xref rid=\"B34\" ref-type=\"bibr\">2019</xref>). For example, PEO polymer combined with vertically aligned inorganic walls (shown in <xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>) shows an RT ionic conductivity level of 10<sup>−4</sup> S cm<sup>−1</sup>, which is three to six times higher than that of the PEO electrolyte with ceramic nanoparticles randomly dispersed inside. For the LLTO-PEO SPEs, the prepercolating structure in Li<sub>0.35</sub>La<sub>0.55</sub>TiO<sub>3</sub> (LLTO) could accelerate Li-ion conduction (σ = 0.88 × 10<sup>−4</sup> S cm<sup>−1</sup>) via its successive ion pathways, which could also further avoid agglomeration of particles (Bae et al., <xref rid=\"B3\" ref-type=\"bibr\">2018</xref>). As a Li-ion fast ionic conductor, LLZO also owns remarkable electrochemical stability (even under 6.0 V). Thus, the Li symmetric cells using LLZO-PEO SPEs can stably cycle for 1,000 h without short-circuiting even at 60°C, which is due to the highly packed LLZO nanowires inside the SPEs and the uniform deposition of lithium metal (Wan et al., <xref rid=\"B32\" ref-type=\"bibr\">2019</xref>). Furthermore, other lithium salts, such as sulfide (Li<sub>3</sub>PS<sub>4</sub>) and borate [lithium bis(modified imidazole)borate] (LiBMB), have been successfully used to ameliorate flexible PEO-based SPEs. Li<sub>3</sub>PS<sub>4</sub> particles exhibit uniformly nanosized morphologies, and the particle diameter of the Li<sub>3</sub>PS<sub>4</sub> is about 400–700 nm. <italic>In situ</italic> synthesis of Li<sub>3</sub>PS<sub>4</sub> nanoparticles within the PEO matrix possesses good distribution of nanofillers. β-Li<sub>3</sub>PS<sub>4</sub> glass-ceramic is a Li superionic conductor whose conductivity is higher than 10<sup>−4</sup> S cm<sup>−1</sup> at RT with a relatively stable electrochemical property (Chen et al., <xref rid=\"B5\" ref-type=\"bibr\">2018</xref>). LiBMB-PEO SPEs display a highly ordered ionic pathways [the quasi-period value (<italic>T</italic><sub>p</sub>) is lower than 100 ns] associated with a high electro-stable potential of up to 7.2 V at 60°C (Yuan et al., <xref rid=\"B41\" ref-type=\"bibr\">2019</xref>). The LiFePO<sub>4</sub>|LiBMB-PEO SPEs|Li cell delivers an initial discharge capacity of 145.5 mA h g<sup>−1</sup> at 0.1 C and ultra-high capacity retention (98.5% after 60 cycles), revealing good reliability of the produced cells.</p><p>Besides the aforementioned lithium salt, several metal oxide solid solutions such as MgAl<sub>2</sub>O<sub>4</sub> have been supplied in the PEO-based composite SPEs (Angulakshmi et al., <xref rid=\"B1\" ref-type=\"bibr\">2013</xref>). In this two-step synthesis case, the self-synthesized MgAl<sub>2</sub>O<sub>4</sub> nanoparticles were incorporated with PEO polymer through a rapid hot press procedure. The assembled all-solid-state cells deliver a discharge capacity approaching 110 mA h g<sup>−1</sup> after prolonged cycling (up to 100 cycles, 70°C), which renders them to be qualified for practical battery applications.</p><p>In short, inorganic-PEO SPEs show considerable superiority with an improved ionic conductivity, enhanced mechanical strength, and obvious accessibility for enlarging the preparation. Along with the introduction of inorganic fillers, the possible conductive mechanisms in the composite SPEs, especially with neotype lithium salts as the fillers, need more comprehensive and in-depth research. Also, improving the RT ionic conductivities of the current inorganic-PEO SPEs to the liquid-electrolyte level (10<sup>−3</sup>-10<sup>−2</sup> S cm<sup>−1</sup>) becomes the most urgent work.</p></sec><sec><title>Organic-PEO Composite SPEs</title><p>Compared with inorganic-PEO composite SPEs, organic-PEO composite SPEs have been widely studied in previous works (Lopez et al., <xref rid=\"B22\" ref-type=\"bibr\">2019</xref>). Related organic fillers can be briefly classified as metal-organic frameworks (MOFs), simple organisms, and molecular polymers.</p><p>MOF materials have been investigated for extensive application in the fields of electrochemical catalysis, electrodes, and electrolyte because of their high specific surface area and ordered microporous structure. For example, Zn<sub>4</sub>O(1,4-benzenedicarboxylate)<sub>3</sub> (MOF-5) nanoparticles with a size of 20–30 nm were first synthesized and then incorporated into the polymer metric to form the MOF-PEO SPEs (Yuan et al., <xref rid=\"B40\" ref-type=\"bibr\">2013</xref>). MOF-5 fillers show a strong absorbing ability for the solvent impurities and help SPEs to prevent those impurities from accumulating at the Li/SPEs interface. The reversible capacities of cells increase after filling MOF-5 particles as a result of reduced cell polarization of the electrode/SPE interface at both 60 and 80°C.</p><p>Also, simple organic fillers such as silicane (Mehdi et al., <xref rid=\"B25\" ref-type=\"bibr\">2017</xref>; Mohanta et al., <xref rid=\"B26\" ref-type=\"bibr\">2017</xref>) and carbonate (He et al., <xref rid=\"B13\" ref-type=\"bibr\">2017</xref>) have been successfully introduced into the PEO-based SPEs, improving the ion conductivity and ameliorating the polymer crystallinity. For example, the monosilylated PEO precursor with the loose networks supplies higher segmental motion and amorphous networks, which are beneficial for a significantly enhanced capacity and faster Li-ion transmission (Mohanta et al., <xref rid=\"B26\" ref-type=\"bibr\">2017</xref>). By a facile transesterification reaction, the carbonate-linked PEO SPEs could achieve a high yield with intrinsic amorphous nature and low glass transition temperature, which would be beneficial for getting a high ionic conductivity. Cycling performance of the cell with carbonate-linked PEO SPEs displays extremely stable capacity as well as coulombic efficiencies close to 100% up to 100 cycles at both 25 and 55°C.</p><p>Moreover, the ionic conductivity of a polymer electrolyte depends on the chain mobility. Various kinds of polymer fillers, such as polystyrene (PSt) (Niitani et al., <xref rid=\"B28\" ref-type=\"bibr\">2005</xref>), PVDF (Deng et al., <xref rid=\"B10\" ref-type=\"bibr\">2015</xref>), polyvinyl alcohol (PVA) (Jinisha et al., <xref rid=\"B16\" ref-type=\"bibr\">2018</xref>), thermoplastic polyurethane (TPU) (Tao et al., <xref rid=\"B31\" ref-type=\"bibr\">2017</xref>), and polystyrene (PS) (Chen et al., <xref rid=\"B6\" ref-type=\"bibr\">2019</xref>), are discussed in the following portion. Copolymer PSt-PEO with microphase separation structure displays high tensile strengths (>3 MPa), which is high enough utilization in battery applications. PSt-PEO SPEs exhibit exceptionally high Li<sup>+</sup> conductivity (3.03 × 10<sup>−3</sup> S cm<sup>−1</sup> at RT) and remarkable electrochemical stability (stable above 5.0 V vs. Li/Li<sup>+</sup>). A similar RT ionic conductivity has been reported in the study of PVA-PEO composite SPEs. The superhigh conductivity value is evaluated in the PSt-PEO and PVA-PEO SPEs, which is quite close to that of the liquid electrolytes.</p><p>Thermogravimetric analysis is constantly used to directly investigate the thermal stability of the polymer electrolytes. In the case of TPU-PEO SPEs (Tao et al., <xref rid=\"B31\" ref-type=\"bibr\">2017</xref>), copolymer electrolyte shows a high conductivity (5.3 × 10<sup>−4</sup> S cm<sup>−1</sup> at 60°C) and notably enhanced thermostability even under 200°C. A similar conductivity and electrochemical improvements could be observed for PS-PEO SPEs (Chen et al., <xref rid=\"B6\" ref-type=\"bibr\">2019</xref>). PS-PEO SPEs have a high storage modulus of 1.4 MPa at 60°C. The Li|PS-PEO SPE|Li cell can be cycled for 400 h at 0.1 mA cm<sup>−2</sup> without any Li dendrite failure. The charge passed is 144 C cm<sup>−2</sup> with an average voltage increase of 0.6 mV C<sup>−1</sup> cm<sup>2</sup>. These results indicate that the Li/SPE interface is stable and that the mechanical strength of the SPEs is capable of suppressing Li dendrite growth. As illustrated in <xref ref-type=\"fig\" rid=\"F1\">Figure 1E</xref>, PS arms would rearrange and entangle after phase separation, and then the lamellar structure would be reformed by alternating self-assembled PS and PEO layers. So far, several types of organic fillers of the PEO-based SPEs have been discussed in this portion, and more organic fillers will be explored and applied for high-performance flexibility SPEs.</p></sec><sec><title>Other Complex Composite SPEs</title><p>Except for simplex compound fillers in PEO-based composite SPEs, multiphase composites, such as PEO-inorganic-organic, PEO-organic-organic, and other complicated ones, have also been widely explored to satisfy the need of practical application. These multiblock copolymers with various fillers show improved properties in different aspects.</p><p>PEO-inorganic-organic SPEs composite possess the combined advantages of PEO, inorganic and organic components. For instance, the PEO–Al<sub>2</sub>O<sub>3</sub>-Pr<sub>4</sub>NI (tetrapropylammonium iodide) composite SPEs show a high conductivity of 4.2 × 10<sup>−4</sup> S cm<sup>−1</sup> and an admirable PEO spherulitic crystallinity at 24°C with 5% Al<sub>2</sub>O<sub>3</sub> filler (Bandara et al., <xref rid=\"B4\" ref-type=\"bibr\">2017</xref>). The ceramic material Li<sub>6.4</sub>La<sub>3</sub>Zr<sub>1.4</sub>Ta<sub>0.6</sub>O<sub>12</sub> (LLZT) was introduced to the PEO–succinonitrile (SN) system (Zha et al., <xref rid=\"B42\" ref-type=\"bibr\">2018</xref>). The one containing 60 wt% of LLZT and 10 wt% of SN shows a high ion conductivity of 1.22 × 10<sup>−4</sup> S cm<sup>−1</sup> at 30°C and a wide electrochemical window of 5.5 V vs. Li/Li<sup>+</sup>. The SPEs composed of aluminate complexes (LiAl)–polyethylene glycol (PEG) and PEO could also be prepared via the common solution casting method (Feng et al., <xref rid=\"B11\" ref-type=\"bibr\">2018</xref>). With PEO additive, the segmental mobility of the ether-chain bonded with Al atoms would be improved, providing PEO–LiAl–PEG hybrid SPEs with extra ionic pathways and high ionic conductivity. The new hopping transport mechanism was verified for the single Li-ion conductor system at the nanoscale.</p><p>PEO-organic-organic multiblock copolymer SPEs have recently garnered increasing attention because of their hard-separated segments and improved mechanical properties with their multiple microphase separated domains. A multiblock copolymer electrolyte composed of poly(butylene terephthalate) (PBT) and PEO alternating multiblock copolymers (mBCPs) (PBT-<italic>b</italic>-PEO-<italic>b</italic>-PBT)<sub><italic>n</italic></sub>, is synthesized by the cascade polycondensation-coupling ring-opening polymerization (PROP) method (Huang et al., <xref rid=\"B15\" ref-type=\"bibr\">2017</xref>). The mBCP electrolyte shows a high ionic conductivity of 8.2 × 10<sup>−4</sup> S cm<sup>−1</sup> at 90°C and considerable mechanical properties. A gel polymer electrolyte composed of PEO, PMMA, and P(VDF-HFP) (PEMVH) is synthesized by adding some inorganic oxide fillers (Shi et al., <xref rid=\"B30\" ref-type=\"bibr\">2018</xref>). The PEMVH-oxide filler electrolyte possesses porous and amorphous structures, in which the oxide fillers can promote the formation of the pores of the polymer matrix and the segmental motion of the polymer chain. This unique structure of the polymer membranes contributes to the high ionic conductivity and interfacial stability of hybrid SPEs.</p><p>In addition, multilayered composite PEO-based SPEs are also developed to give excellent mechanical stability, ionic conductivity, and interfacial compatibility for the all-solid-state lithium batteries. The flexible composite SPEs composed of PEO layers on both sides of the PEO–perovskite composite are actually an integrated sandwich structure, which is denoted as PEO|PEO-perovskite|PEO (shown in <xref ref-type=\"fig\" rid=\"F1\">Figure 1F</xref>). The synthesis routes PEO and LiTFSI were dissolved into acetonitrile. The solution was then dropped onto the surface of the Li<sub>0.33</sub>La<sub>0.557</sub>TiO<sub>3</sub> (LLTO) nanofiber mat. The wetted LLTO mat was predried in air for 6 h before drying in vacuum for 24 h at 65°C. Then, the membrane was turned to the other side, followed by repeating the wetting and drying processes to obtain the PEO–perovskite SPEs with an integrated sandwich structure. The 3D perovskite nanofiber network can enhance the ionic conductivity by offering Li-ion with abundant channels and improve the mechanical strength of the membrane (Liu K. et al., <xref rid=\"B18\" ref-type=\"bibr\">2019</xref>). Besides, multilayered SPEs with a differentiated salt-based multilayered solid polymer electrolyte were synthesized via a facile slurry casting-drying method, which helped the Li symmetric battery to achieve a high capacity retention of 79.0% (after 300 cycles at 60 and 2°C) (Wang C. et al., <xref rid=\"B33\" ref-type=\"bibr\">2019</xref>). The schematic of multilayered SPEs are displayed in <xref ref-type=\"fig\" rid=\"F1\">Figure 1G</xref>. Every layer is purposefully designed to make full use of their respective advantages (i.e., the middle layer can provide a good ionic conductivity for the electrolyte), while the outer layers can enhance the interfacial contact and promote the formation of steady solid electrolyte interface (SEI) and cathode electrolyte interface (CEI) films. As compared with conventional PEO-based SPEs, the multilayer strategy is powerful to achieve high performance and solve electrolyte/electrode interfacial problems of the solid-state batteries.</p><p>A summary of the electrochemical window and the ionic conductivity of PEO-based SPEs is presented in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Table S1</xref>. Most of them exhibit high electrochemical stable voltages (≥4.5 V) and ionic conductivity (≥1 × 10<sup>−4</sup> S cm<sup>−1</sup>). Generally, PEO-organic and multiple complex SPEs show superior high-temperature conductivity and mechanical property. In general, the multiple complex electrolyte with high ionic transport and electrode–electrolyte interfacial stability is expected for future commercial application of solid-state LMBs.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Calculated electrochemical stability windows for the polyethylene oxide (PEO)-based solid-state polymer electrolyte (SPE) candidates in a lithium metal battery. The inorganic-PEO hybrid SPEs are marked by green and dark cyan bars; the organic-PEO hybrid SPEs are marked with an orange bar; and the complex PEO-based hybrid SPEs are marked with a rose bar.</p></caption><graphic xlink:href=\"fchem-08-00640-g0002\"/></fig></sec></sec><sec sec-type=\"conclusions\" id=\"s3\"><title>Conclusions</title><p>PEO-based SPEs have gained wide attention as promising electrolytes for lithium metal batteries (LMBs) because of their superior processing flexibility and electrode–electrolyte contact. Considerable research efforts have been devoted to improving the SPEs' limited room-temperature ionic conductivity and electrochemical stability of PEO. Inorganic and organic components have been introduced to the PEO-based SPEs to widen its electrochemical window of PEO. The inorganic fillers possess the advantages of low cost and high safety, while the organic ones can improve the SPEs' flexibility and change the polymer crystallinity with a crosslink or copolymerization approach. Moreover, by using multiple complex PEO-based SPEs, the LMBs can achieve much enhanced mechanical strength and electrode-electrolyte interfacial stability. At present, developing suitable PEO-based composite SPEs remains a challenge for the practical applications of high-performance LMBs with high safety and electro-stability.</p></sec><sec id=\"s4\"><title>Author Contributions</title><p>LY supervised the implementation of the project. LY and SZ conceived the idea. SZ, QW, WM, and LY analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s5\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This research was supported by the National Natural Science Foundation of China (21805083), Natural Science Foundation of Hunan Province (2018JJ3331), Science and Technology Planning Project of Hunan Province (2018TP1017), and Scientific Research Fund of Hunan Provincial Education Department (19K058).</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s6\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fchem.2020.00640/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fchem.2020.00640/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Table_1.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Angulakshmi</surname><given-names>N.</given-names></name><name><surname>Nahm</surname><given-names>K. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Chem</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Chem</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Chem.</journal-id><journal-title-group><journal-title>Frontiers in Chemistry</journal-title></journal-title-group><issn pub-type=\"epub\">2296-2646</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850665</article-id><article-id pub-id-type=\"pmc\">PMC7431672</article-id><article-id pub-id-type=\"doi\">10.3389/fchem.2020.00652</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Chemistry</subject><subj-group><subject>Mini Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Electrolyte Technologies for High Performance Sodium-Ion Capacitors</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Meng</surname><given-names>Fancheng</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/858169/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Long</surname><given-names>Tao</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1042010/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Xu</surname><given-names>Bin</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1041758/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zhao</surname><given-names>Yixin</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1041148/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Hu</surname><given-names>Zexuan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1042139/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Luxian</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Liu</surname><given-names>Jiehua</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/860486/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>School of Materials Science and Engineering, Hefei University of Technology</institution>, <addr-line>Hefei</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Guangde Tianyun New Tech. Co. Ltd.</institution>, <addr-line>Xuancheng</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Kwan San Hui, University of East Anglia, United Kingdom</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Liqiang Mai, Wuhan University of Technology, China; Guanglei Cui, Qingdao Institute of Bioenergy and Bioprocess Technology (CAS), China</p></fn><corresp id=\"c001\">*Correspondence: Jiehua Liu <email>liujh@hfut.edu.cn</email></corresp><corresp id=\"c002\">Fancheng Meng <email>fancheng.meng@hfut.edu.cn</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Electrochemistry, a section of the journal Frontiers in Chemistry</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>652</elocation-id><history><date date-type=\"received\"><day>07</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>23</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Meng, Long, Xu, Zhao, Hu, Zhang and Liu.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Meng, Long, Xu, Zhao, Hu, Zhang and Liu</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Bridging the energy gap between batteries and capacitors, while in principle delivering a supercapacitor-like high power density and long lifespan, sodium-ion capacitors (SIC) have been considered promising energy storage devices that could be commercialized in the near future due to the natural abundance of sodium sources and the performance superiority of SIC devices. Therefore, in the past decade, substantial research efforts have been devoted to their structure and property improvements. With regard to the electrochemical performance of an ion capacitor, except for the electrode, the composition and structure of the electrolytes are also of great importance. Thus, in this mini review, we present a brief summary of the electrolytes developed recently for high performance SIC, including aqueous, organic, and ionic liquid based electrolytes. The influence factors such as ionic conductivities, electrolyte concentrations, electrochemical stable windows, as well as the cost and safety issues are discussed. Furthermore, the future perspectives and challenges in the science and engineering of new electrolytes are also considered. We hope that this review may be helpful to the energy storage community regarding the electrolytes of advanced SIC systems.</p></abstract><kwd-group><kwd>electrolyte</kwd><kwd>sodium-ion capacitor</kwd><kwd>sodium salt</kwd><kwd>aqueous</kwd><kwd>organic</kwd><kwd>ionic liquid</kwd><kwd>gel polymer</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Natural Science Foundation of Anhui Province<named-content content-type=\"fundref-id\">10.13039/501100003995</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"1\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"42\"/><page-count count=\"6\"/><word-count count=\"4752\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>With the in-depth development and extensive application of stationary and portable power systems, people have higher and higher requirements on energy storage equipment. At present, the most frequently used secondary power-supply units are batteries and capacitors. Among them, the lithium-ion batteries (LIB) have a high energy density (150–200 W h kg<sup>−1</sup>) and a low power density (<350 W kg<sup>−1</sup>) (Han et al., <xref rid=\"B8\" ref-type=\"bibr\">2018</xref>), while the electrochemical capacitors (EC), especially supercapacitor, usually has a high power density (>10 kW kg<sup>−1</sup>) and a low energy density (<10 W h kg<sup>−1</sup>) (Gao et al., <xref rid=\"B7\" ref-type=\"bibr\">2018</xref>; Zhang et al., <xref rid=\"B41\" ref-type=\"bibr\">2020</xref>). Their respective defectiveness restricts the application in certain areas. To bridge the gap, lithium ion capacitors (LIC) and sodium-ion capacitors (SIC) that have both high energy density and high power density have attracted extensive research interest. However, since the huge exploration of Li resources in portable electronics, electric vehicles, large grids, and the limited reserves of Li resources on earth (Wang et al., <xref rid=\"B33\" ref-type=\"bibr\">2015</xref>), the development of the analogous SIC comes to be a well acknowledged solution due to the abundant sodium reserves and the reasonable redox potential (Na/Na<sup>+</sup> = −2.7 V) (Zhang et al., <xref rid=\"B41\" ref-type=\"bibr\">2020</xref>). Moreover, sodium does not react with aluminum, making it possible to replace the expensive current collector copper. It is also well known that the Na<sup>+</sup> and Li<sup>+</sup> ions share similar physical/chemical properties (Mendes et al., <xref rid=\"B20\" ref-type=\"bibr\">2018</xref>; Jia et al., <xref rid=\"B11\" ref-type=\"bibr\">2020</xref>) and some of the conclusions involving LIC are also applicable to SIC (Ding et al., <xref rid=\"B4\" ref-type=\"bibr\">2018</xref>). However, the radius of a Na<sup>+</sup> (1.02Å) is larger than that of a Li<sup>+</sup> (0.76Å). The electrochemical behavior of Na<sup>+</sup> in the electrolyte is different from that of Li<sup>+</sup> under the same conditions (Qu et al., <xref rid=\"B26\" ref-type=\"bibr\">2008</xref>; Gao et al., <xref rid=\"B7\" ref-type=\"bibr\">2018</xref>). As a result, it is not difficult to understand that the exploration of high performance SIC requires new effort and investment.</p><p>A SIC shares the same structure as a LIC, which is composed of mainly anode, cathode, electrolyte, separator, and collector (Jia et al., <xref rid=\"B11\" ref-type=\"bibr\">2020</xref>; <xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). Since the majority of research work has been centered on the material and structure of electrodes, the study on electrolyte of SIC is relatively limited. However, an electrolyte is the carrier and transport channel of charges, and the influence of electrolyte on the final device performance can never be overlooked. Generally, the electrolyte applied in SIC consists of sodium salt and solvent, and sometimes certain additives. The optimization of electrolyte for desirable electrochemical properties can be realized by careful selection and rational matching of these components.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p><bold>(A)</bold> A schematic illustration showing the structure of a SIC. <bold>(B)</bold> CV curves and <bold>(C)</bold> cycle stability of a SIC with organic electrolyte. The inset shows10 red LEDs powered by a charged SIC (reproduced with permission from Li et al., <xref rid=\"B18\" ref-type=\"bibr\">2020</xref>, Copyright 2020 Springer).</p></caption><graphic xlink:href=\"fchem-08-00652-g0001\"/></fig><p>In this mini review, the properties of different electrolytes, including the composition and concentration, cost and safety issues, as well as their influences on SIC performance are presented. The prospect and challenge of the future development of SIC electrolytes are also reviewed.</p></sec><sec id=\"s2\"><title>Sodium Salts</title><p>Sodium salt is one of the most important components of SIC electrolyte that is originally selected on the basis of lithium salt by replacing the cations. However, their chemical stability, electrochemical activity, and ionic conductivity (IC) are different. The general principles for selecting an appropriate SIC electrolyte salt include: (i) It should have a high solubility in the corresponding solvent to produce sufficient charges; (ii) Stable in a certain voltage range, no decomposition and redox reactions; (iii) Good chemical stability and will not react with the solvent, electrode, and collector; (iv) Environmental friendliness, safe, non-toxic, and so on.</p><p>Since the cation is fixed to Na<sup>+</sup>, the choice of anions becomes pretty important. As can be seen from <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, anions largely determine the properties of the salts. During the charge/discharge process, the anion is most often the electrolyte part that is oxidized first. Thus, it determines the upper limit of electrochemical stability window (ESW) (Jónsson and Johansson, <xref rid=\"B12\" ref-type=\"bibr\">2015</xref>; Ponrouch et al., <xref rid=\"B24\" ref-type=\"bibr\">2015</xref>). The higher the working voltage, the higher the energy flow density. Among the exemplified anions, <inline-formula><mml:math id=\"M2\"><mml:msubsup><mml:mrow><mml:mtext>ClO</mml:mtext></mml:mrow><mml:mrow><mml:mn>4</mml:mn></mml:mrow><mml:mrow><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula> is highly oxidized. Thus, it is more likely to react with other materials, limiting its application in SIC (Vidal-Abarca et al., <xref rid=\"B31\" ref-type=\"bibr\">2012</xref>). The interaction between <inline-formula><mml:math id=\"M3\"><mml:msubsup><mml:mrow><mml:mtext>BF</mml:mtext></mml:mrow><mml:mrow><mml:mn>4</mml:mn></mml:mrow><mml:mrow><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula> and cations is generally strong, resulting in a relatively small number of free ions and poor IC (Ponrouch et al., <xref rid=\"B24\" ref-type=\"bibr\">2015</xref>). While <inline-formula><mml:math id=\"M4\"><mml:msubsup><mml:mrow><mml:mtext>PF</mml:mtext></mml:mrow><mml:mrow><mml:mn>6</mml:mn></mml:mrow><mml:mrow><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula> poses a serious safety problem because it easily hydrolyzes into PF<sub>5</sub>, POF<sub>3</sub>, and HF at elevated temperatures or water containing conditions, bringing in a strong corrosive environment (Lee et al., <xref rid=\"B16\" ref-type=\"bibr\">2005</xref>). The ionic conductivity of Tf<sup>−</sup> in solvent EC/DMC is generally too poor to be used in SIC electrolytes. However, as the solvent is changed to tetra (ethylene glycol) dimethyl ether (TEGDME), the resultant SIC using NaTf<sup>−</sup> based electrolyte reveals the highest discharge capacity, the lowest interface impedance, and a good cycling performance (250 mA h g<sup>−1</sup> after 40 cycles) compared with using the salts of NaPF<sub>6</sub> and NaClO<sub>4</sub> (Kim et al., <xref rid=\"B13\" ref-type=\"bibr\">2011</xref>). Because the radius of FSI<sup>−</sup> is smaller than that of TFSI<sup>−</sup>, it is more soluble in solvents, and results in a higher-IC electrolyte (Kühnel et al., <xref rid=\"B15\" ref-type=\"bibr\">2017</xref>). However, it should be noted that NaFSI and NaTFSI have a problem of aluminum corrosion (Otaegui et al., <xref rid=\"B22\" ref-type=\"bibr\">2015</xref>), which should be taken into account when designing a specific SIC. According to the report (Senthilkumar et al., <xref rid=\"B27\" ref-type=\"bibr\">2014</xref>), the ionic solvation radius of OH<sup>−</sup>, <inline-formula><mml:math id=\"M5\"><mml:msubsup><mml:mrow><mml:mtext>NO</mml:mtext></mml:mrow><mml:mrow><mml:mn>3</mml:mn></mml:mrow><mml:mrow><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula>, Cl<sup>−</sup>, and <inline-formula><mml:math id=\"M6\"><mml:msubsup><mml:mrow><mml:mtext>SO</mml:mtext></mml:mrow><mml:mrow><mml:mn>4</mml:mn></mml:mrow><mml:mrow><mml:mn>2</mml:mn><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula> in water are 3.00, 3.35, 3.32, and 3.79 Å, respectively. Thus, the NaOH based aqueous electrolyte demonstrates obvious advantages in IC and ionic diffusion rate. As a result, the highest capacity of 390 F g<sup>−1</sup> has been achieved. To sum up, the choice of sodium salt has a crucial effect on the property of electrolytes for high performance SIC.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Basic properties of some commonly used sodium salts and organic solvents.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Salt</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>M<sub>w</sub> [g mol<sup>−1</sup>]</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>T<sub>m</sub> [°C]</bold></th><th valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\"><bold>σ<sub>max</sub> [mS cm<sup>−1</sup>] (solvent)</bold></th><th valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\"><bold>References</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Na<sub>2</sub>SO<sub>4</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">142.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">884</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">125 (H<sub>2</sub>O)</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Wu et al., <xref rid=\"B36\" ref-type=\"bibr\">2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NaClO<sub>4</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">122.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">468</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">180 (H<sub>2</sub>O)</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Wu et al., <xref rid=\"B36\" ref-type=\"bibr\">2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NaBF<sub>4</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">109.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">384</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">1.5 (PC)</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Herlem et al., <xref rid=\"B10\" ref-type=\"bibr\">2002</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NaPF<sub>6</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">167.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">300</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">6.8 (EC/DMC)</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Bhide et al., <xref rid=\"B2\" ref-type=\"bibr\">2014</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NaTf</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">172.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">248</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">3.7 (EC/DMC)</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Bhide et al., <xref rid=\"B2\" ref-type=\"bibr\">2014</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NaTFSI</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">303.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">257</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">8.8 (PC)</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Vogl et al., <xref rid=\"B32\" ref-type=\"bibr\">2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NaFSI</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">203.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">118</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">15.1 (DME)</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Lee et al., <xref rid=\"B17\" ref-type=\"bibr\">2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Solvent</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>T</bold><sub><bold>m</bold></sub>\n<bold>[°C]</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>T</bold><sub><bold>b</bold></sub><break/>\n<bold>[°C]</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>T</bold><sub><bold>f</bold></sub>\n<bold>[°C]</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>η</bold><break/>\n<bold>(cP) 25°C</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>ε 25°C</bold></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>References</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">36.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">248</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">160</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19 (40°C)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">89.78</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Xu, <xref rid=\"B37\" ref-type=\"bibr\">2004</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−48.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">242</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">132</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.53</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">64.92</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Xu, <xref rid=\"B37\" ref-type=\"bibr\">2004</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DMC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.59</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.107</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ponrouch et al., <xref rid=\"B24\" ref-type=\"bibr\">2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DEC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−74.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">126</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.75</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.805</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Xu, <xref rid=\"B37\" ref-type=\"bibr\">2004</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DME</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">84</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.46</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.18</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ponrouch et al., <xref rid=\"B24\" ref-type=\"bibr\">2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DEGDME</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−64</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">162</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">57</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.06</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ponrouch et al., <xref rid=\"B24\" ref-type=\"bibr\">2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TEGDME</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−46</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">216</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">111</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.53</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ponrouch et al., <xref rid=\"B24\" ref-type=\"bibr\">2015</xref></td></tr></tbody></table><table-wrap-foot><p><italic>M<sub>w</sub>, molecular weight; T<sub>m</sub>, melting temperature; σ<sub>max</sub>, maximum conductivity; T<sub>b</sub>, boiling point; T<sub>f</sub>, flash point; η, viscosity; ε, dielectric constant; Tf<sup>−</sup>, CF<sub>3</sub><inline-formula><mml:math id=\"M1\"><mml:msubsup><mml:mrow><mml:mtext>SO</mml:mtext></mml:mrow><mml:mrow><mml:mstyle class=\"text\"><mml:mtext class=\"textit\" mathvariant=\"italic\">3</mml:mtext></mml:mstyle></mml:mrow><mml:mrow><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula>; TFSI<sup>−</sup>, [N(CF<sub>3</sub>SO<sub>2</sub>)<sub>2</sub>]<sup>−</sup>; FSI<sup>−</sup>, [N(SO<sub>2</sub>F)<sub>2</sub>] <sup>−</sup>; EC, ethylene carbonate; PC, propylene carbonate; DMC, dimethyl-carbonate; DEC, diethyl carbonate; DME, 1, 2-dimethoxyethane; DEGDME, diethylene glycol dimethyl ether; TEGDME, triethylene glycol dimethyl ether</italic>.</p></table-wrap-foot></table-wrap></sec><sec id=\"s3\"><title>Solvents</title><p>As the carrier of sodium salt, solvent is another main component of SIC electrolyte. The solvent not only affects the diffusion rate of ions but also often determines the lower limit of ESW of a SIC (Ponrouch et al., <xref rid=\"B24\" ref-type=\"bibr\">2015</xref>). Depending on the hydrophilicity and composition differences, the solvent employed in SIC electrolyte can be mainly divided into aqueous, organic, and ionic liquid (IL), and so is the resultant electrolyte.</p><sec><title>Aqueous Electrolyte</title><p>An aqueous electrolyte usually has the advantages of high ionic conductivity, low cost, and high safety. Among the acidic, basic, and neutral electrolytes, neutral ones are more attractive owing to the safety issues (Whitacre et al., <xref rid=\"B35\" ref-type=\"bibr\">2012</xref>). One prominent problem of aqueous electrolyte is that its ESW is narrow due to the low decomposition voltage of 1.23 V for the solvent water (Kühnel et al., <xref rid=\"B15\" ref-type=\"bibr\">2017</xref>). Fortunately, asymmetric assembly structure can compensate this problem to a certain extent. For instance, asymmetric capacitors based on activated carbon//NaMnO<sub>2</sub> and Fe<sub>3</sub>O<sub>4</sub>//rGO electrode pairs with 0.5 M NaSO<sub>4</sub> aqueous electrolyte extended the operational voltage window to 1.9 and 1.80 V, respectively (Qu et al., <xref rid=\"B25\" ref-type=\"bibr\">2009</xref>; Lu et al., <xref rid=\"B19\" ref-type=\"bibr\">2015</xref>), far exceeding the decomposition voltage of water.</p><p>Another commonly employed technology to enlarge the ESW of aqueous electrolyte is increasing the concentration of sodium salt. A high salt concentration is more likely to form a passivation layer on the electrode surface, which can prevent the redox process from happening at low voltages. For example, when 17 mol kg<sup>−1</sup> NaClO<sub>4</sub> aqueous electrolyte is utilized, the ESW of the resultant SIC can be extended to 2.75 V (Zhang Y. et al., <xref rid=\"B42\" ref-type=\"bibr\">2018</xref>). A high ESW indicates more electrochemical energy can be reserved in a certain SIC. It is also reported the ESW of 2.6 V in a sodium ion battery with 35 mol kg<sup>−1</sup> NaFSI aqueous electrolyte (Kühnel et al., <xref rid=\"B15\" ref-type=\"bibr\">2017</xref>). The wide operational voltage window was also attributed to the high concentration sodium salt. Nevertheless, the higher the concentration of salt, the higher the viscosity of electrolyte, and so is the ionic diffusion resistance. Therefore, the concentration of salt also influences the energy density of a SIC and the total device cost (Zhang P. et al., <xref rid=\"B40\" ref-type=\"bibr\">2018</xref>). Therefore, it is necessary to seek a balance between the electrolyte concentration and the final electrochemical performance.</p></sec><sec><title>Organic Electrolyte</title><p>Organic electrolyte is the most commonly used electrolyte which has a wide voltage window of up to 4 V. The drawbacks of this kind of electrolyte lie in the poor conductivity and the safety risks like volatile poisonousness and low flash point (Beguin et al., <xref rid=\"B1\" ref-type=\"bibr\">2014</xref>).</p><p>Organic solvents such as EC, PC, DMC, DEC, DME, DEGDME, and TEGDME, etc., have been explored in SIC electrolytes, and their basic properties are shown in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>. Since sodium salts are generally strong electrolytes, most of the Na<sup>+</sup> can be dissociated before reaching saturation to participate into the charge storage process. Thus, the higher the solubility of salt in certain solvent, the more the free Na<sup>+</sup> can be obtained in the electrolyte. However, it is revealed that in organic solvent with low dielectric constant, the relative ratio of free ions decreases with the increase of concentration (Okoshi et al., <xref rid=\"B21\" ref-type=\"bibr\">2013</xref>; Park et al., <xref rid=\"B23\" ref-type=\"bibr\">2018</xref>). Therefore, the ionic conductivity and the resultant device's rate performance will be different. For example, at the temperature of 25°C, electrolytes with 1 M NaClO<sub>4</sub> in solvents of EC/DEC (1:1), EC/PC (1:1), PC, DME, DEGME, and TEGDME showed the corresponding ionic conductivities of 6.8, 7.8, 6, 6.5, 5.7, and 1.8 mS cm<sup>−1</sup>, respectively (Park et al., <xref rid=\"B23\" ref-type=\"bibr\">2018</xref>). The resultant SIC with an electrolyte of NaClO<sub>4</sub>-EC/PC reached a maximum discharge capacity of 63.1 mA h g<sup>−1</sup> at the rate of 1 A g<sup>−1</sup> due to the highest IC in EC/PC composite solvents. Hybrid electrolytes can also adapt to a wide range of temperature owing to the melting point difference of different organic solvents (Ding et al., <xref rid=\"B5\" ref-type=\"bibr\">2015</xref>). Besides ionic conductivity, it is also important to consider the electrochemical stability of the electrolyte when screening potential solvents. Good electrochemical stability and high IC will no doubt improve the capacitive performance and extend the device application field. For example, it is reported that with the electrolyte of 1 M NaClO<sub>4</sub> dissolved in EC/DMC (1:1), the operation voltage window of the resultant SIC ranges from 0.5–4 V (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). In addition, a stable cycling performance with the capacity retention of 84.5% over 3,000 cycles at 3 A g<sup>−1</sup> has been achieved, and the Coulomb efficiency is nearly 100% as shown in <xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref> (Li et al., <xref rid=\"B18\" ref-type=\"bibr\">2020</xref>).</p><p>It is worth mention that additives are usually introduced to organic electrolyte in small amounts to compensate for the defects of the existing electrolyte (Chen et al., <xref rid=\"B3\" ref-type=\"bibr\">2018</xref>). Main functions of additives in the electrochemical process of Na<sup>+</sup> storage (including in sodium-ion batteries) are as follows: modify SEI film, protect overcharging, change the ionic conductivity, wet interfaces, etc. (Ponrouch et al., <xref rid=\"B24\" ref-type=\"bibr\">2015</xref>; Soto et al., <xref rid=\"B28\" ref-type=\"bibr\">2017</xref>; Sun et al., <xref rid=\"B29\" ref-type=\"bibr\">2019</xref>). Certain electrolytic additives like fluoroethylene carbonate (FEC), vinylene carbonate, and biphenyl have been involved in the previous studies of sodium-ion batteries (Sun et al., <xref rid=\"B29\" ref-type=\"bibr\">2019</xref>). However, in case of a SIC system, the research on electrolytic additives is seldom reported except for FEC, and the main contribution of FEC rests with the SEI film formation (Jia et al., <xref rid=\"B11\" ref-type=\"bibr\">2020</xref>). For example, FEC additive (usually 2–5 wt%) has been added to the electrolyte of a SIC that proves in quickly repairing the damaged SEI film to revival the device performance (Soto et al., <xref rid=\"B28\" ref-type=\"bibr\">2017</xref>). However, too much FEC addition might lead to emergence of the passivation layer or too thick SEI film which will increase the device's internal resistance (Komaba et al., <xref rid=\"B14\" ref-type=\"bibr\">2011</xref>).</p></sec><sec><title>Ionic Liquid Electrolyte</title><p>Non-flammable IL electrolytes usually have the features of high viscosity and low IC. In addition, due to the relative scarcity and difficulty in material synthesis, the cost of ionic liquid based electrolyte is higher than those of other electrolytes (Ponrouch et al., <xref rid=\"B24\" ref-type=\"bibr\">2015</xref>). However, IL electrolyte can provide a large potential window and a stable solid electrolyte interphase film (SEI) in the electrochemical storage systems (Hasa et al., <xref rid=\"B9\" ref-type=\"bibr\">2016</xref>) that would be an attractive benefit for high energy density SIC devices. In the field of Na<sup>+</sup> storage, IL anion is commonly the same as sodium salt, while the cations are generally large organic cations. Thus, the currently reported IL electrolytes for SIC are relatively few.</p><p>One impressive IL-based electrolyte is that 0.8 mol L<sup>−1</sup> sodium bis (fluorosulfonyl) imide (Na-TFSI) dissolved in the IL of 1-methyl-1-propylpyrrolidinium bis (trifluoromethyl-sulfonyl) imide (PMPyrr-TFSI) (Fleischmann et al., <xref rid=\"B6\" ref-type=\"bibr\">2019</xref>). The resultant Li<sub>4</sub>Ti<sub>5</sub>O<sub>12</sub>//AC based SIC (Na-AHSC) can be charged/discharged between the voltage of 1.0–4.0 V. As a result, a high energy density of 90 W h kg<sup>−1</sup> was obtained with an energy efficiency of 78% and a Coulombic efficiency of 100%. Moreover, the electrolyte demonstrated a high temperature resistant property that can be stably performed in a SIC system at 80°C for 3,000 cycles. Electrolyte with sodium salt dissolved in IL has also been employed in the Na//Carbon based SIC system (Mendes et al., <xref rid=\"B20\" ref-type=\"bibr\">2018</xref>). When operated under the potential of 3.8 V, an energy density of 263 W h kg<sup>−1</sup> (based on the mass of electrodes) was obtained at room temperature, which further increased to 270 W h kg<sup>−1</sup> at 50°C. There are also some attempts to incorporate IL into polymer structures to fabricate composite gel-state electrolyte, and thus flexible SIC can be readily achieved with the advantage of ease-of-use except for the wide ESW and good thermal stability (Wang et al., <xref rid=\"B34\" ref-type=\"bibr\">2020</xref>). Comparing with the aqueous and organic electrolytes, IL electrolytes usually have a better safety performance at higher temperatures, which indicates a broad prospect in certain application fields.</p></sec></sec><sec id=\"s4\"><title>Gel Polymer Electrolyte (GPE)</title><p>Due to the inherent instability of the above liquid state electrolytes in terms of flammability, leakage, and internal short circuit problems, GPE in the form of quasi-solid-state with good ionic conductivity and high mechanical flexibility is attracting extensive interest in the areas of electrochemical energy storage (Yang et al., <xref rid=\"B39\" ref-type=\"bibr\">2019</xref>).</p><p>For example, Wang et al. reported the first quasi-solid-state SIC with a Na<sup>+</sup> conducting GPE poly (vinylidene difluoride-co-hexafluoropropylene). The resultant SIC can be operated at 4.2 V, delivering an energy density of 168 W h kg<sup>−1</sup>, which showed a stable cycling performance of 85% capacitance retention over 1,200 cycles (Wang et al., <xref rid=\"B33\" ref-type=\"bibr\">2015</xref>). Xu et al. reported a hydroxyethyl cellulose-polyethylene oxide based Na<sup>+</sup> conducting GPE, demonstrating a high energy storage property of 181 W h kg<sup>−1</sup> at 150 W kg<sup>−1</sup> in the SIC device (Xu et al., <xref rid=\"B38\" ref-type=\"bibr\">2019</xref>). Different polymer based gel-state Na<sup>+</sup> conducting electrolytes are also demonstrated recently even with a higher potential window of 4.4 V, which greatly enhanced the electrochemical properties of SIC beyond the handling safety issues (Zhang et al., <xref rid=\"B41\" ref-type=\"bibr\">2020</xref>).</p><p>Despite various electrolytes being applicable to SIC, the choice of appropriate electrolyte depends much on the electrode materials. For example, the electrolyte should not corrode or react with the electrodes. To facilitate the free and fast Na<sup>+</sup> transport, proper electrolyte is anticipated to well wet the electrode materials all the time. In that the redox potential of different materials varies, the electrolyte is also required to have an ESW that matches the redox potential of the electrode to give out a desired electrochemical property (Tang et al., <xref rid=\"B30\" ref-type=\"bibr\">2020</xref>). It should be noted that the computer aided simulation can be employed to match the specific electrode materials with suitable electrolytic systems for high performance SIC (Zhang P. et al., <xref rid=\"B40\" ref-type=\"bibr\">2018</xref>).</p></sec><sec id=\"s5\"><title>Conclusion and Outlook</title><p>In conclusion, the development of metal ion-based capacitors is still in its infancy, and especially the SIC. In view of that most of the efforts have been centered on electrodes, the study of electrolyte is of urgency now owing to the fact that any component in the electrolyte plays an important role to the whole device performance. It can be seen that the matching of specific sodium salt, solvent, and additive should improve the energy storage performance of SIC on the one hand; on the other hand, all components of the electrolytes should be safe and chemically and thermally stable. These are the focus and also the difficult points of the future direction of SIC electrolytes. In general, SIC has attractive advantages over other electrochemical energy storage system in terms of the cooperative features of both high energy density and high power density. Therefore, advanced electrolyte science and technologies for high performance SIC deserve in-depth studies and further development.</p></sec><sec id=\"s6\"><title>Author Contributions</title><p>All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.</p></sec><sec id=\"s7\"><title>Conflict of Interest</title><p>FM and LZ were employed by the company Guangde Tianyun New Tech. Co. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by the Natural Science Foundation of Anhui province (JZ2018AKZR0058), the Fundamental Research Funds for the Central Universities (PA2020GDGP0054), the National Natural Science Foundation of China (U1832136 and 21303038), and Hundred Talents Program of Anhui Province.</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Beguin</surname><given-names>F.</given-names></name><name><surname>Presser</surname><given-names>V.</given-names></name><name><surname>Balducci</surname><given-names>A.</given-names></name><name><surname>Frackowiak</surname><given-names>E.</given-names></name></person-group> (<year>2014</year>). <article-title>Carbons and electrolytes for advanced supercapacitors</article-title>. <source>Adv. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Bioeng Biotechnol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Bioeng Biotechnol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Bioeng. Biotechnol.</journal-id><journal-title-group><journal-title>Frontiers in Bioengineering and Biotechnology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-4185</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850771</article-id><article-id pub-id-type=\"pmc\">PMC7431673</article-id><article-id pub-id-type=\"doi\">10.3389/fbioe.2020.00962</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Bioengineering and Biotechnology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Microbial Culture in Minimal Medium With Oil Favors Enrichment of Biosurfactant Producing Genes</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Araújo</surname><given-names>W. J.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/993342/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Oliveira</surname><given-names>J. S.</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/959462/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Araújo</surname><given-names>S. C. S.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Minnicelli</surname><given-names>C. F.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/939777/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Silva-Portela</surname><given-names>R. C. B.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>da Fonseca</surname><given-names>M. M. B.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/952464/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Freitas</surname><given-names>J. F.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/939962/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Silva-Barbalho</surname><given-names>K. K.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/939326/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Napp</surname><given-names>A. P.</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Pereira</surname><given-names>J. E. S.</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Peralba</surname><given-names>M. C. R.</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Passaglia</surname><given-names>L. M. P.</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/445636/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Vainstein</surname><given-names>M. H.</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/22565/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Agnez-Lima</surname><given-names>L. F.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/303556/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Laboratório de Biologia Molecular e Genômica, Departamento de Biologia Celular e Genética, Centro de Biociências, Universidade Federal do Rio Grande do Norte</institution>, <addr-line>Natal</addr-line>, <country>Brazi</country></aff><aff id=\"aff2\"><sup>2</sup><institution>INESC-ID/IST – Instituto de Engenharia de Sistemas e Computadores/Instituto Superior Técnico, Universidade de Lisboa</institution>, <addr-line>Lisbon</addr-line>, <country>Portugal</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Laboratório de Fungos de Importância Médica e Biotecnológica, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul</institution>, <addr-line>Porto Alegre</addr-line>, <country>Brazil</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Laboratório de Química Analítica e Ambiental, Departamento de Química, Instituto de Química, Universidade Federal do Rio Grande do Sul</institution>, <addr-line>Porto Alegre</addr-line>, <country>Brazil</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Laboratório de Genética Molecular Vegetal, Departamento de Genética, Instituto de Biociência, Universidade Federal do Rio Grande do Sul</institution>, <addr-line>Porto Alegre</addr-line>, <country>Brazil</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Rudolf Hausmann, University of Hohenheim, Germany</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Alif Chebbi, University of Milano-Bicocca, Italy; Fengjie Cui, Jiangsu University, China</p></fn><corresp id=\"c001\">*Correspondence: L. F. Agnez-Lima, <email>lfagnez@ufrnet.br</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Industrial Biotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>962</elocation-id><history><date date-type=\"received\"><day>21</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>24</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Araújo, Oliveira, Araújo, Minnicelli, Silva-Portela, da Fonseca, Freitas, Silva-Barbalho, Napp, Pereira, Peralba, Passaglia, Vainstein and Agnez-Lima.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Araújo, Oliveira, Araújo, Minnicelli, Silva-Portela, da Fonseca, Freitas, Silva-Barbalho, Napp, Pereira, Peralba, Passaglia, Vainstein and Agnez-Lima</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>The waste produced by petrochemical industries has a significant environmental impact. Biotechnological approaches offer promising alternatives for waste treatment in a sustainable and environment-friendly manner. Microbial consortia potentially clean up the wastes through degradation of hydrocarbons using biosurfactants as adjuvants. In this work, microbial consortia were obtained from a production water (PW) sample from a Brazilian oil reservoir using enrichment and selection approaches in the presence of oil as carbon source. A consortium was obtained using Bushnell-Haas (BH) mineral medium with petroleum. In parallel, another consortium was obtained in yeast extract peptone dextrose (YPD)-rich medium and was subsequently compared to the BH mineral medium with petroleum. Metagenomic sequencing of these microbial communities showed that the BH consortium was less diverse and predominantly composed of <italic>Brevibacillus</italic> genus members, while the YPD consortium was taxonomically more diverse. Functional annotation revealed that the BH consortium was enriched with genes involved in biosurfactant synthesis, while the YPD consortium presented higher abundance of hydrocarbon degradation genes. The comparison of these two consortia against consortia available in public databases confirmed the enrichment of biosurfactant genes in the BH consortium. Functional assays showed that the BH consortium exhibits high cellular hydrophobicity and formation of stable emulsions, suggesting that oil uptake by microorganisms might be favored by biosurfactants. In contrast, the YPD consortium was more efficient than the BH consortium in reducing interfacial tension. Despite the genetic differences between the consortia, analysis by a gas chromatography-flame ionization detector showed few significant differences regarding the hydrocarbon degradation rates. Specifically, the YPD consortium presented higher degradation rates of C12 to C14 alkanes, while the BH consortium showed a significant increase in the degradation of some polycyclic aromatic hydrocarbons (PAHs). These data suggest that the enrichment of biosurfactant genes in the BH consortium could promote efficient hydrocarbon degradation, despite its lower taxonomical diversity compared to the consortium enriched in YPD medium. Together, these results showed that cultivation in a minimal medium supplemented with oil was an efficient strategy in selecting biosurfactant-producing microorganisms and highlighted the biotechnological potential of these bacterial consortia in waste treatment and bioremediation of impacted areas.</p></abstract><kwd-group><kwd>biodegradation</kwd><kwd>biotechnology</kwd><kwd>petrochemical waste</kwd><kwd>consortia</kwd><kwd>metagenomics</kwd><kwd>production water</kwd><kwd>biosurfactant</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Agência Nacional do Petróleo, Gás Natural e Biocombustíveis<named-content content-type=\"fundref-id\">10.13039/501100006487</named-content></funding-source></award-group><award-group><funding-source id=\"cn002\">Conselho Nacional de Desenvolvimento Científico e Tecnológico<named-content content-type=\"fundref-id\">10.13039/501100003593</named-content></funding-source></award-group><award-group><funding-source id=\"cn003\">Coordenação de Aperfeiçoamento de Pessoal de Nível Superior<named-content content-type=\"fundref-id\">10.13039/501100002322</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"7\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"97\"/><page-count count=\"16\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>During oil production, processing, and storage operations, large volumes of waste are generated such as oily sludge, waste rock, and production water (PW), which exhibit highly toxic hydrocarbon concentrations and pose a serious environmental risk, demanding treatment before disposal (<xref rid=\"B59\" ref-type=\"bibr\">Neff et al., 2011</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Cordes et al., 2016</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Al-Ghouti et al., 2019</xref>). PW reinjection is an oil recovery method that increases oil production by 15–25% (<xref rid=\"B84\" ref-type=\"bibr\">Shibulal et al., 2014</xref>). Despite this reuse, contaminated water continues to be produced, requiring treatment before disposal (<xref rid=\"B3\" ref-type=\"bibr\">Al-Ghouti et al., 2019</xref>).</p><p>Therefore, the growth in oil production increases the demand for alternative treatments (sustainable and ecofriendly) for both waste management and environmental accidents resulting from the petrochemical industry. Bioremediation is a viable alternative to remove contaminants, since biological treatments are cheaper than chemical and physical treatments, and occasionally result in complete mineralization (<xref rid=\"B91\" ref-type=\"bibr\">Van Hamme et al., 2003</xref>; <xref rid=\"B16\" ref-type=\"bibr\">Cappello et al., 2019</xref>). Bioremediation techniques for the recovery of polluted environments, such as autochthonous bioaugmentation, use only organisms that are indigenous to the community (<xref rid=\"B34\" ref-type=\"bibr\">Hassanshahian et al., 2014</xref>; <xref rid=\"B74\" ref-type=\"bibr\">Radwan et al., 2019</xref>).</p><p>Microbial consortia are preferred for the bioremediation of contaminants due to the presence of a large number of diverse metabolic functionalities (<xref rid=\"B53\" ref-type=\"bibr\">McGenity et al., 2012</xref>). The consortia can be constructed in defined or undefined ways (<xref rid=\"B5\" ref-type=\"bibr\">Alvarez and Polti, 2014</xref>). A defined consortium is an association of previously known microorganisms with a specific function, such as biodegradation (<xref rid=\"B85\" ref-type=\"bibr\">Smith et al., 2013</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Alvarez and Polti, 2014</xref>). In contrast, undefined consortia result from enrichment procedures of environmental samples, including different organisms that may already be known (<xref rid=\"B87\" ref-type=\"bibr\">Sugiura et al., 1997</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Hosokawa et al., 2009</xref>; <xref rid=\"B28\" ref-type=\"bibr\">Escalante et al., 2015</xref>; <xref rid=\"B79\" ref-type=\"bibr\">Santisi et al., 2015</xref>). While creating indefinite consortia, the samples are first grown in “rich or generic medium” with different carbon sources, and then submitted to a selection phase in minimal medium (<xref rid=\"B87\" ref-type=\"bibr\">Sugiura et al., 1997</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Budzinski et al., 1998</xref>). Therefore, the undefined consortium approach may favor the selection of several organisms directly or indirectly involved in the biotechnological functions of interest, such as hydrocarbon degradation and production of biosurfactants (<xref rid=\"B92\" ref-type=\"bibr\">Venkateswaran, 1991</xref>).</p><p>Biosurfactants are amphipathic molecules that can be classified in different categories, one of which is by their molecular weight (<xref rid=\"B41\" ref-type=\"bibr\">Karlapudi et al., 2018</xref>). Low molecular weight biosurfactants are usually glycolipids (as rhamnolipids) or lipopeptides (as surfactin), while high molecular weight biosurfactants include amphipathic polysaccharides, lipopolysaccharides, proteins, and lipoproteins (<xref rid=\"B41\" ref-type=\"bibr\">Karlapudi et al., 2018</xref>; <xref rid=\"B62\" ref-type=\"bibr\">Nurfarahin et al., 2018</xref>). Most biosurfactants are synthesized by non-ribosomal pathways, however, the mechanisms that control their synthesis and production are poorly understood, which demands investigation (<xref rid=\"B62\" ref-type=\"bibr\">Nurfarahin et al., 2018</xref>). The recent discovery of a ribosomal protein with surfactant properties, named MBSP1, emphasizes the advantage of furthering the knowledge about biosurfactant biosynthesis (<xref rid=\"B9\" ref-type=\"bibr\">Araújo et al., 2020</xref>). Although various studies have described microbial consortia capable of bioremediation petrochemical waste through biosurfactant production, there are many questions about the role and production of biosurfactants for bioremediation (<xref rid=\"B44\" ref-type=\"bibr\">Ławniczak et al., 2013</xref>; <xref rid=\"B66\" ref-type=\"bibr\">Patowary et al., 2016</xref>; <xref rid=\"B45\" ref-type=\"bibr\">Lee et al., 2018</xref>).</p><p>In the current work, we used metagenomics to compare undefined microbial consortia, obtained by two different cultivation approaches using oil as a carbon source. We aimed to identify the factors that favor the enrichment of species, genes, and metabolic pathways related to petroleum degradation and biosurfactants production for biotechnological applications.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Obtaining Microbial Consortia</title><p>The PW sample used in this work was kindly provided by Petrogal Brasil S/A from onshore oil reservoir located in Aracajú, Sergipe, Brazil (Latitude: −10.9111099; Longitude: −37.0716705). Based on protocols adapted from <xref rid=\"B96\" ref-type=\"bibr\">Wu et al. (2013)</xref> and <xref rid=\"B33\" ref-type=\"bibr\">Guerra et al. (2018)</xref>, the consortia were obtained in Erlenmeyer flasks containing 50 mL of the PW sample and culture medium in a 1:1 (v/v) of volume proportion, and were incubated at 30°C at 180 rpm for 7 days (1 cycle). After the first incubation cycle, 5% (v/v) of the culture was transferred to a fresh culture medium containing 1% (v/v) sterile petroleum by autoclaving at 135°C for 30 min. Incubation was performed under the same conditions and repeated twice, totaling three cycles. At the end of the third cycle, an aliquot (1% v/v) was transferred to Bushnell-Haas (BH) culture medium (g/L: 0.2 MgSO<sub>4</sub>, 0.02 CaCl<sub>2</sub>, 1 KH<sub>2</sub>PO<sub>4</sub>, 1 K<sub>2</sub>HPO<sub>4</sub>, 1 (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 0.05 FeCl<sub>3</sub>, pH 7) containing 1% (v/v) petroleum. Similar to the first three cycles described above, this phase was also repeated three times under the same conditions. The culture medium differentiated the consortia. The yeast extract peptone dextrose (YPD) consortium was obtained from the enrichment of the PW sample culture in the presence of the carbon-rich Peptone Dextrose Yeast Extract medium (g/L: 10 yeast extract, 20 peptone, 20 glucose) during the first three cycles and then transferred in the last three cycles for the selection phase with BH medium. The BH consortium was cultured for 6 weeks only with BH medium. The consortia were stored in a 1:1 (v/v) ratio of 100% glycerol (Synth<sup>®</sup>, Diadema, SP, BR) at −80°C for subsequent experiments. The petroleum used as a carbon source was sterilized in an autoclave for 3 cycles of 30 min.</p></sec><sec id=\"S2.SS2\"><title>Growth Curves</title><p>Microbial growth was measured by optical density (OD<sub>600nm</sub>) in a spectrophotometer Global Analyzer, (Global Trade Technology, Monte Alto, SP, BRA) and performed in 250 mL Erlenmeyer flasks containing 50 mL of BH medium with 1% petroleum (v/v) as the sole carbon source, in triplicates. The initial OD<sub>600nm</sub> was adjusted to 0.1. The consortia were grown over 7 days in the same conditions of incubation (30°C and stirring at 180 rpm) and verified every 24 h. All experiments with the consortia were standardized to be performed after 72 h of growth, which corresponds to the stationary growth phase of these consortia.</p></sec><sec id=\"S2.SS3\"><title>DNA Extraction and Sequencing</title><p>DNA extraction of the consortia was performed during the stationary phase of the consortia by the commercial UltraClean Microbial DNA Isolation Kit (MoBio<sup>®</sup>, Carlsbad, CA, United States). For the DNA extraction of microorganisms present in crude PW, 5 L of sample were vacuum filtered in a 0.22 μm filter, and the membrane containing the retained microorganisms was cut into small pieces and used for DNA extraction by the commercial UltraClean Microbial DNA Isolation Kit (MoBio<sup>®</sup>, Carlsbad, CA, United States). The quality and quantity of DNA from all extractions (consortia and crude sample) were estimated using the Qubit 2.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, United States).</p><p>All sequencings were performed according to the manufacturer’s instruction, suitable for the Ion Personal Genome Machine (PGM) platform (Thermo Fisher Scientific, Waltham, MA, United States). For shotgun metagenomic sequencing, 400 bp-fragment libraries were obtained using the Ion Xpress Plus Fragment library kit (Thermo Fisher Scientific, Waltham, MA, United States). The size selection of the library was performed by E-Gel SizeSelect 2% agarose (Invitrogen, United States) and purification steps using magnetic beads on Agencourt<sup>®</sup> XP Kit (Beckman Coulter, United States), always following the manufacturer’s protocols. For 16S rDNA sequencing of the PW sample, 16S rRNA gene regions were amplified with 16S Ion Metagenomics Kit<sup>TM</sup> (Thermo Fisher Scientific, Waltham, MA, United States). For each sample, two PCR reactions were carried out using primer sets V2-4-8 or V3-6,7-9. Ion Plus Fragment Library Kit<sup>TM</sup> was used to obtain the DNA library with the adapter-ligated and nick-repaired DNA. In both sequencing methods, the Ion Xpress Barcode Adapters 1-32 kit<sup>TM</sup> (Thermo Fisher Scientific, Waltham, MA, United States) was used to identify each sample. Subsequently, for both libraries (fragment and amplicon), emulsions were independently created and enriched using an Ion OneTouch<sup>TM</sup> 2 System (Thermo Fisher Scientific, Waltham, MA, United States) and Ion PGM<sup>TM</sup> Hi-Q View OT2 Kit (Thermo Fisher Scientific, Waltham, MA, United States). Sequencing was conducted according to Ion PGM<sup>TM</sup> Hi-Q<sup>TM</sup> View Sequencing Kit protocol on Ion 318 Chip v2 (Thermo Fisher Scientific, Waltham, MA, United States), following the manufacturer’s protocols. All runs were programmed to include 850 nucleotide flows.0</p></sec><sec id=\"S2.SS4\"><title>Bioinformatics Analysis</title><p>Shotgun metagenomic raw sequences were uploaded to Metagenome Rapid Annotation using the subsystem Technology (MG-RAST)<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup> server (<xref rid=\"B42\" ref-type=\"bibr\">Keegan et al., 2016</xref>). Taxonomic analyses were performed using MG-RAST, with the default parameters, against the SILVA Databases (<xref rid=\"B73\" ref-type=\"bibr\">Quast et al., 2013</xref>). The biodegradation functional analyses of hydrocarbons and biosurfactant production were performed using the BLAST program (<xref rid=\"B32\" ref-type=\"bibr\">Gish et al., 1990</xref>) against the protein database of the Biosurfactant and Biodegradation Database (BioSurfDB) (<xref rid=\"B63\" ref-type=\"bibr\">Oliveira et al., 2015</xref>). For this analysis, Trimmomatic (<xref rid=\"B12\" ref-type=\"bibr\">Bolger et al., 2014</xref>) was used to remove quality index bases < 20, as well as fragments of sizes less than 30 bp. Read ends with high variation in nucleotide proportions were removed by the FastX trimmer, and duplicate reads were removed using FastQ/A collapser<sup><xref ref-type=\"fn\" rid=\"footnote2\">2</xref></sup>. For the BioSurfDB alignment, the default BLAST parameters were used. All proteins identified in the metagenomes are available at <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.biosurfdb.org/#/table/protein\">http://www.biosurfdb.org/#/table/protein</ext-link>.</p><p>The program TrimGalore was used to pre-process amplicon sequences of 16S rRNA genes. Taxonomic determination was performed by QIIME (<xref rid=\"B15\" ref-type=\"bibr\">Caporaso et al., 2010</xref>), based on data generated by sequencing the V2–V9 region of the 16S rRNA. Chimera sequences were then excluded with USEARCH 6.1 (<xref rid=\"B26\" ref-type=\"bibr\">Edgar, 2018</xref>). Reads were clustered into OTUs at 97 % sequence identity using the UCLUST algorithm (<xref rid=\"B26\" ref-type=\"bibr\">Edgar, 2018</xref>). Representative sequences from each operational taxonomic unit (OTU) were mapped to the SILVA v132 database (<xref rid=\"B73\" ref-type=\"bibr\">Quast et al., 2013</xref>) to determine potential taxonomic identities.</p><p>We performed comparative bioinformatics analyses between the consortia obtained from this study, microbial consortia obtained from drill cutting samples (<xref rid=\"B33\" ref-type=\"bibr\">Guerra et al., 2018</xref>; <xref rid=\"B57\" ref-type=\"bibr\">Napp et al., 2018</xref>), and non-oil samples (<xref rid=\"B46\" ref-type=\"bibr\">Leinonen et al., 2010</xref>). From drill cutting samples, <xref rid=\"B57\" ref-type=\"bibr\">Napp et al. (2018)</xref> and <xref rid=\"B33\" ref-type=\"bibr\">Guerra et al. (2018)</xref> obtained consortia only with enriched culture medium. Both authors obtained consortia from the Luria Broth culture medium, named LBE consortium in <xref rid=\"B57\" ref-type=\"bibr\">Napp et al. (2018)</xref> and BHLBL consortium in <xref rid=\"B33\" ref-type=\"bibr\">Guerra et al. (2018)</xref>. Furthermore, <xref rid=\"B57\" ref-type=\"bibr\">Napp et al. (2018)</xref> obtained a consortium in the Potato Dextrose medium (PDE consortium) and <xref rid=\"B33\" ref-type=\"bibr\">Guerra et al. (2018)</xref> from the YPD medium (BHYPDL consortium). Two consortia obtained without oil as carbon source were used as control. The metagenomic sequences of the phototrophic (SRX375283) and cellulose degradation (SRX474425) consortia were obtained from the public database Sequence Read Archive (SRA)<sup><xref ref-type=\"fn\" rid=\"footnote3\">3</xref></sup> (<xref rid=\"B46\" ref-type=\"bibr\">Leinonen et al., 2010</xref>) and used for comparison, being control 1 and 2, respectively. The complete description of these consortia is available in the <xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S1</xref>. The consortia were aligned using the BLAST program (<xref rid=\"B32\" ref-type=\"bibr\">Gish et al., 1990</xref>) with the protein database BioSurfDB, and the cluster analysis was performed by studio R (version X). All these consortia were deposited in the online database BioSurfDB.</p></sec><sec id=\"S2.SS5\"><title>Emulsification Index (E<sub>24%</sub>) Determination</title><p>The production of emulsifiers was assessed during the stationary phase. Then, emulsification activity was measured according to <xref rid=\"B18\" ref-type=\"bibr\">Cooper and Goldenberg (1987)</xref> with minor adaptations. In brief, 2 mL of different hydrocarbons (kerosene, naphthalene, decane, hexadecane, dichloromethane, and hexacosane) were added to 2 mL of cell-free supernatant from cultures grown in BH medium with petroleum as the only carbon source. The mixture was agitated in vortex at high speed (3,000 rpm) for 2 min and then left to stand for 24 hat room temperature. Emulsification activity was calculated as the height of the emulsion layer divided by the total height and multiplied by 100. As naphthalene and hexacosane were diluted in dichloromethane, the emulsification index (E24%) dichloromethane was subtracted from naphthalene and hexacosane indexes. Culture medium BH and SDS 1% were used as negative and positive controls, respectively.</p></sec><sec id=\"S2.SS6\"><title>Interfacial Tension</title><p>The evaluation of interfacial tension was performed according to <xref rid=\"B9\" ref-type=\"bibr\">Araújo et al. (2020)</xref> in a tensiometer, model DVT50 (Krüss, Hamburg, DEU) using the Rising Drop method, as recommended by the manufacturer. The assay was performed between two phases, the interfacial force between the liquid containing surfactant (bulk phase), and the oil droplet formed in dispense phase is evaluated. Culture supernatant of BH medium was the bulk phase, and petroleum was the other phase. The consortia were cultivated for 5 days until the late stationary phase. Cell-free supernatant (15 mL) was obtained by centrifugation at 5,583 × <italic>g</italic> for 15 min. SDS (1 %) and distilled water were used in the bulk phase as positive and negative controls, respectively.</p></sec><sec id=\"S2.SS7\"><title>Cell Hydrophobicity</title><p>The hydrophobicity of cells was evaluated as described by <xref rid=\"B77\" ref-type=\"bibr\">Rosenberg (1984)</xref> with modifications. The consortia were cultivated 5 days until the late stationary phase. The pellet was obtained by centrifugation at 5,583 × <italic>g</italic> for 15 min and was then washed and resuspended in PUM buffer (g/L) (22.2 K<sub>2</sub>HPO<sub>4</sub>3H<sub>2</sub>O, 7.26 KH<sub>2</sub>PO<sub>4</sub>, 1.8 urea, 0.2 MgSO<sub>4</sub>7H<sub>2</sub>O, pH 7). The culture was diluted in PUM buffer to OD<sub>600nm</sub> 0.5, and 3 mL was added to a penicillin flask with 500 μL of chloroform. The flask was homogenized in a vortex at high speed (3,000 rpm) for 2 min and incubated at room temperature for 1 h in rest. The triplicates of aqueous phase were read at OD<sub>600nm</sub>, and the hydrophobicity was estimated based on the following equation 01: Cell hydrophobicity (%) = (OD before−OD after/OD before) × 10.</p></sec><sec id=\"S2.SS8\"><title>Biodegradation Percentage Determination</title><p>The assay was performed with a consortia pre-inoculum after a correction of the initial OD<sub>600nm</sub> to 0.1 in Erlenmeyer flasks of 250 mL containing 50 mL of BH medium with 1% of petroleum as the sole source of carbon and cultivated for 24 days at 30°C with stirring at 180 rpm. After the incubation period, the biodegradation percentage of both aliphatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs) by microbiological consortia from oil reservoirs were determined by a gas chromatography-flame ionization detector (GC-FID) on a Clarus<sup>®</sup> 600 Chromatograph Adapter (PerkinElmer, Inc., Waltham, MA, United States). The samples were subjected to a liquid-liquid extraction process and to preparative liquid chromatography to clean up the aliphatic and PAH fractions for carrying out quantitative analysis by GC-FID based on (<xref rid=\"B71\" ref-type=\"bibr\">Pugazhendi et al., 2017</xref>; <xref rid=\"B57\" ref-type=\"bibr\">Napp et al., 2018</xref>; <xref rid=\"B67\" ref-type=\"bibr\">Pereira et al., 2019</xref>). The identification of aliphatic hydrocarbons and PAH constituents was performed according to <xref rid=\"B22\" ref-type=\"bibr\">Dörr de Quadros et al. (2016)</xref> and <xref rid=\"B57\" ref-type=\"bibr\">Napp et al. (2018)</xref>. Quantitative analyses were performed using the modified external standardization method according to <xref rid=\"B67\" ref-type=\"bibr\">Pereira et al. (2019)</xref> and <xref rid=\"B9\" ref-type=\"bibr\">Araújo et al. (2020)</xref>. Biodegradation percentage was calculated based on the following equation: 02. B<sub><italic>p</italic></sub> = [(C<sub><italic>i</italic></sub>−C<sub><italic>f</italic></sub>)/C<sub><italic>i</italic></sub>] × 100, where <italic>B</italic><sub><italic>p</italic></sub>, <italic>C</italic><sub><italic>i</italic></sub>, and <italic>C</italic><sub><italic>f</italic></sub> are the biodegradation percentages at the end of the incubation time, the amount of contaminant at the start of incubation, and the amount of contaminant at the end of incubation, respectively (<xref rid=\"B9\" ref-type=\"bibr\">Araújo et al., 2020</xref>).</p></sec><sec id=\"S2.SS9\"><title>Statistical Analysis</title><p>Comparative taxonomic and functional analyses were performed using statistical analyses of taxonomic and functional profiles (STAMP) (<xref rid=\"B65\" ref-type=\"bibr\">Parks et al., 2014</xref>). The significant differences between the relative proportions were calculated using the two-sided Fisher’s exact test with the Newcombe–Wilson confidence interval method, using Storey’s false discovery rate (FDR) and the Benjamin–Hochberg FDR for correction. Results with <italic>q</italic> < 0.05 (corrected <italic>p</italic>-value) were considered significant; unclassified reads were removed from the analysis. For all other experimental assays, a two-way ANOVA, <italic>t</italic>-test, or two-sided Fisher’s exact test was used when appropriate and considered significant at <italic>p</italic> < 0.05.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Consortia Growth Behavior</title><p>In this work, enrichment and selection approaches were used to obtain hydrocarbon-degrading microbial consortia, derived from a Brazilian reservoir PW sample. The first approach consisted of using a rich medium (YPD) in the enrichment step, previously to the selection step with mineral medium (BH), while in the second approach, both steps were performed only in BH medium, containing oil as the only carbon source. In order to assess the hydrocarbon assimilation potential, microbial growth behavior of undefined consortia with or without the addition of petroleum was evaluated. The consortia presented different growth dynamics until the stationary phase in the presence or absence of oil. The stationary phase of the YPD consortium was reached after 24 h, while the population density of the BH consortium stabilized only after 72 h of growth. Therefore, subsequent experiments were carried out after 3 days. It was observed that the consortia also grew in the absence of petroleum. The BH consortium presented similar growth regardless of the presence or absence of oil, and the YPD consortium showed a significant difference between growth in the presence of oil compared to the medium without oil (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Consortia growth curves in mineral medium with 1% (v/v) sterile petroleum. <bold>(A)</bold> Growth curve of the BH consortium + petroleum, compared to the negative controls, one containing only mineral medium and autoclaved oil (Abiotic control), and the other containing BH consortium in mineral medium without oil (BH consortium – petroleum). <bold>(B)</bold> Growth curve of the YPD consortium + petroleum, compared to the negative controls (Abiotic control and YPD consortium – petroleum). The statistical significance was analyzed by a two-way ANOVA, and <italic>p</italic> < 0.05 was considered significant (*<italic>p</italic> < 0.05).</p></caption><graphic xlink:href=\"fbioe-08-00962-g001\"/></fig></sec><sec id=\"S3.SS2\"><title>Metagenomic Diversity</title><p>In order to access the taxonomic and metabolic diversity profiles of the PW crude sample and consortia obtained, metagenomic analyses were carried out. General information regarding each sample submitted for shotgun metagenome sequencing is available in the <xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S2</xref>. Rarefaction curves were obtained, and the plateau was reached for all metagenomic samples, meaning that sequencing was deep enough to cover the diversity of species in each community (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>). Both consortia showed a reduction in diversity in relation to the environmental PW sample (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). Although the BH consortium presented a higher number of species (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>), these predominantly (over 90%) belonged to the genus <italic>Brevibacillus</italic> (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>), in accordance with the low Shannon index (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). On the other hand, the YPD consortium presented several genera in equitable proportions (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>). In addition, the BH consortium had the larger proportion of annotated proteins compared to YPD consortium (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S2</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Taxonomic diversity of the samples. <bold>(A)</bold> Rarefaction curve of samples sequenced by the shotgun metagenomics analysis method and analyzed using MG-RAST. Blue curve = production water (PW); red curve = BH consortium; and yellow curve = YPD consortium. <bold>(B)</bold> Comparison of the Shannon’s diversity indices (red line) between the three sequenced samples. Automatic annotation performed by the MG-RAST pipeline. <bold>(C)</bold> Most abundant genera identified in the production water (PW), YPD consortium, and BH consortium.</p></caption><graphic xlink:href=\"fbioe-08-00962-g002\"/></fig></sec><sec id=\"S3.SS3\"><title>Taxonomic Abundance</title><p>The microbial community of the PW sample was analyzed by sequencing of the 16S rDNA gene and shotgun metagenome (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). The most abundant genera in the PW metagenome were <italic>Marinobacter</italic>, <italic>Halanaerobium</italic>, <italic>Desulfohalobium</italic>, <italic>Desulfovibrio</italic>, and <italic>Alcanivorax</italic> (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>), but these predominant genera were not enriched in the consortia. Furthermore, the most enriched genera in the consortia were present in low abundance in the PW sample. Therefore, consortium cultivation favored the growth of rare genera, since non-abundant genera in the PW sample were found to be among the most prevalent in cultivation.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Relative abundance of genera in production water by shotgun metagenomic and rDNA 16S sequencing methods.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"center\" colspan=\"4\" rowspan=\"1\"><bold>Abundance (%)</bold></td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"4\" rowspan=\"1\"><hr/></td></tr><tr><td valign=\"top\" align=\"justify\" colspan=\"2\" rowspan=\"1\"><bold>Production water (Shotgun sequencing)</bold></td><td valign=\"top\" align=\"justify\" colspan=\"2\" rowspan=\"1\"><bold>Production water (16S rDNA sequencing)</bold></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Marinobacter</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">52.74</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Halanaerobium</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">27.85</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Halanaerobium</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">20.83</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Marinobacter</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">11.82</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Desulfohalobium</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">6.34</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Alcanivorax</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">9.38</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Desulfovibrio</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4.07</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Desulfovibrio</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3.77</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Alcanivorax</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2.84</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Brevibacillus</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.31</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Halothermothrix</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.98</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Ochrobactrum</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.30</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Halothiobacillus</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.85</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Bacillus</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.27</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Arsenophonus</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.50</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Desulfohalobium</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.21</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Streptomyces</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.45</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rhizobium</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">unclassified (derived from Bacteria)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.26</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Phyllobacterium</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.01</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Bartonella</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.08</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Paenibacillus</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.01</td></tr></tbody></table></table-wrap><p>Significant differences (<italic>q</italic> < 0.05) at the domain, phyla, and genera level between consortia were presented in <xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>. There is a predominance of the Bacteria domain in both consortia, similarly to that observed for the crude sample. However, at the phylum level there is a predominance of Proteobacteria in the YPD consortium, while Firmicutes predominated in the BH consortium. As described above, the BH consortium was predominantly composed of representatives of the genus <italic>Brevibacillus</italic> (99,73%). Low abundance genera (frequency < 1%), such as <italic>Bacillus</italic>, <italic>Spiroplasma</italic>, <italic>Lactobacillus</italic>, <italic>Arthrobacter</italic>, and <italic>Corynebacterium</italic>, were also detected in the BH consortium. In the YPD consortium, <italic>Brucella</italic> was the most abundant genus (28,11%), but there was no predominance over the other bacterial genera, such as <italic>Bacillus</italic> (28,11%), <italic>Bartonella</italic> (23,14%), <italic>Bacillus</italic> (19,94%), and <italic>Ochrobactrum</italic> (12,34%) (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). In addition, YPD cultivation favored the enrichment of genera classified as non-cultivable or unclassified genera derived from Bacteria.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Differences in taxonomic profiles between consortia. <bold>(A)</bold> Domain; <bold>(B)</bold> Phylum; and <bold>(C)</bold> Genus levels. <italic>q</italic> < 0.05 was considered statistically significant, using the two-sided Fisher’s exact test.</p></caption><graphic xlink:href=\"fbioe-08-00962-g003\"/></fig><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Frequency of the 10 most abundant genera in the consortia identified by shotgun metagenomic sequencing.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"center\" colspan=\"4\" rowspan=\"1\"><bold>Abundance (%)</bold></td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"4\" rowspan=\"1\"><hr/></td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"2\" rowspan=\"1\"><bold>YPD consortium</bold></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\"><bold>BH consortium</bold></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Brucella</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.11</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Brevibacillus</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.73</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Bartonella</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.14</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Bacillus</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.11</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Bacillus</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19.94</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Spiroplasma</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Ochrobactrum</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.34</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Lactobacillus</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.02</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Phyllobacterium</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.72</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Anoxybacillus</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.01</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rhizobium</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.29</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Arthrobacter</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.01</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Candidatus Liberibacter</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.21</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Corynebacterium</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.01</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Arsenophonus</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.07</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Lysinibacillus</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.01</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>unclassified (derived from Viruses)</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.07</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Mycoplasma</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.01</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>unclassified (derived from Bacteria)</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.79</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Tolypocladium</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.01</td></tr></tbody></table></table-wrap></sec><sec id=\"S3.SS4\"><title>Comparative Functional Profiles</title><p>In order to identify genes and metabolic pathways related to hydrocarbon degradation and production of biosurfactants, the metagenomes obtained were aligned against the BioSurfDB database. The results suggested that YPD medium favored the growth of a consortium enriched in hydrocarbon degradation genes, compared to the BH consortium. The main pathways enriched are involved in the degradation of aromatic compounds, nitrogen metabolism, methane, and metabolism of xenobiotic compounds by cytochrome P450 (<xref ref-type=\"fig\" rid=\"F4\">Figures 4A,C</xref>). In contrast, biosurfactant biosynthesis pathways, such as iturin A, liquenisin, surfactin, and plipastatin, were enriched in the BH consortium, except for the Putisolvin biosurfactant, which was more abundant in YPD consortium (<xref ref-type=\"fig\" rid=\"F4\">Figures 4B,C</xref>). Although both consortia were obtained under the same functional selection in BH with oil, the functional profiles analyzed by principal component analysis (PCA) showed that the consortia are not metabolically similar (<xref ref-type=\"fig\" rid=\"F4\">Figure 4D</xref>). Thus, the YPD culture media exerted different selective pressures on the YPD consortium.</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Functional annotation of shotgun metagenome sequencing performed using the BioSurfDB database. <bold>(A)</bold> Statistical differences in metabolic pathways between YPD consortia and PW. <bold>(B)</bold> Statistical differences in metabolic pathways between the consortia BH and PW. <bold>(C)</bold> Functional comparison between the consortia BH and YPD. <bold>(D)</bold> Principal component analysis between BH and YPD consortia and PW. The graph shows the statistical differences between functional profiles observed in the metagenomes. <italic>q</italic> < 0.05 was considered statistically significant, using the two-sided Fisher’s exact test.</p></caption><graphic xlink:href=\"fbioe-08-00962-g004\"/></fig><p>To identify the set of genes that specifically responded to selection in oil as the sole carbon source, we performed a comparative analysis between the consortia we obtained in this work and other consortia obtained in previous works of our group, <xref rid=\"B57\" ref-type=\"bibr\">Napp et al. (2018)</xref>, and <xref rid=\"B33\" ref-type=\"bibr\">Guerra et al. (2018)</xref>, as well as a control without oil from the databank SRA (SRX375283 and SRX474425). The heatmap (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>) shows a vertical cluster formed by the metagenomes of BHYPDL, BHLBL, BH, and YPD consortia, which all underwent the common selection step in BH medium. This cluster presented an enrichment of pathways related to degradation of more complex hydrocarbons, and the Peroxisome proliferator-activated receptor (PPAR) signaling pathway was exclusive for this cluster. In fact, this pathway (PPAR) is finder in a eukaryotic cell. The alkane hydroxylase (<italic>AlkB</italic> gene) is the only bacterial enzyme associated with this pathway due to a similar activity to that of the non-heme integral-membrane acyl coenzyme A (CoA) desaturases and acyl lipid desaturases (<xref rid=\"B81\" ref-type=\"bibr\">Shanklin and Whittle, 2003</xref>). In addition, there is a horizontal cluster formed by degradation pathways of simple hydrocarbons, such as fatty acids, benzoate, and chloroalkanes, with no differences between consortia grown with or without oil, regardless of the culture medium.</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Cluster analysis of biodegradation and biosurfactant biosynthesis pathways in the consortia. The influence of the consortia obtainment medium on the abundance of biodegradation and biosurfactant genes was analyzed. <bold>(A)</bold> Clustering of genes involved in hydrocarbon biodegradation pathways. <bold>(B)</bold> Clustering of genes involved in biosurfactant biosynthesis pathways. The clustering was based on the abundance of reads annotated in each pathway. Heatmap colors are generated with log<sub>10</sub> among the individual samples. A heatmap was generated using the heatmap package in the statistical program R.</p></caption><graphic xlink:href=\"fbioe-08-00962-g005\"/></fig><p>The metagenomes of the BH, BHLBL, and BHYPDL consortia also formed a cluster with regard to the abundance of genes involved in biosurfactant biosynthesis (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>). The step through BH as minimal medium, proved to be more effective in the selection of biosurfactant biosynthesis pathways, since the consortium obtained in rich media presented a reduced abundance of these genes. The lipopeptide, serrawettin, trehalolipid, and rhamnolipid biosynthesis pathways were mainly enriched in the consortium that were selected only in BH medium with hydrophobic substrate, such as oil (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>).</p></sec><sec id=\"S3.SS5\"><title>Production of Biosurfactants</title><p>To assess the presence of extracellular surfactant produced by the consortia, cell-free supernatant was analyzed for the formation of stable emulsion and the ability to reduce interfacial tension. The YPD consortium presented emulsification only in kerosene and hexacosane, whereas the BH consortium showed a significant emulsification capacity in all hydrocarbons tested and was the only one to emulsify an aromatic hydrocarbon (naphthalene) (<xref ref-type=\"fig\" rid=\"F6\">Figure 6A</xref>). This result is in line with the greater abundance of biosurfactant genes observed in the BH consortium (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>).</p><fig id=\"F6\" position=\"float\"><label>FIGURE 6</label><caption><p>Production of biosurfactants by consortia. <bold>(A)</bold> Emulsification indexes (E24%) for BH and YPD consortia in different hydrocarbons (naphthalene, kerosene, decane, hexadecane, and hexacosane). The statistical significance was analyzed by a two-way ANOVA (*<italic>p</italic> < 0.05). <bold>(B)</bold> Cell hydrophobicity in YPD and BH consortia. These were analyzed using a <italic>t</italic>-test (*<italic>p</italic> < 0.05). <bold>(C)</bold> Evaluation of the interfacial tension in relation to the time of release of the oil droplet in contact with the supernatant of the BH and YPD consortia. This result was analyzed by a two-way ANOVA (*<italic>p</italic> < 0.05 compared to the control).</p></caption><graphic xlink:href=\"fbioe-08-00962-g006\"/></fig><p>Some biosurfactants are found adherent to cell membranes giving high hydrophobicity to the cell membrane (<xref rid=\"B75\" ref-type=\"bibr\">Ramkrishna, 2010</xref>; <xref rid=\"B86\" ref-type=\"bibr\">Soberón-Chávez and Maier, 2011</xref>). Therefore, cellular hydrophobicity was measured in consortia. The BH consortium presented a microbial community that was significantly more hydrophobic than the YPD consortium, 52 and 4%, respectively (<xref ref-type=\"fig\" rid=\"F6\">Figure 6B</xref>). In contrast, the YPD consortium showed greater capacity to reduce interfacial tension when compared to the BH consortium (<xref ref-type=\"fig\" rid=\"F6\">Figure 6C</xref>).</p></sec><sec id=\"S3.SS6\"><title>Biodegradation of Aliphatic and Polycyclic Aromatics Hydrocarbons</title><p>The biodegradation of aliphatic and PAHs by the consortia was evaluated using GC-FID. The comparison of aliphatic hydrocarbon biodegradation in general showed no statistical difference between consortia (<xref ref-type=\"fig\" rid=\"F7\">Figures 7A–D</xref> and <xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Figure S1</xref>). Regarding the alkane degradation, statistical differences were only observed in C12 to C14 degradation, in which YPD showed a significant increase compared to BH consortium. The biodegradation of PAHs, acetylene (Acy), fluorene (Flu), pyrene (Pyr), also occurs in both consortia, and there is no statistical difference for the first two PAHs. However, exclusive biodegradation of phenanthrene (Phe) occurs in the YPD consortium, whereas biodegradation of fluoranthene (Flo) and benzo[a]anthracene (BaA) occurs in the BH consortium (<xref ref-type=\"fig\" rid=\"F7\">Figure 7E</xref>). Therefore, there was more biodegradation of different types of PAHs in the consortium cultivated only in mineral BH medium.</p><fig id=\"F7\" position=\"float\"><label>FIGURE 7</label><caption><p>Hydrocarbon biodegradation profiles of the BH and YPD consortia. <bold>(A)</bold> Comparison of biodegradation of short aliphatic hydrocarbons (from 7C to 14C) in percentages; <bold>(B)</bold> Biodegradation of aliphatic hydrocarbons (from 15C to 21C); <bold>(C)</bold> Biodegradation of long aliphatic hydrocarbons (from 22C to 28C); <bold>(D)</bold> Biodegradation of long aliphatic hydrocarbons (from 29C to 36C); and <bold>(E)</bold> Biodegradation of total polycyclic aromatic hydrocarbons (PAHs), in percentage, compared to the negative control only with mineral medium and petroleum analyzed by chromatography. All data were analyzed using two-way ANOVA (*<italic>p</italic> < 0.05).</p></caption><graphic xlink:href=\"fbioe-08-00962-g007\"/></fig></sec></sec><sec id=\"S4\"><title>Discussion</title><p>In the current work, two consortia from the same PW sample were obtained using different methods in order to test the medium influence on microbial diversity, hydrocarbon biodegradation, and biosurfactant production genes. Our data demonstrated that the YPD consortium was taxonomically more diverse and enriched in hydrocarbon degradation genes, while the BH consortium showed <italic>Brevibacillus</italic> predominance and enrichment in several biosurfactants production genes. These findings might improve the production efficiency of low molecular weight biosurfactants from hydrophobic substrates such as oil. Few studies focus on the interactions between hydrocarbons biodegradation and biosurfactant families simultaneously. Some works described that bacteria compete for hydrocarbon substrates through biosurfactants production, which have antimicrobial activity and improve their ability to biodegrade hydrocarbons (<xref rid=\"B20\" ref-type=\"bibr\">Cray et al., 2013</xref>; <xref rid=\"B58\" ref-type=\"bibr\">Ndlovu et al., 2017</xref>). However, there are studies that demonstrate that microbial consortia can present higher rates of hydrocarbon degradation and biosurfactants production than isolated species (<xref rid=\"B17\" ref-type=\"bibr\">Cerqueira et al., 2011</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Araújo et al., 2020</xref>).</p><p>The presence of biosurfactant favors a better efficiency of hydrocarbon degradation in microbial consortia with a slow growth rate. In general, slow-degrading consortia (such as BH consortium) perform hydrocarbon absorption from the aqueous phase through their solubilization using biosurfactants (<xref rid=\"B44\" ref-type=\"bibr\">Ławniczak et al., 2013</xref>). In addition, BH consortium growth was observed even in the absence of oil, which suggests the occurrence of CO<sub>2</sub> sequestering species. This was corroborated by the data obtained from the sequencing, which showed the presence of reads annotated as carbonic anhydrase attributed to the genus <italic>Brevibacillus</italic>. This enzyme catalyzes the interconversion between carbon dioxide and bicarbonate, being used by bacteria to capture CO<sub>2</sub> (<xref rid=\"B13\" ref-type=\"bibr\">Boone et al., 2013</xref>; <xref rid=\"B88\" ref-type=\"bibr\">Supuran and Capasso, 2017</xref>). The CO<sub>2</sub> sequestration capacity has been described for the <italic>Bacillus</italic> sp. SS105. The authors observed activity of carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) associated with CO<sub>2</sub> sequestration and increased production of biosurfactants, suggesting that the use of this strain for CO<sub>2</sub> fixation as a strategy to mitigate CO<sub>2</sub> emissions (<xref rid=\"B49\" ref-type=\"bibr\">Maheshwari et al., 2017</xref>, <xref rid=\"B50\" ref-type=\"bibr\">2018</xref>). Considering our results, it is plausible to propose that <italic>Brevibacillus</italic> has the same capacity, which should be investigated in the future.</p><p>In contrast to slow-growing bacteria, fast-growing consortia, such as YPD, tend to form biofilms at the interfacial frontier, which suggests that direct capture mechanisms are prevalent. In this case, biosurfactants may be synthesized as secondary metabolites after degradation of the oil-water interface (<xref rid=\"B44\" ref-type=\"bibr\">Ławniczak et al., 2013</xref>). However, in this work, the analysis of biosurfactants was made when both consortia were in the stationary phase, i.e., during the highest production of biosurfactants (<xref rid=\"B47\" ref-type=\"bibr\">Lin, 1996</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Fontes et al., 2012</xref>; <xref rid=\"B78\" ref-type=\"bibr\">Rufino et al., 2014</xref>).</p><p>Low-abundant genera in the PW were enriched in both consortia. This proportion may be due to the PW anaerobic conditions in the oil reservoir (<xref rid=\"B94\" ref-type=\"bibr\">Wang et al., 2011</xref>), contrasting with the culture aerobic conditions used in this work. Although anaerobic taxa are prevalent, there is a great abundance of aerobic bacteria in oil reservoirs (<xref rid=\"B6\" ref-type=\"bibr\">An et al., 2013</xref>). We observed the predominance of the <italic>Brevibacillus</italic> genus in BH consortia. This genus has been reported in oil reservoir samples and associated to oil degradation and biosurfactant production (<xref rid=\"B54\" ref-type=\"bibr\">Mnif et al., 2011</xref>; <xref rid=\"B83\" ref-type=\"bibr\">She et al., 2014</xref>), which has allowed for its application in biotechnological approaches for microbial enhanced oil recovery (MEOR) (<xref rid=\"B38\" ref-type=\"bibr\">Hou et al., 2005</xref>, <xref rid=\"B37\" ref-type=\"bibr\">2008</xref>, <xref rid=\"B36\" ref-type=\"bibr\">2011</xref>; <xref rid=\"B82\" ref-type=\"bibr\">She et al., 2012</xref>; <xref rid=\"B72\" ref-type=\"bibr\">Purwasena et al., 2018</xref>). However, the genes and metabolic pathways involved in these processes are poorly understood.</p><p>The comparison between consortia from this work and publicly available consortia showed that the presence of oil during the microbial growth imposed weak selective pressure for the enrichment of simple hydrocarbon degradation pathways, such as fatty acid, aliphatic alkane, and monoaromatic hydrocarbons, since all consortia showed a similar abundance of genes involved in these degradation pathways. Thus, the presence of oil in the environment does not seem to be a limiting factor for the enrichment of simple hydrocarbon degradation genes, since these genes are part of the basal lipid metabolism (<xref rid=\"B97\" ref-type=\"bibr\">Yao and Rock, 2017</xref>). An exception was the <italic>AlkB</italic> gene (alkane hydroxylase), associated with both the fatty acid and the PPAR pathways, which was enriched only in consortia that had undergone the selection stage in BH medium. AlkB is an integral-membrane di-iron enzyme that plays a pivotal role in aerobic degradation of alkanes and is widely distributed in bacteria (<xref rid=\"B60\" ref-type=\"bibr\">Nie et al., 2014</xref>).</p><p>Although the YPD consortium presented a greater predominance of genes related to the hydrocarbon degradation, few significant differences in the aliphatic hydrocarbon degradation were observed when compared to BH consortium. However, the BH consortium degraded more types of aromatic hydrocarbons than the YPD consortium, suggesting a synergistic effect caused by biosurfactants (<xref rid=\"B43\" ref-type=\"bibr\">Kim et al., 2009</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Alvarez and Polti, 2014</xref>; <xref rid=\"B68\" ref-type=\"bibr\">Perera et al., 2019</xref>). Biosurfactants allow hydrophobic molecule solubilization, increasing their bioavailability (<xref rid=\"B61\" ref-type=\"bibr\">Nitschke and Costa, 2007</xref>). Different biosurfactants can solubilize more hydrocarbons types, promoting the assimilation of hydrocarbons onto the cell membrane by changes of cell hydrophobicity and bioavailability (<xref rid=\"B69\" ref-type=\"bibr\">Phale et al., 1995</xref>; <xref rid=\"B70\" ref-type=\"bibr\">Prabhu and Phale, 2003</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Andreoni et al., 2004</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Kim et al., 2009</xref>). In addition, it has been described that Firmicutes, abundant in the BH consortium, are an indicator of the later stages of hydrocarbon degradation when more recalcitrant compounds, such as PAHs, are present (<xref rid=\"B11\" ref-type=\"bibr\">Beazley et al., 2012</xref>). As described by <xref rid=\"B21\" ref-type=\"bibr\">Dasari et al. (2014)</xref>, biosurfactants are useful for PAH biodegradation, which is in line with our results, since we observed PAH degradation and occurrence of emulsifying activity in the BH consortium. Altogether, these results suggest that the BH consortium, although less diverse and presenting less reads related to hydrocarbon degradation, has a community capable of degrading oil due to the production of biosurfactants.</p><p>The YPD and BH consortia also differ in their ability to reduce interfacial tension and emulsify different hydrocarbons. The YPD consortium was more efficient in reducing interfacial tension. This consortium stands out for the higher occurrence of genes related to Putisolvin biosynthesis. Low molecular weight biosurfactants, such as the lipopeptide Putisolvin, are more effective in reducing interfacial tension (<xref rid=\"B2\" ref-type=\"bibr\">Al-Araji et al., 2007</xref>; <xref rid=\"B80\" ref-type=\"bibr\">Satpute et al., 2010</xref>; <xref rid=\"B90\" ref-type=\"bibr\">Uzoigwe et al., 2015</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Jahan et al., 2020</xref>). This biosurfactant has been described in <italic>Pseudomonas</italic> species, with no report of its production by other genera (<xref rid=\"B24\" ref-type=\"bibr\">Dubern et al., 2005</xref>, <xref rid=\"B25\" ref-type=\"bibr\">2006</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Dubern and Bloemberg, 2006</xref>). In this work, <italic>Pseudomonas</italic> is not among the most abundant genera in the YPD consortium, suggesting that other genera may be responsible for the production of Putisolvin. In fact, the BLAST analysis indicated occurrence of Putisolvin genes in <italic>Bacillus</italic>, which is one of the most abundant genera in the YPD consortium.</p><p>The BH consortium can emulsify more hydrocarbon types compared to the YPD consortium and shows enrichment of several biosurfactant classes. It has been proposed that high molecular weight biosurfactants and bioemulsifiers form better emulsions (<xref rid=\"B2\" ref-type=\"bibr\">Al-Araji et al., 2007</xref>; <xref rid=\"B80\" ref-type=\"bibr\">Satpute et al., 2010</xref>; <xref rid=\"B90\" ref-type=\"bibr\">Uzoigwe et al., 2015</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Jahan et al., 2020</xref>). As an example, the cluster formed by BH, BHLBL, and BHYPDL consortia showed enrichment of some high molecular weight biosurfactants such as alasan, which is an anionic polysaccharide that possesses effective PAH emulsifying activity (<xref rid=\"B89\" ref-type=\"bibr\">Toren et al., 2002</xref>). <xref rid=\"B93\" ref-type=\"bibr\">Viramontes-Ramos et al. (2010)</xref> observed that strains grown in mineral medium supplemented with olive oil showed a higher rate of emulsification in kerosene, compared to strains grown in mineral medium supplemented with glucose. Together, the data indicate that cultivation in a minimal medium supplemented with oil is a more effective strategy for the selection of microorganisms producing biosurfactants with emulsifying activity.</p><p>The biosynthesis pathways for low molecular weight biosurfactants, such as lipopeptides, serrawettin, trehalolipid and rhamnolipids are also more prevalent in the BH consortium and may explain the high cell hydrophobicity in this consortium, since some biosurfactants can bind to the cell membrane and alter its hydrophobicity due to the presence of hydrocarbons (<xref rid=\"B76\" ref-type=\"bibr\">Rosenberg et al., 1988</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Franzetti et al., 2008</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Banat et al., 2010</xref>). Trehalolipid biosynthesis is induced by the presence of hydrophobic substrates, resulting in both membrane-bound as well as trehalolipids secreted to the extracellular medium promoting greater cellular hydrophobicity and hydrocarbon emulsification (<xref rid=\"B31\" ref-type=\"bibr\">Franzetti et al., 2010</xref>; <xref rid=\"B64\" ref-type=\"bibr\">Pacheco et al., 2010</xref>; <xref rid=\"B48\" ref-type=\"bibr\">Liu and Liu, 2011</xref>; <xref rid=\"B95\" ref-type=\"bibr\">White et al., 2013</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Edosa et al., 2018</xref>). Lipopeptides and rhamnolipids are produced both in the presence of hydrophobic and hydrophilic substrates in mineral medium (<xref rid=\"B55\" ref-type=\"bibr\">Mukherjee and Das, 2005</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Abdel-Mawgoud et al., 2011</xref>). By using oil as a carbon source, the production of rhamnolipids was 80% higher, compared to other hydrophobic substrates and almost 100% higher than with glucose (<xref rid=\"B52\" ref-type=\"bibr\">Mata-Sandoval’ et al., 2001</xref>). Rhamnolipids may be attached to the cell membrane allowing a more efficient hydrocarbon uptake by rendering the cell surface more hydrophobic (<xref rid=\"B4\" ref-type=\"bibr\">Al-Tahhan et al., 2000</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Banat et al., 2010</xref>). Serrawettin, trehalolipid and rhamnolipid were not yet identified in <italic>Brevibacillus</italic>. However, the genes related to the production of these biosurfactants are more abundant in the BH consortium, suggesting that <italic>Brevibacillus</italic> species can produce these low molecular weight biosurfactant. The extraction and characterization of the biosurfactants produced by BH consortium will confirm this hypothesis.</p><p>In terms of yields, studies show that the production of biosurfactants and hydrocarbons biodegradation in consortia is greater than in culture of isolated bacteria (<xref rid=\"B40\" ref-type=\"bibr\">Jing et al., 2007</xref>; <xref rid=\"B56\" ref-type=\"bibr\">Mukred et al., 2008</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Cerqueira et al., 2011</xref>). In contrast, <xref rid=\"B8\" ref-type=\"bibr\">Antoniou et al. (2015)</xref> using a defined consortia showed that the isolates produces more low-molecular weight biosurfactants, specially rhamnolipids and sophorolipids, than bacterial consortia. However, the latter produces a greater diversity of low-molecular weight biosurfactants, which may reflect the growth competition between microorganisms. Therefore, the success in the production of biosurfactants seems to depend on the interactions established between microorganisms present in the environment, such as synergism or competition, which needs to be further investigated.</p><p>Several studies have analyzed the influence of biosurfactants on bioremediation processes, mainly in terms of increased efficiency, which has been associated with better solubilization of pollutants, resulting in greater bioavailability and consequently higher rates of biodegradation (<xref rid=\"B51\" ref-type=\"bibr\">Makkar and Rockne, 2003</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Araújo et al., 2020</xref>). In general, most of the works that enrich microorganisms for biosurfactant production use hydrophilic carbon sources. Simple sugar, starch, and plant sugar-based carbohydrates are the major carbon sources used as substrates (<xref rid=\"B62\" ref-type=\"bibr\">Nurfarahin et al., 2018</xref>). In this work, a consortium obtained only in mineral medium with oil as carbon source showed greater efficiency and competitiveness in the biodegradation of hydrocarbons through the production of biosurfactants.</p></sec><sec id=\"S5\"><title>Conclusion</title><p>The microbial enrichment in minimal medium containing petroleum as carbon source was shown to be effective for the selection of microorganisms that produce biosurfactants. The BH consortium obtained proved to be efficient in hydrocarbon degradation, being superior to the YPD consortium in relation to the degradation of some PAHs. The selective pressure exerted by minimal medium containing oil led to the obtainment a consortium with low diversity in bacterial genera, constituted predominantly by representatives of the genus <italic>Brevibacillus</italic>, indicating that this is an efficient producer of different biosurfactant classes. Finally, our data indicate the biotechnological potential of consortia selected in a minimal medium containing oil, applicable for approaches as MEOR, CO<sub>2</sub> capture, and bioremediation of waste and impacted areas.</p></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>The datasets presented in this study can be found in online repositories. The names of the repositories and accession numbers can be found at: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.mg-rast.org/\">http://www.mg-rast.org/</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"4726099.3\">4726099.3</ext-link>; <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.mg-rast.org/\">http://www.mg-rast.org/</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"4827355.3\">4827355.3</ext-link>; <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.mg-rast.org/\">http://www.mg-rast.org/</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"4826970.3\">4826970.3</ext-link>; <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.mg-rast.org/\">http://www.mg-rast.org/</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"4643476.3\">4643476.3</ext-link>; <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.mg-rast.org/\">http://www.mg-rast.org/</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"4643480.3\">4643480.3</ext-link>; <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.mg-rast.org/\">http://www.mg-rast.org/</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"4583777.3\">4583777.3</ext-link>; <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.mg-rast.org/\">http://www.mg-rast.org/</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"4583773.3\">4583773.3</ext-link>; <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/\">https://www.ncbi.nlm.nih.gov/</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SRX375283\">SRX375283</ext-link>; <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/\">https://www.ncbi.nlm.nih.gov/</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SRX474425\">SRX474425</ext-link>.</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>WA, JO, MF, SA, CM, RS-P, KS-B, AN, JP, and JF carried out the experiment, data analysis, and wrote the manuscript. MP, LP, MV, and LA-L were responsible for the conception, experimental design, and supervision of the project, as well as data analysis and wrote the manuscript. All authors read and approved the final version of the manuscript.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This study was supported by Petrogal Brasil S/A, Agência Nacional de Petróleo, Gás Natural e Biocombustíveis (ANP-Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico – Brazil (CNPq).</p></fn></fn-group><ack><p>The authors are grateful to Petrogal Brasil S/A and Agência Nacional de Petróleo, Gás Natural e Biocombustíveis (ANP-Brazil) for providing the PW sample, enabling the development of the research.</p></ack><fn-group><fn id=\"footnote1\"><label>1</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://metagenomics.anl.gov\">http://metagenomics.anl.gov</ext-link></p></fn><fn id=\"footnote2\"><label>2</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://hannonlab.cshl.edu/fastx_toolkit/\">http://hannonlab.cshl.edu/fastx_toolkit/</ext-link></p></fn><fn id=\"footnote3\"><label>3</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/sra/\">https://www.ncbi.nlm.nih.gov/sra/</ext-link></p></fn></fn-group><sec id=\"S10\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fbioe.2020.00962/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fbioe.2020.00962/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"DS1\"><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Abdel-Mawgoud</surname><given-names>A. 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Pollut.</italic></source>\n<volume>178</volume>\n<fpage>152</fpage>–<lpage>158</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.envpol.2013.03.004</pub-id>\n<pub-id pub-id-type=\"pmid\">23570783</pub-id></mixed-citation></ref><ref id=\"B97\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yao</surname><given-names>J.</given-names></name><name><surname>Rock</surname><given-names>C. O.</given-names></name></person-group> (<year>2017</year>). <article-title>Exogenous fatty acid metabolism in bacteria.</article-title>\n<source><italic>Biochimie</italic></source>\n<volume>141</volume>\n<fpage>30</fpage>–<lpage>39</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.biochi.2017.06.015</pub-id>\n<pub-id pub-id-type=\"pmid\">28668270</pub-id></mixed-citation></ref></ref-list><glossary><title>Abbreviations</title><def-list id=\"DL1\"><def-item><term>Acy</term><def><p>acetylene</p></def></def-item><def-item><term>BaA</term><def><p>benzo[a]anthracene</p></def></def-item><def-item><term>BH</term><def><p>Bushnell-Haas</p></def></def-item><def-item><term>E<sub>24%</sub></term><def><p>emulsification index</p></def></def-item><def-item><term>Flo</term><def><p>fluoranthene</p></def></def-item><def-item><term>Flu</term><def><p>fluorene</p></def></def-item><def-item><term>GC-FID</term><def><p>gas chromatography-flame ionization detector</p></def></def-item><def-item><term>MEOR</term><def><p>microbial enhanced oil recovery</p></def></def-item><def-item><term>OD<sub>600nm</sub></term><def><p>optical density</p></def></def-item><def-item><term>PAHs</term><def><p>polycyclic aromatic hydrocarbons</p></def></def-item><def-item><term>PCA</term><def><p>principal component analysis</p></def></def-item><def-item><term>PGM</term><def><p>Personal Genome Machine</p></def></def-item><def-item><term>Phe</term><def><p>phenanthrene</p></def></def-item><def-item><term>PPAR</term><def><p>peroxisome proliferator-activated receptor</p></def></def-item><def-item><term>PW</term><def><p>production water</p></def></def-item><def-item><term>Pyr</term><def><p>pyrene</p></def></def-item><def-item><term>YPD</term><def><p>yeast extract peptone dextrose.</p></def></def-item></def-list></glossary></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Clin Pract Epidemiol Ment Health</journal-id><journal-id journal-id-type=\"iso-abbrev\">Clin Pract Epidemiol Ment Health</journal-id><journal-id journal-id-type=\"publisher-id\">CPEMH</journal-id><journal-title-group><journal-title>Clinical Practice and Epidemiology in Mental Health : CP & EMH</journal-title></journal-title-group><issn pub-type=\"epub\">1745-0179</issn><publisher><publisher-name>Bentham Open</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32874192</article-id><article-id pub-id-type=\"pmc\">PMC7431682</article-id><article-id pub-id-type=\"publisher-id\">CPEMH-16-174</article-id><article-id pub-id-type=\"doi\">10.2174/1745017902016010174</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Clinical Practice Epidemiology in Mental Health</subject></subj-group></article-categories><title-group><article-title>Health Related Quality of Life in Patients with Onco-hematological Diseases</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>La Nasa</surname><given-names>Giorgio</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Caocci</surname><given-names>Giovanni</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref><xref ref-type=\"corresp\" rid=\"cor1\">*</xref></contrib><contrib contrib-type=\"author\"><name><surname>Morelli</surname><given-names>Emanuela</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Massa</surname><given-names>Elena</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Farci</surname><given-names>Antonio</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Deiana</surname><given-names>Laura</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Pintus</surname><given-names>Elisa</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Scartozzi</surname><given-names>Mario</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sancassiani</surname><given-names>Federica</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">2</xref></contrib><aff id=\"aff1\">\n<label>1</label>Ematologia e CTMO, Ospedale Businco, Azienda Ospedaliera Brotzu, <addr-line><city>Cagliari</city></addr-line>, <country>Italy</country></aff><aff id=\"aff2\">\n<label>2</label>Dipartimento di Scienze Mediche e Sanita Pubblica, <institution>Universita di Cagliari</institution>, Cagliari, <country>Italy</country></aff></contrib-group><author-notes><corresp id=\"cor1\"><label>*</label>Address correspondence to this author at the Ematologia e CTMO, Ospedale Businco, Azienda Ospedaliera Brotzu, Via Jenner, sn, 09124 Cagliari, Italy; Tel: +39-70-52964901; Fax: +39-70-52965317; E-mail: <email xlink:href=\"giovanni.caocci@unica.it\">giovanni.caocci@unica.it</email></corresp></author-notes><pub-date pub-type=\"epub\"><day>30</day><month>7</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>16</volume><fpage>174</fpage><lpage>179</lpage><history><date date-type=\"received\"><day>10</day><month>5</month><year>2020</year></date><date date-type=\"rev-recd\"><day>3</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>6</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 La Nasa <italic>et al</italic>.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>La Nasa.</copyright-holder><license license-type=\"open-access\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/legalcode\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (<uri xlink:href=\"https://creativecommons.org/licenses/by/4.0/legalcode\">https://creativecommons.org/licenses/by/4.0/legalcode</uri>). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract><sec><title>Background:</title><p>HRQoL is generally conceptualized as a broad multidimensional construct that refers to patients' perceptions of the impact of disease and its treatment on their physical, psychological, and social functioning and well-being. Little is known in patients with onco-hematological cancer in comparison with the general population and other chronic diseases.</p></sec><sec><title>Objective:</title><p>We assessed HRQoL in patients diagnosed with haematological cancers in comparison with the general population and other chronic diseases.</p></sec><sec><title>Methods:</title><p>The questionnaire Short Form (SF)-12 was administered to 62 patients with onco-hematological disease and results were compared with 702 controls (184 healthy people, 37 Major Depression, 201 Multiple Sclerosis; 23 Wilson disease; 46 Carotidal Atherosclerosis; 60 Celiac disease; 151 solid tumours).</p></sec><sec><title>Results:</title><p>HRQoL in patients diagnosed with a haematological cancer was significantly worse in comparison with the general population (F= 43.853, p <0.00001) but similar when compared with solid tumour and other chronic diseases such as Major Depression and Carotid Atherosclerosis. In addition, HRQoL in patients diagnosed with a haematological cancer was significantly higher than that due to Celiac disease (p <0.00001) and Wilson's disease (p= 0.02), and lower than that due to Multiple Sclerosis (p= 0.032).</p></sec><sec><title>Conclusion:</title><p>This study confirmed that haematological cancers negatively affects overall HRQoL. The results showed an impact of haematological cancers on HRQoL that is similar to what found in patients with solid tumors, Major Depression and Carotid Atherosclerosis. Current successful therapeutic strategy achieved in the treatment of haematological cancers not only positively impact on survival rate but also could improve the overall HRQoL.</p></sec></abstract><kwd-group kwd-group-type=\"author\"><title>Keywords</title><kwd>HRQoL</kwd><kwd>Hematological cancer</kwd><kwd>Mood depression</kwd><kwd>SF-12</kwd><kwd>Multiple Sclerosis</kwd><kwd>Tumors</kwd><kwd>Celiac disease</kwd></kwd-group></article-meta></front><body><sec sec-type=\"intro\" id=\"sec1\"><label>1</label><title>INTRODUCTION</title><p>Cancer is the second leading cause of death in Italy, second only to cardiovascular disease [<xref rid=\"r1\" ref-type=\"bibr\">1</xref>]. Nonetheless, in 2017 survival has been consistently increasing in Italy after the diagnosis of cancer, specifically 3.304.648 people out of a population of around 60 million [<xref rid=\"r1\" ref-type=\"bibr\">1</xref>]. Early detection, screening, and diagnosis have demonstrated to significantly improve patient survival rates and also increase awareness of the benefit of prompt therapies and healthy lifestyles. In this context, it emerges the joint relevance of the health related quality of life (HRQoL) as a prognostic factor of clinical outcome and adherence to treatment [<xref rid=\"r2\" ref-type=\"bibr\">2</xref>-<xref rid=\"r4\" ref-type=\"bibr\">4</xref>].</p><p>HRQoL is a complex, multifaceted construct that includes' illness perception and self-evaluation of physical, mental, and social health status of the individuals [<xref rid=\"r5\" ref-type=\"bibr\">5</xref>]. HRQoL in patients with cancer is considered a critical prognostic factor for predicting survival, regardless of the clinical and socio-demographic characteristics well-known to have an important impact on the progress of the disease [<xref rid=\"r6\" ref-type=\"bibr\">6</xref>, <xref rid=\"r7\" ref-type=\"bibr\">7</xref>]. Moreover, clinicians and patients now frequently face difficult choices concerning therapies that are often comparable in regard to efficacy, especially making medical complex decisions . In addition, cancer treatments can affect several dimensions of quality of life: body image, fatigue and mental health [<xref rid=\"r8\" ref-type=\"bibr\">8</xref>].</p><p>So far, little attention has been paid to the effects on HRQoL in patients diagnosed with haematological cancer [<xref rid=\"r9\" ref-type=\"bibr\">9</xref>-<xref rid=\"r13\" ref-type=\"bibr\">13</xref>]. Life expectancy in patients with haematological cancer is generally increased and sometimes is now close to that observed in the general population for all ages [<xref rid=\"r14\" ref-type=\"bibr\">14</xref>]. In particular circumstances, therapy can be suspended and patients can be monitored during the phase of remission free of treatment [<xref rid=\"r15\" ref-type=\"bibr\">15</xref>, <xref rid=\"r16\" ref-type=\"bibr\">16</xref>]. In this context, the application of patient-reported outcomes (PRO), an instrument that evaluates health outcomes from the patient's perspective, plays a crucial role [<xref rid=\"r17\" ref-type=\"bibr\">17</xref>-<xref rid=\"r21\" ref-type=\"bibr\">21</xref>].</p><p>The primary endpoint of our research was to assess self-perceived HRQoL in patients diagnosed with haematological cancers and compare the results with the general population and with different chronic pathologies.</p></sec><sec sec-type=\"materials|methods\" id=\"sec2\"><label>2</label><title>MATERIALS AND METHODS</title><sec id=\"sec2.1\"><label>2.1</label><title>Sample and Setting</title><p>Patients were recruited during the period of June-July 2018 from the Day-Hospital Service (DH) of the Hematology Unit and Stem Cell Transplantation Center, Hospital Businco, Azienda Ospedaliera Brotzu, Cagliari, Italy. The study population included patients receiving active treatment with age >18 years, both sexes and histologic confirmation of malignant neoplasm.The study was approved by the ethics committee of the Italian National Health Institute, Rome (\"Istituto Superiore della Sanita\"). All procedures were carried out in accordance with the 1964 Helsinki declaration and its later amendments. Each participant was provided with full descriptions of the aims and procedures of the study before signing the informed consent form. Participants were also made aware of data protection and ensured that they could terminate the interview at any time.</p></sec><sec id=\"sec2.2\"><label>2.2</label><title>Questionnaire</title><p>The questionnaire SF-12 (Short Form Health Survey - 12 item) was administered to evaluate the HRQoL [<xref rid=\"r22\" ref-type=\"bibr\">22</xref>]. The instrument consisting of 12 items was used to investigate the two sub-dimensions, physical and psychosocial health. The total score (range: 12-47 points) consisted of asking subjects to indicate the extent to which they agreed or disagreed with individual items. The resulting answers were then scored to obtain a Likert Scale value. Higher scores reflect a better perceived HRQoL; a score <36 indicates low perceived HRQoL levels [<xref rid=\"r22\" ref-type=\"bibr\">22</xref>].</p><p>In addition, data were collected through a socio-demographic and clinical-oncological data collection form, countenancing information on marital and employment status, education level, time of follow-up, cancer staging, toxicity of treatments, intent of treatment, response and adherence to treatment. The diffusion of hematological cancer was scored from 1 to 4 considering 1 as a unique localization in one nodal station or extra-nodal; 2 as two or more localizations from the same side of diaphragm, 3 as localizations from both side of diaphragm and 4 diffuse disease. The toxicity of treatment was scored from 1 (mild) to 5 (death), according to Common Toxicities Criteria (CTC), version 4.0 [<xref rid=\"r23\" ref-type=\"bibr\">23</xref>].</p></sec><sec id=\"sec2.3\"><label>2.3</label><title>Statistical Analysis</title><p>For each individual with onco-haematological disease, four healthy control subjects matched by gender and age were extracted from a database of a community survey on well-being and mental health that used a similar methodology [<xref rid=\"r24\" ref-type=\"bibr\">24</xref>]. The Control Sample (CS) extracted from the general population, through simple random sampling and matched for age and gender, consists of 88 females and 160 males for a total of 248 people. The impact of the pathology on the HRQoL was calculated by the difference between the mean SF-12 score obtained in the group of patients in comparison with HRQoL scores obtained from the healthy general population. Data on different chronic diseases coming from previously published studies were used for comparison [<xref rid=\"r25\" ref-type=\"bibr\">25</xref>-<xref rid=\"r31\" ref-type=\"bibr\">31</xref>].</p><p>The scores obtained in the SF-12 scales were compared using analysis of variance (ANOVA)test. Differences with a p-value of less than 0.05 were considered statistically significant.</p></sec></sec><sec sec-type=\"results\" id=\"sec3\"><label>3</label><title>RESULTS</title><p>The Study Sample (SS) consists of 62 people, including 22 females (35.5%) and 40 males (65.5%). The socio-demographic and clinical-oncological characteristics of the study sample are illustrated in Table <bold><xref rid=\"T1\" ref-type=\"table\">1</xref></bold>. The mean age of the sample for the SS was 57.23±16.5 years and 57.40±15.5 years for the CS. In the SS the average of the SF-12 total score was 32.60±5.43; in the CS the average of the SF-12 total score is 38.04±5.87. HRQoL in patients diagnosed with a haematological cancer was found significantly worse in comparison with the general population (F= 43.853, p <0.00001, df 1, 308.309). The impact on HRQoL of the burden attributable to the cancer was measured in 5.44 ± 2.93 points on the SF-12 scale without differences between genders(5.19±3.18 in females and 5.53±3.02 in males, p=0.6).</p><p>The HRQoL profile in patients diagnosed with a haematological cancer was comparable to that with solid tumour (F = 0.770; df= 1,211,212; p= 0.381) and other chronic diseases such as Major Depression (F= 0.255; df= 1.95.96; p= 0.615), and Carotid Atherosclerosis (F= 0.115; df= 1.106.107; p= 0.735). The impact on HRQoL was significantly greater than that due to Celiac disease (F= 67.959; df= 1.120.121; p <0.00001) and Wilson's disease (F= 5.623; df= 1.83.84; p= 0.02) and significantly lower than that due to Multiple Sclerosis (F= 4,673; df= 1,261.9262; p= 0.032) (Table <bold><xref rid=\"T2\" ref-type=\"table\">2</xref></bold>).</p></sec><sec sec-type=\"discussion\" id=\"sec4\"><label>4</label><title>DISCUSSION</title><p>This study suggests that patients with haematological cancers have significantly worse HRQoL outcomes than their peers in the general population [<xref rid=\"r9\" ref-type=\"bibr\">9</xref>, <xref rid=\"r10\" ref-type=\"bibr\">10</xref>, <xref rid=\"r24\" ref-type=\"bibr\">24</xref>]. Results are confirmed in males and females although it is recognized that within the general population women have poorer HRQoL [<xref rid=\"r24\" ref-type=\"bibr\">24</xref>]. The results of our study showed that both haematological and solid cancers have a similar impact on the patient HRQoL, along with other chronic diseases such as Major Depression, and Carotid Atherosclerosis; a significantly higher impact of haematological cancers on HRQoL was reported compared with Celiac and Wilson's disease but lower compared with Multiple Sclerosis [<xref rid=\"r26\" ref-type=\"bibr\">26</xref>-<xref rid=\"r28\" ref-type=\"bibr\">28</xref>].</p><p>In this regard, it must be taken into consideration that the \"raw\" score on the respective SF-12 scale is comparable among cases and controls but not among pathologies since the different gender and age distribution as well as other risk factors, such as schooling and dissimilar comorbid conditions can have an impact on the outcomes. For example, atherosclerosis is classed as a disease of aging, such that increasing age is a risk factor for the development of atherosclerosis. Nonetheless, comparing SF12 scores across different diseases and the respective age and gender controls scores could be considered correct.</p><p>It is thus important to take into account all comorbidities and their relative effect on quality of life state when we consider specific conditions. It is also important to ensure an accurate calculation of the \"Attributable Burden\" of a specific diagnosis. An example is that about 30% of patients with cardiovascular diseases experience an excess of mood disturbances [<xref rid=\"r24\" ref-type=\"bibr\">24</xref>]. Instead, if these conditions are not typically related to the diagnosis but intrinsic to the population characteristics, therefore independent from the diagnosis itself, the effect is cancelled by matching gender and age variables. The calculation of the burden of the disease allows a comparison across different pathologies.</p><p>In this respect, we reported that in a sample of patients with haematological cancers, in which the vast majority have a course disease of more than one year and advanced stage, the patients share the same HRQoL profile as in the case of other chronic diseases. Interestingly, lower HRQoL was reported when compared with patients with multiple sclerosis although patients with haematological cancers experience a prolonged expectancy of life. This indicates that the current therapeutic regime to treat haematological cancers not only lead to a better survival rate but also improve acceptance and quality of life.</p><p>This study has limitations. One limitation is relative to the observational nature of cross-sectional study that does not permit estimating the perceived HRQoL differences over time as instead longitudinal study would permit. Also, the small sample size decreases the power of the difference between HRQoL and both socio-demographic and clinical-oncological anamnestic variables. Furthermore, SF-12 questionnaire, as health generic scale, may not express adequately the changes across different pathological conditions examined in this study. Nevertheless, considering the paucity of HRQoL reports in hematological malignancies, this study contributes to new insight in this field.</p></sec><sec sec-type=\"conclusions\"><title>CONCLUSION</title><p>This study suggests that haematological cancers negatively affect overall HRQoL. The patients diagnosed with haematological cancer have poorer perceived HRQoL than their peers in the general population.</p><p>The results showed an impact of haematological cancers on HRQoL that is similar to what found in patients with solid tumors, Major Depression and Carotid Atherosclerosis. Differently, the impact of haematological cancer on HRQoL compared with other strongly disabling disorders such as multiple sclerosis was reported to be lower.</p><p>Current successful therapeutic strategy achieved in the treatment of haematological cancers not only positively impacts on survival rate but could also improve the overall HRQoL.</p></sec></body><back><ack><title>ACKNOWLEDGEMENTS</title><p>We are deeply grateful to the patients who participated in this study.</p></ack><glossary><title>LIST OF ABBREVIATIONS</title><def-list><def-item><term>HRQoL</term><def><p> = Health Related Quality of Life</p></def></def-item><def-item><term>SS</term><def><p> = Study Sample</p></def></def-item><def-item><term>CS</term><def><p> = Control Sample</p></def></def-item><def-item><term>CTX</term><def><p> = Common Toxicities Criteria</p></def></def-item></def-list></glossary><sec sec-type=\"competing-interests\"><title>ETHICS APPROVAL AND CONSENT TO PARTICIPATE</title><p>The study was approved by the ethics committee of the Italian National Health Institute, Rome (“Istituto Superiore della Sanità”).</p></sec><sec sec-type=\"competing-interests\"><title>HUMAN AND ANIMAL RIGHTS</title><p>No animals were used in this research. All human research procedures followed were in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2013.</p></sec><sec sec-type=\"competing-interests\"><title>CONSENT FOR PUBLICATION</title><p>Each participant was provided with full descriptions of the aims and procedures of the study before signing the informed consent form. Participants were also made aware of data protection and ensured that they could terminate the interview at any time.</p></sec><sec sec-type=\"financial-disclosure\"><title>AVAILABILITY OF DATA AND MATERIALS</title><p>Data are available in the medical charts at Ematologia, Ospedale Businco, Cagliari, Italy.</p></sec><sec sec-type=\"competing-interests\"><title>FUNDING</title><p>None</p></sec><sec sec-type=\"competing-interests\"><title>CONFLICT OF INTEREST</title><p>The author declares no conflict of interest, financial or otherwise.</p></sec><ref-list><title>REFERENCES</title><ref id=\"r1\"><label>1</label><element-citation publication-type=\"web\"><source>Benvenuti nel sito dell’AIRTUM | Associazione Italiana Registri Tumori</source><comment>Available from: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.registri-tumori.it/cms/\">https://www.registri-tumori.it/cms/</ext-link></comment></element-citation></ref><ref id=\"r2\"><label>2</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scalzulli</surname><given-names>E.</given-names></name><name><surname>Molica</surname><given-names>M.</given-names></name><name><surname>Alunni</surname><given-names>F.D.</given-names></name><name><surname>Colafigli</surname><given-names>G.</given-names></name><name><surname>Rizzo</surname><given-names>L.</given-names></name><name><surname>Mancini</surname><given-names>M.</given-names></name><name><surname>Efficace</surname><given-names>F.</given-names></name><name><surname>Latagliata</surname><given-names>R.</given-names></name><name><surname>Fo?�</surname><given-names>R.</given-names></name><name><surname>Breccia</surname><given-names>M.</given-names></name></person-group><article-title>Identification of predictive factors for overall survival at baseline and during azacitidine treatment in high-risk myelodysplastic syndrome patients treated in the clinical practice.</article-title><source>Ann. 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The toxicity of treatment was scored from 1 (mild) to 5 (death), according to common toxicities criteria (CTC), version 4.0 *data not available</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th rowspan=\"1\" scope=\"colgroup\" align=\"center\" valign=\"top\" colspan=\"2\">\n<bold>VARIABLES</bold>\n</th><th colspan=\"1\" rowspan=\"1\" scope=\"col\" align=\"center\" valign=\"top\">\n<bold>(N°)</bold>\n</th><th colspan=\"1\" rowspan=\"1\" scope=\"col\" align=\"center\" valign=\"top\">\n<bold>(%)</bold>\n</th></tr></thead><tbody><tr><td colspan=\"1\" scope=\"row\" align=\"center\" valign=\"top\" rowspan=\"4\"><bold>Marital status</bold></td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">Single</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">17</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">27.4</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">Married/Living with partner</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">41</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">66.1</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">Divorced</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1.6</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">Widow</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4.8</td></tr><tr><td colspan=\"1\" scope=\"row\" align=\"center\" valign=\"top\" rowspan=\"5\"><bold>Employment status</bold></td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">Housewife</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">9.7</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">Unemployed</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">5</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">8.1</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">Employed</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">19</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">30.6</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">Retired</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">29</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">46.7</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">Student</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4.8</td></tr><tr><td colspan=\"1\" scope=\"row\" align=\"center\" valign=\"top\" rowspan=\"5\"><bold>Education level</bold></td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">Primary school</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6.5</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">Secondary school</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">24</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">38.7</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">High school</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">24</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">38.7</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">University degree</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">8</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">12.9</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">Higher</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3.2</td></tr><tr><td colspan=\"1\" rowspan=\"1\" scope=\"row\" align=\"center\" valign=\"top\"><bold>Outpatient service</bold></td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">Outpatient</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">62</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">78,2</td></tr><tr><td colspan=\"1\" scope=\"row\" align=\"center\" valign=\"top\" rowspan=\"5\"><bold>Follow-up</bold></td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">First admission</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1.6</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\"><6 months</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">7</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">11.3</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">6-12 months</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">7</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">11.3</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">>12 months</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">46</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">74.2</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">NA*</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1.6</td></tr><tr><td colspan=\"1\" scope=\"row\" align=\"center\" valign=\"top\" rowspan=\"5\"><bold>Cancer stage</bold></td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1.6</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">2</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">7</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">11.3</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">3</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">9.7</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">4</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">44</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">71</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">NA*</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6.5</td></tr><tr><td colspan=\"1\" scope=\"row\" align=\"center\" valign=\"top\" rowspan=\"6\"><bold>Toxicity of treatments</bold></td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1.6</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">2</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">5</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">8.1</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">3</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">14</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">22.6</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">4</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">25</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">40.3</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">5</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">13</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">21</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">NA*</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6.5</td></tr><tr><td colspan=\"1\" scope=\"row\" align=\"center\" valign=\"top\" rowspan=\"4\"><bold>Intent of treatment</bold></td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">Curative</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">31</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">50</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">maintenance</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">22</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">35.5</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">Supportive</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">5</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">8.1</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">NA*</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6.5</td></tr><tr><td colspan=\"1\" scope=\"row\" align=\"center\" valign=\"top\" rowspan=\"5\"><bold>Response to treatment</bold></td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">Complete response</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">9.7</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">Partial response</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">27</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">43.5</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">Progression</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4.8</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">Ongoing evaluation</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">22</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">35.5</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">NA*</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6.5</td></tr><tr><td colspan=\"1\" scope=\"row\" align=\"center\" valign=\"top\" rowspan=\"4\"><bold>Adherenceat 3 months of follow-up</bold></td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">Yes</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">54</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">87.1</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">No</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">1.6</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">Not evaluated</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">3</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4.8</td></tr><tr><td rowspan=\"1\" scope=\"row\" align=\"center\" colspan=\"1\" valign=\"top\">NA*</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6.5</td></tr></tbody></table></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><title>Attributable burden on HRQoL in patients with onco-hematological disease: comparison with chronic tumors or diseases.</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th colspan=\"1\" rowspan=\"1\" scope=\"col\" align=\"justify\" valign=\"top\">\n<bold>Disease</bold>\n</th><th colspan=\"1\" rowspan=\"1\" scope=\"col\" align=\"center\" valign=\"top\">\n<bold>SF-12 score</bold>\n<break/>\n<bold>Average±sd</bold>\n</th><th colspan=\"1\" rowspan=\"1\" scope=\"col\" align=\"center\" valign=\"top\">\n<bold>Attributable Burden on HRQoL</bold>\n</th><th colspan=\"1\" rowspan=\"1\" scope=\"col\" align=\"center\" valign=\"bottom\">\n<bold>Comparison with Onco-hematologic Disease</bold>\n<break/>\n<bold>ANOVA 1 way</bold>\n</th><th colspan=\"1\" rowspan=\"1\" scope=\"col\" align=\"center\" valign=\"top\">\n<bold>p</bold>\n</th></tr></thead><tbody><tr><td colspan=\"1\" rowspan=\"1\" scope=\"row\" align=\"center\" valign=\"bottom\">Major Depression<break/>(N=37)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">33.8±9.2</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">5.6±3.6</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">df 1,95,96<break/>F=0.255</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0.615</td></tr><tr><td colspan=\"1\" rowspan=\"1\" scope=\"row\" align=\"center\" valign=\"bottom\">Multiple Sclerosis<break/>(N=201)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">29.5± 7.3</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">7.0±3.5</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">df 1,261,9262<break/>F=4.673</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"><bold>0.032</bold></td></tr><tr><td colspan=\"1\" rowspan=\"1\" scope=\"row\" align=\"center\" valign=\"top\">Wilson Disease<break/>(N=23)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">33.8±9.0</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4.4±1.7</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"bottom\">df 1,83,84<break/>F=5.623</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"><bold>0.020</bold></td></tr><tr><td colspan=\"1\" rowspan=\"1\" scope=\"row\" align=\"center\" valign=\"top\">Carotidal atherosclerosis<break/>(N=46)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">30.6±8.1</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">6.2±5.0</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">df 1,106,107<break/>F=0.115</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0.735</td></tr><tr><td colspan=\"1\" rowspan=\"1\" scope=\"row\" align=\"center\" valign=\"top\">Celiac disease<break/>(N=60)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">35.83±5.72</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">2.4±1.0</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">df 1,120,121<break/>F= 67.959</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"><bold><0.00001</bold></td></tr><tr><td colspan=\"1\" rowspan=\"1\" scope=\"row\" align=\"center\" valign=\"top\">Solid tumors<break/>(N=151)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">32.34±6.764</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">4.67±6.64</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">df 1,211,212<break/>F= 0.770</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">0.381</td></tr><tr><td colspan=\"1\" rowspan=\"1\" scope=\"row\" align=\"center\" valign=\"top\">Onco-hematological tumors<break/>(N=62)</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">32.60±5.43</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\">5.44±2.93</td><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/><td colspan=\"1\" rowspan=\"1\" align=\"center\" valign=\"top\"/></tr></tbody></table></table-wrap></floats-group></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"discussion\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Mol Biosci</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Mol Biosci</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Mol. Biosci.</journal-id><journal-title-group><journal-title>Frontiers in Molecular Biosciences</journal-title></journal-title-group><issn pub-type=\"epub\">2296-889X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850958</article-id><article-id pub-id-type=\"pmc\">PMC7431683</article-id><article-id pub-id-type=\"doi\">10.3389/fmolb.2020.00165</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Molecular Biosciences</subject><subj-group><subject>Opinion</subject></subj-group></subj-group></article-categories><title-group><article-title>COVID-19: Looking Into the Overlooked</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Petersen</surname><given-names>Fernanda Cristina</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/338482/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Dahle</surname><given-names>Ulf Reidar</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1012228/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Nicolau</surname><given-names>Belinda</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Casals-Pascual</surname><given-names>Climent</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>University of Oslo</institution>, <addr-line>Oslo</addr-line>, <country>Norway</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Centre for Antimicrobial Resistance, Norwegian Institute of Public Health</institution>, <addr-line>Oslo</addr-line>, <country>Norway</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Faculty of Dentistry, McGill University</institution>, <addr-line>Montreal, QC</addr-line>, <country>Canada</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Laboratorio de Microbiología, Centro de Diagnóstico Biomédico, Hospital Clínic de Barcelona</institution>, <addr-line>Barcelona</addr-line>, <country>Spain</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Hospital Clínic de Barcelona</institution>, <addr-line>Barcelona</addr-line>, <country>Spain</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Paola Patrignani, University of Studies G. d'Annunzio Chieti and Pescara, Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Stefania Tacconelli, University of Studies G. d'Annunzio Chieti and Pescara, Italy; Uma Gaur, University of Macau, China</p></fn><corresp id=\"c001\">*Correspondence: Fernanda Cristina Petersen <email>f.c.petersen@odont.uio.no</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Molecular Diagnostics and Therapeutics, a section of the journal Frontiers in Molecular Biosciences</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>11</day><month>8</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>7</volume><elocation-id>165</elocation-id><history><date date-type=\"received\"><day>27</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Petersen, Dahle, Nicolau and Casals-Pascual.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Petersen, Dahle, Nicolau and Casals-Pascual</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><kwd-group><kwd>COVID- 19</kwd><kwd>microbiome</kwd><kwd>antibiotic resistance</kwd><kwd>SARS-CoV- 2</kwd><kwd>biomarker</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Norges Forskningsråd<named-content content-type=\"fundref-id\">10.13039/501100005416</named-content></funding-source><award-id rid=\"cn001\">273833</award-id><award-id rid=\"cn001\">274867</award-id></award-group></funding-group><counts><fig-count count=\"1\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"24\"/><page-count count=\"4\"/><word-count count=\"2470\"/></counts></article-meta></front><body><p>Viral infections have plagued humanity since the beginning of time causing numerous deaths through specific pathogenic events directly related to the virus and indirectly, through secondary infections accompanying or following the viral episode. Highly pathogenic bacteria, such as <italic>Neisseria meningitidis, Streptococcus pneumoniae</italic>, and <italic>Haemophilus influenzae</italic> can overgrow during viral infections, and cause severe bacterial infections. During the 1918 Spanish influenza pandemic, for instance, the majority of influenza fatalities were likely caused by secondary pneumococcal pneumonia (Morens et al., <xref rid=\"B14\" ref-type=\"bibr\">2008</xref>), but we were not aware of it until 2008, when tissue biopsy investigations revealed this important association. Secondary bacterial infections also accounted for 25–50% of deaths during the 2009 H1N1 influenza pandemic (Macintyre et al., <xref rid=\"B10\" ref-type=\"bibr\">2018</xref>). In this instance, the association with secondary infections, particularly with <italic>S. pneumoniae</italic>, was acknowledged only almost 10 years after the pandemic.</p><p>In the current COVID-19 pandemic, the role of co-infectiveness remains unclear. At the peak of the pandemic, broad-spectrum antibiotics have been administered to the majority of patients admitted with COVID-19 to hospital to prevent secondary infections but also, some antibiotics, like teicoplanin, have been used due to their alleged antiviral properties. In an early report, secondary infections were detected in 1 out of 7 patients. Among those that died, 50% had secondary infections (Zhou F. et al., <xref rid=\"B23\" ref-type=\"bibr\">2020</xref>). Other reports indicate that bacterial infections were documented in <10% of COVID-19 patients (Zhou P. et al., <xref rid=\"B24\" ref-type=\"bibr\">2020</xref>). In light of this uncertainty, antibiotic use has been reported to be as high as 74% among patients with COVID-19 who were admitted to ICUs (Cox et al., <xref rid=\"B1\" ref-type=\"bibr\">2020</xref>). Some infections may have been caused by changes in colonization resistance, others due to increase use of corticosteroids, parenteral nutrition or just intravenous catheter infections. The administration of broad-spectrum antibiotics in the absence of high suspicion or documented infection challenges the current dogma of antimicrobial stewardship. This problem is further compounded by the fact that acute respiratory syndromes due to infection or non-infection are notoriously difficult to discriminate. However, the risk and potential severity of secondary infections associated with COVID-19, particularly in overcrowded clinical settings and in immune-compromised patients, has in many cases reduced the compliance with the local prescription practice guidelines.</p><p>A quick look at host factors may now be more relevant than ever. As the first wave of the pandemic is tailing off in Europe, we have gathered substantial and actionable evidence that suggests that both severity and mortality associated with COVID-19 is due to host factors, ranging from endothelial and coagulation disturbances leading to the formation of micro thrombi to a major dysregulation of the host immunity and inflammatory response. Here, a recent study has shown significant survival benefits of corticosteroids administration to patients with severe COVID-19 infection.</p><p>How is the host contributing to the severity of COVID-19? It was a matter of time that a genome wide analysis study (GWAS) showed some association with disease severity. Indeed, we have recently learnt that particular blood groups (group A) may be linked to respiratory failure in COVID-19 patients (Ellinghaus et al., <xref rid=\"B3\" ref-type=\"bibr\">2020</xref>). However, with a moderate increase severity risk (odds ratio: 1.45 with respect to other blood groups), it is unlikely that this association explain all the variability of disease severity. Here we should not forget that one of the major contributors that regulate individual immune/inflammatory responses is the human microbiome. In particular, the gut microbiome may influence directly and indirectly the immune response generated. Accordingly, the lymphocyte populations may adopt a more pro-inflammatory phenotype when microbiome diversity is reduced, as typically observed in old age patients (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>) and recently reviewed by Dhar and Mohanty (<xref rid=\"B2\" ref-type=\"bibr\">2020</xref>).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Host-microbiota-virus triumvirate. Individual microbiome taxonomic profile and metabolic capacities may affect the balance between pro- and anti- inflammatory responses, and between suppression or enhancement of viral dissemination.</p></caption><graphic xlink:href=\"fmolb-07-00165-g0001\"/></fig><p>Broad spectrum antibiotics are well-known to promote major disturbances in the microbiome. For critically ill patients, secondary infections and lack of response to antibiotics may be all that is necessary to shift the balance toward irreversible clinical deterioration and death. Also for some patients, antibiotics may do more harm than good. At least two of the antibiotics that are being used for treatment or prophylaxis in COVID-19 patients, ceftriaxone and teicoplanin (Martinez, <xref rid=\"B11\" ref-type=\"bibr\">2020</xref>), have been known since the 1990s to promote a substantial increase in the population of resistant enterococci in the gut microbiota (Meijer-Severs et al., <xref rid=\"B13\" ref-type=\"bibr\">1990</xref>; Van Der Auwera et al., <xref rid=\"B21\" ref-type=\"bibr\">1996</xref>). Azithromycin is another antibiotic that, like teicoplanin, has been repurposed for use in COVID-19 patients due to its possible anti-SARS-CoV2 activity. Although there are few studies focusing on microbiota disturbances by azithromycin in adults, there are reports of macrolide resistance enrichment and reduction in diversity among patients with asthma (Taylor et al., <xref rid=\"B18\" ref-type=\"bibr\">2019</xref>). Examples of promotion and suppression of viral infections in combination with antibiotics have been studied mostly in animal models. In humans, seminal studies have shown the impact on antibody response by approaches using antibiotic depletion of the microbiota in combination with live viral vaccines (Harris et al., <xref rid=\"B5\" ref-type=\"bibr\">2018</xref>; Hagan et al., <xref rid=\"B4\" ref-type=\"bibr\">2019</xref>).</p><p>The broad use of antibiotics during COVID-19 is <italic>per se</italic> an important reason to monitor changes in the microbiota of the respiratory or gastrointestinal tract. Moreover, in COVID-19, there are a number of striking observations in the course of the disease that we cannot yet explain, like healthy patients with minor or no co-morbidities that suddenly become irreversibly ill (Team, <xref rid=\"B19\" ref-type=\"bibr\">2020</xref>). There are also more and more reports of patients cured with negative PCRs and positive antibodies that later on develop severe thrombotic events with reactivation of the virus (Oxley et al., <xref rid=\"B15\" ref-type=\"bibr\">2020</xref>). Inflammatory responses are exacerbated in several cases, as indicated by cytokine storms. Understanding the link between microbiome, immunity and inflammatory response in SARS-CoV-2 infected patients could help explaining such unexpected outcomes. A plethora of mechanisms by which the gut microbiota modulates the immune system have been described in the literature, and reviewed in several articles (Maynard et al., <xref rid=\"B12\" ref-type=\"bibr\">2012</xref>; Honda and Littman, <xref rid=\"B6\" ref-type=\"bibr\">2016</xref>; Levy et al., <xref rid=\"B7\" ref-type=\"bibr\">2016</xref>). One relevant example are short chain fatty acids provided by the gut microbiota, which have a critical role in promoting expansion and differentiation of regulatory T cells (Tregs), thus impacting maintenance of immune homeostasis (Tanoue et al., <xref rid=\"B17\" ref-type=\"bibr\">2016</xref>). In the airways, Tregs induced by short chain fatty acids contribute among other effects, to suppression of lung inflammation (Trompette et al., <xref rid=\"B20\" ref-type=\"bibr\">2014</xref>). Both pro- and anti- inflammatory stimulation, and responses promoting or suppressing viral infections have been reported as directly modulated by members of the microbiota (Li et al., <xref rid=\"B8\" ref-type=\"bibr\">2019</xref>). Of note, old age and comorbidities such as diabetes and cardiovascular diseases, which are linked to COVID-19 severity and mortality are also both associated with dysbiotic states (Peterson et al., <xref rid=\"B16\" ref-type=\"bibr\">2015</xref>; Yang et al., <xref rid=\"B22\" ref-type=\"bibr\">2015</xref>; Li et al., <xref rid=\"B9\" ref-type=\"bibr\">2017</xref>).</p><p>Quantifying microbiota-host imbalances would have the potential to yield novel diagnostics for risk stratification and improved clinical management. However, implementing such clinical tools within a reasonable time frame depends on collecting this information from patients now. International consortia and multicenter studies have in record time organized initiatives to create and explore existing biobanks for COVID-19, as to understand the diversity in disease manifestations. Several risk indexes are created or combined with those commonly used in ICUs, and numerous initiatives to identify human genetic factors, antibody responses, and viral changes are underway, with virtually no mention on the microbiota. After two decades of major milestone discoveries in the field, this is somehow surprising. So far, search for COVID-19 biobank initiatives at Biobanks Europe (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.bbmri-eric.eu\">https://www.bbmri-eric.eu</ext-link>) do not retrieve any hits for fecal samples. Actually, as of May 27th 2020, only seven in 1706 registries of COVID-19 studies in the <ext-link ext-link-type=\"uri\" xlink:href=\"https://ClinicalTrials.gov\">ClinicalTrials.gov</ext-link> refers to microbiome or microbiota as an outcome measurement.</p><p><bold>“The art of being wise is the art of knowing what to overlook”- William James, 1890</bold></p><p>Focusing either on the host or on the virus during pandemics may be the wise way to go when resources are scarce and priorities must be established. On the other hand, the history of human infections, and the accumulated knowledge on the interplay of the human microbiota and the immune system indicate that we may be overlooking one of the three core elements in the present COVID-19 pandemic. With COVID-19 varying in severity from asymptomatic to lethal, the microbiota could provide valuable biomarkers to predict which individuals are most at risk of suffering severe disease.</p><sec id=\"s1\"><title>Author Contributions</title><p>FP wrote the paper with inputs from all authors. All authors conceived the presented ideas and approved the final manuscript.</p></sec><sec id=\"s2\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by a grant from the Norwegian Research Council (Grant numbers−273833 and 274867), and the Olav Thon Foundation. BN holds a Canada Research Chair in Life Course Oral Epidemiology.</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cox</surname><given-names>M. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Clin Pract Epidemiol Ment Health</journal-id><journal-id journal-id-type=\"iso-abbrev\">Clin Pract Epidemiol Ment Health</journal-id><journal-id journal-id-type=\"publisher-id\">CPEMH</journal-id><journal-title-group><journal-title>Clinical Practice and Epidemiology in Mental Health : CP & EMH</journal-title></journal-title-group><issn pub-type=\"epub\">1745-0179</issn><publisher><publisher-name>Bentham Science Publishers</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32874191</article-id><article-id pub-id-type=\"pmc\">PMC7431684</article-id><article-id pub-id-type=\"publisher-id\">CPEMH-16-165</article-id><article-id pub-id-type=\"doi\">10.2174/1745017902016010165</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Clinical Practice Epidemiology in Mental Health</subject></subj-group></article-categories><title-group><article-title>PTSD and Burnout are Related to Lifetime Mood Spectrum in Emergency Healthcare Operator</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Carmassi</surname><given-names>Claudia</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Bertelloni</surname><given-names>Carlo Antonio</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref><xref ref-type=\"corresp\" rid=\"cor1\">*</xref></contrib><contrib contrib-type=\"author\"><name><surname>Avella</surname><given-names>Maria Teresa</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Cremone</surname><given-names>Ivan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Massimetti</surname><given-names>Enrico</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Corsi</surname><given-names>Martina</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Dell’Osso</surname><given-names>Liliana</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref></contrib><aff id=\"aff1\">\n<label>1</label>Department of Clinical and Experimental Medicine, <institution>University of Pisa</institution>, <addr-line><city>Pisa</city></addr-line>, <country>Italy</country></aff><aff id=\"aff2\">\n<label>2</label>ASST, Bergamo Ovest, SSD Servizio Psichiatrico diagnosi e cura, Treviglio, <country>Italy</country></aff></contrib-group><author-notes><corresp id=\"cor1\"><label>*</label>Address correspondence to this author at the Department of Clinical and Experimental Medicine, University of Pisa, Via Roma 67, 56100 Pisa, Italy; Tel: +39-050-2219760; Fax: +39-050-2219787; E-mail: <email xlink:href=\"carlo.ab@hotmail.it\">carlo.ab@hotmail.it</email></corresp></author-notes><pub-date pub-type=\"epub\"><day>30</day><month>7</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>16</volume><fpage>165</fpage><lpage>173</lpage><history><date date-type=\"received\"><day>12</day><month>2</month><year>2020</year></date><date date-type=\"rev-recd\"><day>21</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>28</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 Carmassi <italic>et al</italic>.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Carmassi</copyright-holder><license license-type=\"open-access\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/legalcode\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: <uri xlink:href=\"https://creativecommons.org/licenses/by/4.0/legalcode\">https://creativecommons.org/licenses/by/4.0/legalcode</uri>. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract><sec><title>Background:</title><p>PTSD and burnout are frequent conditions among emergency healthcare personnel because exposed to repeated traumatic working experiences. Increasing evidence suggests high comorbidity between PTSD and mood symptoms, particularly depression, although the real nature of this relationship still remains unclear. The purpose of this study was to investigate the relationship between PTSD, burnout and lifetime mood spectrum, assessed by a specific scale, among health-care professionals of a major University Hospital in Italy.</p></sec><sec><title>Methods:</title><p>N=110 Emergency Unit workers of the Azienda Ospedaliero-Universitaria Pisana (Pisa, Italy) were assessed by the TALS-SR, MOODS-SR lifetime version and the ProQOL R-IV.</p></sec><sec><title>Results:</title><p>Approximately 60% of participants met at least one PTSD symptom criterion (criterion B, 63.4%; criterion C, 40.2%; criterion D 29.3%; criterion E, 26.8%), according to DSM-5 diagnosis. Almost sixteen percent of the sample reported a full symptomatic DSM-5 PTSD (work-related) diagnosis, and these showed significantly higher scores in all MOODS-SR depressive domains, as well as in the rhythmicity domain, compared with workers without PTSD. Further, mood-depressive and cognition-depressive MOODS-SR domains resulted to be predictive for PTSD. Significant correlations emerged between either PTSD diagnosis and criteria or ProQOL subscales and all the MOOD-SR domains.</p></sec><sec><title>Conclusion:</title><p>A significant association emerged among PTSD, burnout and lifetime MOOD Spectrum, particularly the depressive component, in emergency health care operators, suggesting this population should be considered at-risk and undergo regular screenings for depression and PTSD.</p></sec></abstract><kwd-group kwd-group-type=\"author\"><title>Keywords</title><kwd>Trauma</kwd><kwd>Emergency</kwd><kwd>Health operators</kwd><kwd>Nurse</kwd><kwd>PTSD</kwd><kwd>Burnout</kwd><kwd>Depression</kwd><kwd>Mood</kwd></kwd-group></article-meta></front><body><sec sec-type=\"intro\" id=\"sec1\"><label>1</label><title>INTRODUCTION</title><p>Healthcare personnel are frequently and repeatedly exposed to stressful situations and traumatic events so they are more likely to develop pathological conditions such as burnout or Post-Traumatic Stress Disorder (PTSD), compared to the general population [<xref rid=\"r1\" ref-type=\"bibr\">1</xref>-<xref rid=\"r3\" ref-type=\"bibr\">3</xref>]. In light of these data, the last edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) significantly modified the diagnostic criteria related to the trauma for the PTSD diagnosis, specifying in Criterion A4 the impact of “experiencing repeated or extreme exposure to aversive details of the traumatic event(s)” addressing special populations such as emergency operators [<xref rid=\"r4\" ref-type=\"bibr\">4</xref>]. Previous studies reported PTSD rates up to ten-fold higher among emergency service workers with respect to the general population [<xref rid=\"r5\" ref-type=\"bibr\">5</xref>]. Particularly, PTSD rates range between 10% and 21% among medical doctors and nurses [<xref rid=\"r6\" ref-type=\"bibr\">6</xref>, <xref rid=\"r7\" ref-type=\"bibr\">7</xref>]. A recent meta-analysis on 18 studies including over 30,000 subjects showed PTSD in 11% of ambulance personnel [<xref rid=\"r8\" ref-type=\"bibr\">8</xref>].</p><p>Several studies described also a frequent occurrence of burnout among health-care professionals, in particular among emergency unit operators with rates as high as 86% [<xref rid=\"r2\" ref-type=\"bibr\">2</xref>, <xref rid=\"r9\" ref-type=\"bibr\">9</xref>]. Burnout of nurses and physicians in the emergency setting was favored by the stress of emotionally-charged decision-making as well as heavy workloads and extended work hours [<xref rid=\"r10\" ref-type=\"bibr\">10</xref>]. On one hand, burnout can affect physical and mental health, causing sleep pattern alteration, fatigue, concentration deficit and irritability; on the other hand, it can produce impairment in quality of care and patients’ health outcomes, besides increased absenteeism [<xref rid=\"r11\" ref-type=\"bibr\">11</xref>].</p><p>Considering the very high frequency of PTSD and burnout in emergency operators, and the detrimental impact of these syndromes not only on well-being but also on work-related functioning and patients outcome [<xref rid=\"r12\" ref-type=\"bibr\">12</xref>, <xref rid=\"r13\" ref-type=\"bibr\">13</xref>], detecting individuals at risk is essential in order to develop effective prevention strategies. Different factors were found to predispose health workers to PTSD or burnout. From a socio-demographic perspective, being a young, female, nurse with low professional education levels are considered negative factors [<xref rid=\"r2\" ref-type=\"bibr\">2</xref>, <xref rid=\"r6\" ref-type=\"bibr\">6</xref>, <xref rid=\"r14\" ref-type=\"bibr\">14</xref>]. For what concerns organizational work factors, employee reward systems, peer and manager support, besides well-designed organizational structures improve resilience to stressful and traumatic events experienced on duties [<xref rid=\"r15\" ref-type=\"bibr\">15</xref>]. Moreover, some operator’s psychological characteristics or pre-existing psychiatric disorders are related to a higher risk. In this regard, a great amount of literature shows a strict but controversial relationship between mood disorders, particularly depression, and trauma and stress-related disorders [<xref rid=\"r16\" ref-type=\"bibr\">16</xref>-<xref rid=\"r18\" ref-type=\"bibr\">18</xref>].</p><p>PTSD and depressive symptoms show a high tendency to co-occur after a traumatic event [<xref rid=\"r19\" ref-type=\"bibr\">19</xref>, <xref rid=\"r20\" ref-type=\"bibr\">20</xref>] and in epidemiological samples about 50% of subjects with PTSD also reported a depressive disorder [<xref rid=\"r18\" ref-type=\"bibr\">18</xref>, <xref rid=\"r21\" ref-type=\"bibr\">21</xref>, <xref rid=\"r22\" ref-type=\"bibr\">22</xref>]. According to a French study based on a large population sample, one-third of patients with moderately PTSD showed comorbid depressive symptoms. This percentage was higher in those with severe PTSD, who presented comorbid depression in more than half of cases [<xref rid=\"r23\" ref-type=\"bibr\">23</xref>]. Furthermore, another study reported higher comorbidity rates between PTSD and Major Depressive Disorder (84.4%) or Dysthimia (41.9%). In particular, female gender and childhood sexual or physical abuse were positively associated with depressive comorbidity in patients with PTSD [<xref rid=\"r24\" ref-type=\"bibr\">24</xref>]. For what concern emergency personnel, the prevalence of PTSD, depression, and high general psychological distress was higher among medical workers (6.6%, 14.3%, and 14.5%, respectively) than among other professional categories such as firefighters (1.6%, 3.8%, and 2.6%, respectively) [<xref rid=\"r25\" ref-type=\"bibr\">25</xref>]. On the whole, PTSD-depression comorbidity is usually associated with a higher functional impairment than a single disorder [<xref rid=\"r26\" ref-type=\"bibr\">26</xref>].</p><p>These high comorbidity rates have various explanations. According to the causality model, PTSD is a causal risk factor for the onset of depressive disorders. Prospective studies on large Veteran samples found that PTSD diagnosis at the basal evaluation was a predictive factor for depression in a 24-month follow-up [<xref rid=\"r27\" ref-type=\"bibr\">27</xref>]. Data on civilian sample corroborate the hypothesis that post-traumatic stress symptomatology could be a risk factor for the onset of depression [<xref rid=\"r16\" ref-type=\"bibr\">16</xref>, <xref rid=\"r21\" ref-type=\"bibr\">21</xref>, <xref rid=\"r28\" ref-type=\"bibr\">28</xref>, <xref rid=\"r29\" ref-type=\"bibr\">29</xref>]. In contrast, other authors suggested that pre-existing depression could predispose to PTSD after a traumatic event [<xref rid=\"r30\" ref-type=\"bibr\">30</xref>-<xref rid=\"r35\" ref-type=\"bibr\">35</xref>]. In a study on 6,744 male twins from the Vietnam Era Twin Registry, the risk of developing PTSD after trauma exposure was increased in those with pre-existing depressive or anxiety disorders [<xref rid=\"r30\" ref-type=\"bibr\">30</xref>]. Similarly, Shiller-Allon <italic>et al.</italic>, reported that depressive symptoms were related to an increase in post-traumatic distress in a sample of 156 Israeli trauma victims followed-up for 12 weeks after being exposed to trauma [<xref rid=\"r31\" ref-type=\"bibr\">31</xref>].</p><p>Exploring, upon <italic>Mood Spectrum Model</italic> approach, the relationship between lifetime mood spectrum symptoms and PTSD, some of us reported manic and depressive lifetime symptoms to be related to the severity of post-traumatic stress symptomatology and suicidality [<xref rid=\"r32\" ref-type=\"bibr\">32</xref>-<xref rid=\"r35\" ref-type=\"bibr\">35</xref>]. These findings support the hypothesis that depressive symptoms may predispose to PTSD after the exposure of a traumatic event.</p><p>A positive correlation between burnout and depression represents a corroborated finding [<xref rid=\"r36\" ref-type=\"bibr\">36</xref>, <xref rid=\"r37\" ref-type=\"bibr\">37</xref>]. Three longitudinal studies specifically described a direct link from depression to burnout [<xref rid=\"r38\" ref-type=\"bibr\">38</xref>-<xref rid=\"r40\" ref-type=\"bibr\">40</xref>]. In a long longitudinal study on 297 university students, Salmela-Aro <italic>et al.</italic> found that participants with a higher depression trajectory showed higher burnout levels in the long period with respect to those with lower depression, suggesting that depression may be a risk factor for burnout [<xref rid=\"r40\" ref-type=\"bibr\">40</xref>].</p><p>Considering the scant data about the role of depressive symptoms as vulnerability factors of post-traumatic stress reactions among emergency personnel, the purpose of this study was to evaluate lifetime mood spectrum symptoms in emergency personnel and their association with PTSD and burnout in order to identify a subject at risk for developing these conditions. To specifically investigating affective symptoms during the lifespan of the participants, a <italic>mood spectrum model</italic> was adopted for this study. There is growing evidence that mood disorders should be regarded as a spectrum of manifestations, rather than categorical disorders. A <italic>mood spectrum model</italic> is a dimensional approach that evaluates not only unipolar and bipolar symptoms, but also sub-threshold manifestations, atypical symptoms and behavioral traits, in the light of a lifetime perspective [<xref rid=\"r41\" ref-type=\"bibr\">41</xref>]. This approach was found to be a valid method to better define mood disorders in terms of severity, prognosis, treatment implications and relationship with other symptoms [<xref rid=\"r41\" ref-type=\"bibr\">41</xref>-<xref rid=\"r43\" ref-type=\"bibr\">43</xref>].</p></sec><sec sec-type=\"materials|methods\" id=\"sec2\"><label>2</label><title>MATERIALS AND METHODS</title><sec id=\"sec2.1\"><label>2.1</label><title>Study Sample</title><p>The study sample included 95 emergency unit workers of the “Azienda Ospedaliero-Universitaria Pisana (AOUP)”, Italy. The participants' enrollment was conducted between September 2013 to March 2014. Full data were available only for 82 participants from the whole sample because the remaining 13 ones (2 males and 11 females; 3 medical doctors, 5 nurses and 5 healthcare assistants) gave partial responses.</p><p>The study was conducted in accordance with the Declaration of Helsinki. The ethics committee of the University of Pisa, Italy, approved all the procedures of evaluation and recruitment. Suitable candidates provided written informed consent after receiving an exhaustive description of the study and after having the opportunity to ask any questions in reference to the study. Participants completed the questionnaires at the end of the work-shift in a stand-alone room to ensure their privacy.</p></sec><sec id=\"sec2.2\"><label>2.2</label><title>Diagnostic Instruments</title><p>Participants were investigated using the Trauma and Loss Spectrum - Self Report (TALS-SR), to investigate PTSD and posttraumatic stress spectrum symptoms related to the work activity [<xref rid=\"r44\" ref-type=\"bibr\">44</xref>, <xref rid=\"r45\" ref-type=\"bibr\">45</xref>], the MOOD Spectrum – Self Report, Lifetime version (MOODS-SR), in order to explore lifetime mood symptomatology [<xref rid=\"r41\" ref-type=\"bibr\">41</xref>] and the Professional Quality of Life Scale - Revision IV (ProQOL R-IV) to examine compassion satisfaction, burn-out and compassion fatigue related to work activities [<xref rid=\"r46\" ref-type=\"bibr\">46</xref>].</p><p>The TALS-SR is a questionnaire developed for assessing posttraumatic stress spectrum symptoms. It includes 116 items exploring the lifetime experience of a range of losses and/or traumatic events and lifetime symptoms, behaviors, and personal characteristics that might represent manifestations and/or risk factors for the development of a stress response syndrome. The instrument is organized into nine domains including <italic>loss events</italic> (I), <italic>grief reactions</italic> (II), <italic>potentially traumatic events</italic> (III), <italic>reactions to losses or upsetting events</italic> (IV), <italic>re-experiencing</italic> (V), <italic>avoidance and numbing</italic> (VI), <italic>maladaptive coping</italic> (VII), <italic>arousal</italic> (VIII), and <italic>personal characteristics/ risk factors</italic> (IX). The responses to the items are coded in a dichotomous way (yes/no), and domain scores are obtained by counting the number of positive answers.</p><p>Due to the sample characteristics, criterion A was considered satisfied. Particularly, the instrument was adapted, in accordance with the aim of the present study, to assess symptoms due to traumatic experience related to the work as emergency personnel (criterion A4).Consequently, only the following TALS-.Sr domains were included in the study: <italic>reactions to losses or upsetting events</italic> (IV), <italic>re-experiencing</italic> (V), <italic>avoidance and numbing</italic> (VI), <italic>maladaptive coping</italic> (VII), <italic>arousal</italic> (VIII). All participants were asked to report symptoms related to work-related trauma exposure. According to previous studies [<xref rid=\"r47\" ref-type=\"bibr\">47</xref>, <xref rid=\"r48\" ref-type=\"bibr\">48</xref>] a DSM-5 diagnosis of PTSD was assessed by using the following matching between DSM-5 symptoms criteria and TALS-SR items:</p><list list-type=\"bullet\" id=\"L1\"><list-item><p>criterion B (B1 =80; B2 =77; B3 =79; B4 =78; B5 =81);</p></list-item><list-item><p>criterion C (C1 =86; C2 =87 and/or 88 and/or 89);</p></list-item><list-item><p>criterion D (D1 =90; D2 =95; D3 =85; D4 =96; D5 =91; D6 =93; D7 =92); and</p></list-item><list-item><p>criterion E (E1 =108; E2 =99 and/or 100 and/or 102; and/or 103 and/or 104; E3 =106; E4 =107; E5 =105; E6 =109).</p></list-item></list><p>The TALS-SR presented good intra-class correlation coefficients (from 0.934 to 0.994) with SCI-TALS; the interview version was used for assessing post-traumatic stress symptomatology. Similarly, SCI-TALS showed a good internal consistency (Kuder-Richardson coefficient exceeding the minimum standard of 0.50 for each domain).</p><p>The MOODS-SR is a 161-item questionnaire coded as present or absent for one or more periods of at least 3-5 days throughout the subject’s lifespan. Not only <italic>manic</italic> and <italic>depressive components</italic> are investigated, but also disturbances in <italic>rhythmicity and vegetative functions</italic> are assessed. Both the manic and the depressive components are subtyped into three domains exploring <italic>mood</italic>, <italic>energy</italic> and <italic>cognition</italic> symptoms respectively. The number of the <italic>mood</italic>-, <italic>energy</italic>- and <italic>cognition-manic</italic> items endorsed by subjects makes up the <italic>manic component</italic> (62 items), while the sum of the <italic>mood</italic>-, <italic>energy</italic>- and <italic>cognition-depressive</italic> items constitutes the <italic>depressive component</italic> (63 items). The <italic>rhythmicity and vegetative functions</italic> domain (29 items) explore alterations in the circadian rhythms and vegetative functions, including changes in energy, physical well-being, mental and physical efficiency related to the weather and season, and changes in appetite, sleep and sexual activities. The questionnaire showed good internal consistency, with Kuder-Richardson's coefficient ranging from 0.79 to0.92 among single domains.</p><p>The ProQOL is a 22 item self-report measure to assess three different dimensions: <italic>compassion satisfaction</italic>, <italic>burnout</italic> and <italic>compassion fatigue</italic> related to work. Respondents were asked to indicate how often (from 0-never to 5-very often) during the last 30 days, each item was experienced [<xref rid=\"r47\" ref-type=\"bibr\">47</xref>-<xref rid=\"r49\" ref-type=\"bibr\">49</xref>]. The <italic>compassion satisfaction</italic> measures pleasure derived from being able to do your work well, whereas high scores represent a greater satisfaction related to your ability to be an effective caregiver. The <italic>burnout</italic> dimension in this scale is associated with feelings of hopelessness and difficulties in dealing with your work. The <italic>compassion fatigue</italic> dimension is about work-related secondary exposure to stressful events. High scores indicate that you are exposed to frightening experiences at work. Cronbach’s alpha values of the three subscales were 0.80 for CF, 0.89 for CS, and 0.71 for BO. The Italian version of the instrument was validated.</p></sec><sec id=\"sec2.3\"><label>2.3</label><title>Statistical Analysis</title><p>All statistical analyses were carried out using the Statistical Package for Social Science, version 23.0 (SPSS Inc., Chicago 2018). The descriptive procedures were used to evaluate the demographic characteristics of the sample as well as to calculate the frequency of the PTSD and the scores of the TALS-SR, MOODS-SR and ProQOL domains. Considering that MOODS-SR domain scores were not normally distributed, the non-parametric Man-Whitney test was computed in order to compare MOODS-SR domains between subjects with PTSD and without PTSD. A logistic regression model was computed to examine the role of the MOODS-SR domains, education level and gender as a predictive variable of PTSD diagnosis, considered as the dependent variable. Spearman's correlation coefficients were calculated to investigate possible associations between the TALS-SR domains or ProQOL dimensions and the MOODS-SR domains.</p></sec></sec><sec sec-type=\"results\" id=\"sec3\"><label>3</label><title>RESULTS</title><p>Most of the participants were nurses (55, 67.1%), followed by health-care assistants (14, 17%) and medical doctors (13, 15.6%, including physicians and residents). Regarding socio-demographic features, 50 (61%) were females and 32 (39%) were males; the mean age was 40.34±8.10 (min 25, max 61), and 59 (72%) were graduated (bachelor’s or advanced degree).</p><p>In the sample, 13 (15.9%) participants were found to be affected by DSM-5 PTSD, while higher percentages of each PTSD criterion were reported: criterion B (re-experiencing) was presented by 50 (63.4%) subjects; criterion C (avoidance) by 33 (40.2%); criterion D (alteration in mood and cognition) by 24 (29.3%); and finally, criterion E (Hyperarousal) by 22 (26.8%) subjects. According to the ProQOL subscales, the mean score of the <italic>compassion satisfaction</italic> was 31.60±5.0, the <italic>burnout</italic> one was 14.89±4.92 and the <italic>compassion fatigue</italic> one was 10.84±4.92. The mean scores of the MOODS-SR depressive domain scores were respectively 4.61±4.59, 1.29±1.64 and 2.73±3.73 for <italic>mood-depressive</italic>, <italic>energy-depressive</italic> and <italic>cognition-depressive</italic>. The <italic>depressive component</italic> score was 8.63±8.66. On the other hand, the scores of the <italic>mood-manic</italic>, <italic>energy-manic</italic> and <italic>cognition-manic</italic> MOODS-SR domain were 6.28±5.12, 2.18±2.38 and 3.34±3.63 respectively, whereas the <italic>manic component</italic> score was 11.80±10.02. Finally, the <italic>rhythmicity</italic> domain mean score was 5.94±5.21.</p><p>Subjects with PTSD reported significantly higher scores in the <italic>mood-depressive</italic> (3.59±3.77 <italic>versus</italic> 10.00±4.90, p<.001), <italic>energy-depressive</italic> (1.12±1.51 <italic>versus</italic> 2.23±2.05, p=.025) <italic>cognition-depressive</italic> (1.83±2.45 <italic>versus</italic> 7.54±5.47, p=.001) and <italic>rhythmicity</italic> (5.1±4.29 <italic>versus</italic> 10.23±7.41, p=.016) domains, besides in the <italic>depressive component</italic> (6.54±6.70 <italic>versus</italic> 19.77±9.56 4.33 p=<.001). (Table <bold><xref rid=\"T1\" ref-type=\"table\">1</xref></bold>)</p><p>In a logistic regression model (sensitivity=53.8% and specificity=95.6%.), considering the gender and education, besides MOODS-SR <italic>mood-depressive</italic>, <italic>energy-depressive</italic>, <italic>cognition-depressive</italic> and <italic>rhythmicity</italic> scores as independent variables, and the PTSD diagnosis as the dependent variables, the <italic>mood-depressive</italic> [b=0.256 (SE=0.108), p=.018] and the <italic>cognition-depressive</italic> [b=0.268 (SE=0.121), p=.026] domains scores presented a significant positive association with the PTSD (Table <bold><xref rid=\"T2\" ref-type=\"table\">2</xref></bold>). The ROC curve and the characteristics of the regression model are shown in Fig. (<bold><xref ref-type=\"fig\" rid=\"F1\">1</xref></bold> and Table <bold><xref rid=\"T2A\" ref-type=\"table\">2a</xref></bold>), respectively.</p><p>Finally, significant correlations emerged between DSM-5 PTSD diagnosis and criteria, or ProQOL subscales and MOOD-SR domains (Table <bold><xref rid=\"T3\" ref-type=\"table\">3</xref></bold>). Particularly, significant positive moderate correlations emerged between the <italic>mood-depressive</italic> MOODS-SR domain and the total PTSD symptoms scores (r=.513) the cognitive and mood alterations criterion symptoms (r=.519) and the hyperarousal criterion symptoms (r=.517); between the <italic>cognition-depressive</italic> MOODS-SR domain and the total PTSD symptoms scores (r=.563) and the cognitive and mood alterations criterion symptoms (r=.560). Furthermore, the MOODS-SR mood-depressive domain showed a significant low positive correlation with ProQOL burnout (r=376) and <italic>compassion fatigue</italic> (r=.338).</p></sec><sec sec-type=\"discussion\" id=\"sec4\"><label>4</label><title>DISCUSSION</title><p>To the best of our knowledge, this is the first study that aimed at exploring the relationship between lifetime mood spectrum symptoms and PTSD or burnout among emergency Healthcare operators in Italy. Almost 16% of the sample reported symptomatic PTSD due to trauma related to healthcare emergency work. This result is in line with previous studies and meta-analysis on a similar sample [<xref rid=\"r5\" ref-type=\"bibr\">5</xref>, <xref rid=\"r6\" ref-type=\"bibr\">6</xref>, <xref rid=\"r49\" ref-type=\"bibr\">49</xref>, <xref rid=\"r50\" ref-type=\"bibr\">50</xref>] and corroborates the data about high risk for trauma-related psychopathology in emergency workers. Thus, specific training and systematic PTSD prevention program are needed for emergency operators in order to improve their mental health and quality of life. The subjects filled the evaluation in a stand-alone room, after the work-shift in order to ensure their privacy</p><p>Furthermore, PTSD diagnosis was found to be associated with lifetime mood spectrum symptoms, especially with the depressive ones. In the National Comorbidity Survey-Replication, more than half of the subjects affected by PTSD presented a lifetime diagnosis of Major Depressive Disorder [<xref rid=\"r51\" ref-type=\"bibr\">51</xref>]. The relationship between post-traumatic stress reactions and depression is considered complex, with overlapping symptomatology, mutual influence, and very high comorbidity rates [<xref rid=\"r16\" ref-type=\"bibr\">16</xref>, <xref rid=\"r19\" ref-type=\"bibr\">19</xref>, <xref rid=\"r24\" ref-type=\"bibr\">24</xref>, <xref rid=\"r52\" ref-type=\"bibr\">52</xref>]. In the last years, the new broad approach to PTSD proposed by DSM-5, encompassing the negative alterations in cognition and mood symptoms, such as guilt, anger or shame feelings, enhanced the conceptual link between these two disorders [<xref rid=\"r4\" ref-type=\"bibr\">4</xref>].</p><p>Despite most of the existing studies are focused on the role of trauma or PTSD as a risk factor for depressive symptoms [<xref rid=\"r28\" ref-type=\"bibr\">28</xref>, <xref rid=\"r29\" ref-type=\"bibr\">29</xref>, <xref rid=\"r53\" ref-type=\"bibr\">53</xref>], some data also showed an inverse trend [<xref rid=\"r30\" ref-type=\"bibr\">30</xref>-<xref rid=\"r35\" ref-type=\"bibr\">35</xref>, <xref rid=\"r54\" ref-type=\"bibr\">54</xref>, <xref rid=\"r55\" ref-type=\"bibr\">55</xref>]. Some authors, in fact, showed how some features related to a depressive state, such as an impairment of coping skills and greater stress perception could lead to vulnerability to traumatic events [<xref rid=\"r56\" ref-type=\"bibr\">56</xref>]. Negative self-evaluation and reduced initiative may determine cognitive and behavioral avoidance in dealing with trauma-related stimuli, compromising fear extinction [<xref rid=\"r57\" ref-type=\"bibr\">57</xref>]. Moreover, high neuroticism and low extroversion represent underlying personality risk factors shared between depression and PTSD [<xref rid=\"r16\" ref-type=\"bibr\">16</xref>]. On one hand, high levels of neuroticism facilitate reaction to stressors and frustrations by means of negative affect; on the other hand, low levels of extroversion reduce the tendency to seek support from others in order to face upsetting events. All these elements could be considered as impairment in the self-efficacy belief that is crucial for the recovery of a traumatic experience [<xref rid=\"r31\" ref-type=\"bibr\">31</xref>].</p><p>The results of the present study suggested how the vulnerability produced by lifetime depressive spectrum symptoms could be particularly relevant in the professional population, like emergency operators, exposed every day to a traumatic experience or stressful situations. In this chronic context, the impairment in coping with traumas due to low self-esteem, negative affectivity or cognitive deficit is a likely high risky condition to PTSD development and maintenance. A recent study conducted in Pakistan, on over 500 emergency medical personnel, indicated depression as a predictive factor for work-related PTSD [<xref rid=\"r58\" ref-type=\"bibr\">58</xref>]. Furthermore, in a German survey on a large sample of emergency physician and paramedic staff conducted with the purpose to explore post-traumatic stress symptoms and depression rates, the authors recommended that those who presented post-traumatic stress symptoms should be investigated for depression and vice versa, considering the strong correlation between these two dimensions [<xref rid=\"r59\" ref-type=\"bibr\">59</xref>].</p><p>The logistic regression model showed that mood and cognitive lifetime depressive symptoms are associated with PTSD diagnosis. These results appear to be in line with the decision of the DSM-5 to introduce the new diagnostic criterion D (negative alteration in mood and cognition) for PTSD diagnosis, highlighting the importance of negative emotions and distorted cognition as a manifestation of the clinical picture of the disorder [<xref rid=\"r4\" ref-type=\"bibr\">4</xref>]. In this regard, in the Spearman analysis, the strongest correlations emerged between mood-depressive and cognition-depressive MOODS-SR domains and the PTSD diagnosis, as well as the PTSD criteria re-experiencing (B), alteration in mood and cognition (D) and Hyperarousal (E). It may be possible that the tendency to low self-esteem, negative feelings like sadness, guilt or shame, anhedonia and other affective or cognitive depressive spectrum symptoms is exacerbated by trauma exposure and evolved in PTSD. Some authors reported a dimensional commonality between depression and some PTSD symptoms, such as detachment, restricted affect, loss of interest, sense of foreshortened future, irritability, and cognitive impairment [<xref rid=\"r60\" ref-type=\"bibr\">60</xref>]. Furthermore, negative emotions like guilt and shame feelings were related to the onset of PTSD and its severity [<xref rid=\"r61\" ref-type=\"bibr\">61</xref>, <xref rid=\"r62\" ref-type=\"bibr\">62</xref>].</p><p>Significant associations also emerged for the burnout and the compassion fatigue subscale of the ProQOL. Several studies showed circular influences between burnout and depression: if on one hand burnout has been hypothesized to represent an initial phase in depression, on the other hand, this latter may negatively influence the experience of work-related stress, leading to burnout [<xref rid=\"r37\" ref-type=\"bibr\">37</xref>, <xref rid=\"r63\" ref-type=\"bibr\">63</xref>]. The nature of this relationship seems to be very similar to that emerged between depression and PTSD.</p><p>It is noteworthy that a significant association with PTSD clusters also emerged for the rhythmicity MOODS-SR domain. This result appears to be in line with existing data about the role of altered vegetative functions and biological rhythms in PTSD [<xref rid=\"r64\" ref-type=\"bibr\">64</xref>]. Sleep disturbances, somatic complaints, impairment in sexual or eating behaviors, as well as seasonal/circadian rhythms alterations were found not only to be associated with post-traumatic stress symptoms [<xref rid=\"r64\" ref-type=\"bibr\">64</xref>-<xref rid=\"r69\" ref-type=\"bibr\">69</xref>], but also to suicidality in PTSD patients [<xref rid=\"r64\" ref-type=\"bibr\">64</xref>].</p><p>This study has several limitations. The first one is the limited sample size and lack of a control group that may affect results. Furthermore, full data were not available from 13 enrolled subjects that were excluded from the study. Secondly, although TALS-SR has been used to assesses symptomatic PTSD according to DSM-5 criteria across studies [<xref rid=\"r47\" ref-type=\"bibr\">47</xref>, <xref rid=\"r48\" ref-type=\"bibr\">48</xref>], the use of a self-report instrument may be considered less reliable and more influenced by co-occurring events than a standardized structured interview. Thirdly, a selection bias may be present, as subjects with PTSD, showing severe avoidant symptoms, may have not been enrolled. Fourthly, the lack of full evaluation for the whole sample. Lastly, participants were not screened for Axis I psychiatric comorbidities, which may impact significantly on work functioning.</p></sec><sec sec-type=\"conclusions\"><title>CONCLUSION</title><p>Despite the limitations mentioned above, this study showed a relevant association between lifetime depressive symptoms and burnout, as well as work-related PTSD in emergency health operators. It is conceivable that this at-risk population should undergo regular screenings for depression and PTSD in order to better manage the symptoms due to stress and trauma exposure during work. We may argue that this issue is even more relevant in the framework of the Covid-19 emergency, and its possible impact on the mental health of the healthcare operators. Detecting specific risk factors for this population could be useful to assess vulnerable subjects and consequently prevent post-traumatic stress sequelae.</p><p>The present study corroborates previous findings on the complex relationship between “mood” and trauma-related psychopathology, and especially highlights it in health emergency operators. Further longitudinal research studies are required to understand the impact of trauma on this population in order to potentiate prevention interventions and therapeutic strategies.</p></sec></body><back><ack><title>ACKNOWLEDGEMENTS</title><p>Declared none.</p></ack><sec sec-type=\"competing-interests\"><title>AUTHORS' CONTRIBUTIONS</title><p>CC, CAB, and LD participated in the conception and design of the study, the interpretation of the data, the draft, and critical revision of the article. MTA, IC, EM, MC, participated in the draft and critical revision of the article. All authors agreed to be cited as co-authors, accepting the order of authorship, and approved the final version of the manuscript and the manuscript submission to Clinical Practice & Epidemiology in Mental Health.</p></sec><glossary><title>LIST OF ABBREVIATIONS</title><def-list><def-item><term>PTSD</term><def><p> = Post-Traumatic Stress Disorder\n</p></def></def-item><def-item><term>DSM-5</term><def><p> = Diagnostic and Statistical Manual of Mental Disorders\n</p></def></def-item></def-list></glossary><sec sec-type=\"competing-interests\"><title>ETHICS APPROVAL AND CONSENT TO PARTICIPATE</title><p>All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee of the AziendaOspedaliero-Universitaria of Pisa, Italy.</p></sec><sec sec-type=\"competing-interests\"><title>HUMAN AND ANIMAL RIGHTS</title><p>No Animals were used in this research. All human research procedures followed were in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1964, as revised in 2013.</p></sec><sec sec-type=\"competing-interests\"><title>CONSENT FOR PUBLICATION</title><p>Written informed consent was obtained from each participant prior to the study.</p></sec><sec sec-type=\"competing-interests\"><title>AVAILABILITY OF DATA AND MATERIALS</title><p>The data supporting the findings of the article are not publicly available, but it can be provided by the corresponding author [C.A.B] on reasonable request.</p></sec><sec sec-type=\"competing-interests\"><title>FUNDING</title><p>None.</p></sec><sec sec-type=\"competing-interests\"><title>CONFLICT OF INTEREST</title><p>The authors declare no conflict of interest financial or otherwise</p></sec><ref-list><title>REFERENCES</title><ref id=\"r1\"><label>1</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jackson</surname><given-names>T.</given-names></name><name><surname>Provencio</surname><given-names>A.</given-names></name><name><surname>Bentley-Kumar</surname><given-names>K.</given-names></name><name><surname>Pearcy</surname><given-names>C.</given-names></name><name><surname>Cook</surname><given-names>T.</given-names></name><name><surname>McLean</surname><given-names>K.</given-names></name><name><surname>Morgan</surname><given-names>J.</given-names></name><name><surname>Haque</surname><given-names>Y.</given-names></name><name><surname>Agrawal</surname><given-names>V.</given-names></name><name><surname>Bankhead-Kendall</surname><given-names>B.</given-names></name><name><surname>Taubman</surname><given-names>K.</given-names></name><name><surname>Truitt</surname><given-names>M.S.</given-names></name></person-group><article-title>PTSD and surgical residents: Everybody hurts… sometimes.</article-title><source>Am. 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publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dell'Osso</surname><given-names>L.</given-names></name><name><surname>Carmassi</surname><given-names>C.</given-names></name><name><surname>Consoli</surname><given-names>G.</given-names></name><etal/></person-group><article-title>Lifetime post-traumatic stress symptoms are related to the health-related quality of life and severity of pain/fatigue in patients with fibromyalgia.</article-title><source>Clin Exp Rheumatol.</source><year>2011</year><volume>29</volume><fpage>S73</fpage><lpage>8</lpage></element-citation></ref></ref-list></back><floats-group><fig id=\"F1\" fig-type=\"figure\" orientation=\"portrait\" position=\"float\"><label>Fig. (1)</label><caption><p>ROC curve of the regression model.</p></caption><graphic xlink:href=\"CPEMH-16-165_F1\"/></fig><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><title>Comparison of the MOODS-SR domains scores between healthcare operators without PTSD (N=69) and those with PTSD (N=13).</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n</th><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>No-PTSD</bold>\n<break/>\n<bold>Mean ± SD</bold>\n</th><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>PTSD</bold>\n<break/>\n<bold>Mean ± SD</bold>\n</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>Cohen’s</bold>\n<break/>\n<bold>d</bold>\n</th><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>Mann-Whitney</bold>\n<break/>\n<bold>z</bold>\n</th><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>p</bold>\n</th></tr></thead><tbody><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Mood-depressive</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.59±3.77</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.00±4.90</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.47</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.07</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><.001</bold></td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Energy-depressive</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.12±1.51</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.23±2.05</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.62</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.24</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>.025</bold></td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Cognition-depressive</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.83±2.45</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.54±5.47</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.35</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.42</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>.001</bold></td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Depressive component</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.54±6.70</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">19.77±9.56</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.60</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.33</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><.001</bold></td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Mood-manic</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.99±5.11</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.85±5.11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.36</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.23</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.218</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Energy-manic</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.97±2.21</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.31±2.97</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.51</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.68</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.093</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Cognition-manic</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.03±3.52</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.00±3.89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.53</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.93</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.054</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Manic component</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.99±9.70</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.15±10.10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.52</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.68</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.093</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Rhythmicity</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.1±4.29</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.23±7.41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.85</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.41</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>.016</bold></td></tr></tbody></table></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><title>Logistic regression model: MOODS-SR depressive and rhythmicity domains, gender and education levels as predictive variables associated with PTSD diagnosis in the total sample.</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>Predictive factors</bold>\n</th><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>B(S.E.)</italic></bold>\n</th><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>O.R.</italic></bold>\n</th><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>CI95%</italic></bold>\n</th><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>p</italic></bold>\n</th></tr></thead><tbody><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Mood-depressive</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.256 (0.108)</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>1.292</bold></td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.045-1.597</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>.018</bold></td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Energy-depressive</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">-0.309 (0.275)</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.734</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.429-1.258</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.261</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Cognition-depressive</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.268 (0.121)</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>1.308</bold></td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.032-1.657</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>.026</bold></td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Rhythmicity</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.051 (0.102)</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.052</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.861-1.286</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.618</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Gender</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.548 (1.024)</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.731</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.232-12.885</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.592</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Education</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.048 (0.967)</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.851</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.428-18.892</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.279</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">K</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">-6.671 (2.237)</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.001</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.003</td></tr></tbody></table><table-wrap-foot><p>Cox R<sup>2</sup> = 0.31; Nagelkerke R<sup>2</sup> = 0.52; Hosmer-Lemeshow test: χ<sup>2</sup> = 7,24, p = .511; Global-goodness-fit percentage = 89,0%. Sensitivity=53.8%; specificity=95.6%.</p></table-wrap-foot></table-wrap><table-wrap id=\"T2A\" orientation=\"portrait\" position=\"float\"><label>Table 2a</label><caption><title>ROC curve characteristics of the regression model.</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>Predictive Factors</bold>\n</th><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>Area (S.E.)</italic></bold>\n</th><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>CI95%</italic></bold>\n</th><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>p</italic></bold>\n</th></tr></thead><tbody><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Mood-depressive</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.855 (.053)</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.752-.959</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.000</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Energy-depressive</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.685 (.076)</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.535-.835</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.035</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Cognition-depressive</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.790 (.091)</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.611-.968</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.001</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Rhythmicity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.711 (.090)</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.535-.887</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.016</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Gender</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.595 (.082)</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.434-.756</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.281</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Education</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.516 (.089)</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.343.690</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.854</td></tr></tbody></table></table-wrap><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><title>Correlations (r) between MOODS-SR depressive domains and PTSD symptoms clusters or ProQOLsubscales.</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">-\n</th><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>Mood Depressive</bold>\n</th><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>Energy Depressive</bold>\n</th><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>Cognition Depressive</bold>\n</th><th valign=\"middle\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>Rhythmicity</bold>\n</th></tr></thead><tbody><tr><td valign=\"middle\" colspan=\"5\" align=\"center\" scope=\"col\" rowspan=\"1\">PTSD and PTSDdiagnostic criteria</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Total PTSD symptoms</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.513**</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.249*</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.563**</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.360*</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">B) Re-experiencing symptoms</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.429**</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.353*</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.328*</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.304*</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">C) Avoidance symptoms</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.343*</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.188</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.335*</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.245*</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">D) Cognitive and mood alterations symptoms</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.549**</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.360*</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.560**</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.437**</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">E) Hyperarousal symptoms</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.517**</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.380*</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.437**</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.316*</td></tr><tr><td valign=\"middle\" colspan=\"5\" align=\"center\" scope=\"col\" rowspan=\"1\">ProQOL Subscale</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Compassion Satisfaction</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.041</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.052</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.075</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.047</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Burnout</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.376*</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.087</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.281*</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.289*</td></tr><tr><td valign=\"middle\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Compassion Fatigue</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.338*</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.152</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.193</td><td valign=\"middle\" align=\"center\" rowspan=\"1\" colspan=\"1\">.150</td></tr></tbody></table><table-wrap-foot><p>Moderate positive correlations (.5 > r < .7); Low positive correlations (.3 > r < .5); *p<.05;**p<.001\"</p></table-wrap-foot></table-wrap></floats-group></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"brief-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Microbiol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Microbiol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Microbiol.</journal-id><journal-title-group><journal-title>Frontiers in Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-302X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849473</article-id><article-id pub-id-type=\"pmc\">PMC7431685</article-id><article-id pub-id-type=\"doi\">10.3389/fmicb.2020.01900</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Microbiology</subject><subj-group><subject>Perspective</subject></subj-group></subj-group></article-categories><title-group><article-title>It Takes a Village: Discovering and Isolating the Nitrifiers</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Sedlacek</surname><given-names>Christopher J.</given-names></name><xref rid=\"c001\" ref-type=\"corresp\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/516881/overview\"/></contrib></contrib-group><aff><institution>Division of Microbial Ecology, Centre for Microbiology and Environmental Systems Science, University of Vienna</institution>, <addr-line>Vienna</addr-line>, <country>Austria</country></aff><author-notes><fn id=\"fn1\" fn-type=\"edited-by\"><p>Edited by: Sebastian Lücker, Radboud University Nijmegen, Netherlands</p></fn><fn id=\"fn2\" fn-type=\"edited-by\"><p>Reviewed by: Hidetoshi Urakawa, Florida Gulf Coast University, United States; Alyson E. Santoro, University of California, Santa Barbara, United States</p></fn><corresp id=\"c001\">*Correspondence: Christopher J. Sedlacek, <email>chris.j.sedlacek@gmail.com</email></corresp><fn id=\"fn3\" fn-type=\"other\"><p>This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1900</elocation-id><history><date date-type=\"received\"><day>07</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>20</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Sedlacek.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Sedlacek</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>It has been almost 150 years since Jean-Jacques Schloesing and Achille Müntz discovered that the process of nitrification, the oxidation of ammonium to nitrate, is a biological process carried out by microorganisms. In the following 15 years, numerous researchers independently contributed paradigm shifting discoveries that formed the foundation of nitrification and nitrification-related research. One of them was Sergei Winogradsky, whose major accomplishments include the discovery of both lithotrophy (in sulfur-oxidizing bacteria) and chemoautotrophy (in nitrifying bacteria). However, Winogradsky often receives most of the credit for many other foundational nitrification discoveries made by his contemporaries. This accumulation of credit over time is at least in part due to the increased attention, Winogradsky receives in the scientific literature and textbooks as a “founder of microbiology” and “the founder of microbial ecology.” Here, some light is shed on several other researchers who are often overlooked, but whose work was instrumental to the emerging field of nitrification and to the work of Winogradsky himself. Specifically, the discovery of the biological process of nitrification by Schloesing and Müntz, the isolation of the first nitrifier by Grace and Percy Frankland, and the observation that nitrification is carried out by two distinct groups of microorganisms by Robert Warington are highlighted. Finally, the more recent discoveries of the chemolithoautotrophic ammonia-oxidizing archaea and complete ammonia oxidizers are put into this historical context.</p></abstract><kwd-group><kwd>nitrification</kwd><kwd>nitrogen cycle</kwd><kwd>ammonia oxidizers</kwd><kwd>nitrite oxidizers</kwd><kwd>comammox</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn1\">Wittgenstein Award of the Austrian Science Fund</funding-source></award-group><award-group><funding-source id=\"cn2\">Austrian Science Fund<named-content content-type=\"fundref-id\">10.13039/501100002428</named-content></funding-source><award-id rid=\"cn2\">P30570-B29</award-id></award-group><award-group><funding-source id=\"cn3\">Comammox Research Platform of the University of Vienna</funding-source></award-group></funding-group><counts><fig-count count=\"0\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"68\"/><page-count count=\"6\"/><word-count count=\"5417\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"sec1\"><title>Introduction</title><p>It has been almost 150 years since the discovery of microbially mediated nitrification (the oxidation of ammonium to nitrate), and ever since scientists have been working toward identifying and characterizing the microorganisms responsible. The ability to regulate the process of nitrification in the environment is essential in a world battling to reduce greenhouse gas emissions, prevent the eutrophication of aquatic ecosystems, and increase the efficiency of drinking and waste water treatment (<xref rid=\"ref21\" ref-type=\"bibr\">Galloway et al., 2003</xref>, <xref rid=\"ref22\" ref-type=\"bibr\">2017</xref>; <xref rid=\"ref16\" ref-type=\"bibr\">Fields, 2004</xref>; <xref rid=\"ref15\" ref-type=\"bibr\">Erisman et al., 2008</xref>; <xref rid=\"ref28\" ref-type=\"bibr\">Houlton et al., 2019</xref>). With that in mind, the study of nitrification and the microbes that carry this process out is arguably more relevant than ever. Here, a look back at several groundbreaking discoveries that form the foundation for nitrification-related research is presented.</p><p>Many independent researchers and research groups contributed to these groundbreaking discoveries, but one researcher in particular receives the lion’s share of the credit for the initial work surrounding nitrification and nitrifying microorganisms, the Russian microbiologist Sergei Winogradsky. Winogradsky is often broadly credited with the discovery of nitrification, the isolation of the first nitrifiers, and the observation that nitrification occurs as a two-step process. Interestingly, he is most likely not responsible for any of these particular discoveries (albeit determining who really had the first pure nitrifier culture is not without controversy). This accumulation of credit is in part a consequence of the increased attention Winogradsky receives in the scientific literature and textbooks – due to his impressive body of work. In fact, Winogradsky’s seminal papers on nitrification cite the foundational work performed by the lesser known researchers highlighted here (<xref rid=\"ref63\" ref-type=\"bibr\">Winogradsky, 1890</xref>, <xref rid=\"ref64\" ref-type=\"bibr\">1891</xref>).</p><p>Winogradsky did of course make numerous pioneering contributions to the field of nitrification and to the microbiological sciences as a whole. While at the Swiss Polytechnic Institute in Zurich, he discovered that nitrifiers were chemoautotrophic (first example of microbial autotrophy), proposed that nitrification was performed by a particular type of microorganism, championed the enrichment culturing technique that is still widely utilized today and possibly isolated the first nitrite-oxidizing bacterium (<xref rid=\"ref63\" ref-type=\"bibr\">Winogradsky, 1890</xref>). Combined with his discovery of microbial lithotrophy while working with the sulfur-oxidizing <italic>Beggiota</italic> at the University of Strasbourg (<xref rid=\"ref62\" ref-type=\"bibr\">Winogradsky, 1887</xref>; <xref rid=\"ref11\" ref-type=\"bibr\">Doetsch, 1960</xref>), and his doctrine of pleomorphism (<xref rid=\"ref65\" ref-type=\"bibr\">Winogradsky, 1936</xref>; <xref rid=\"ref12\" ref-type=\"bibr\">Doolittle, 2013</xref>), it is easy to see why he is referred to as a “founder of microbiology” and “the founder of microbial ecology.” Consequently, his life and scientific discoveries have been well documented several times over (<xref rid=\"ref54\" ref-type=\"bibr\">Waksman, 1953</xref>; <xref rid=\"ref41\" ref-type=\"bibr\">Penn and Dworkin, 1976</xref>; <xref rid=\"ref67\" ref-type=\"bibr\">Zavarzin, 2006</xref>; <xref rid=\"ref1\" ref-type=\"bibr\">Ackert, 2007</xref>; <xref rid=\"ref13\" ref-type=\"bibr\">Dworkin, 2011</xref>). So, while the works of Winogradsky will be mentioned here for context, his (and his longtime research assistant, Viselli Omelianski’s) work will not be the focus of this piece.</p><p>Instead, some light will be shed on a few of the other less well known but nonetheless critical researchers responsible for key nitrification-related discoveries between 1877 and 1892, which often get credited to, or grouped with, Winogradsky’s accomplishments. Their discoveries are put into a timeline highlighting some of the large scientific leaps made in nitrification research during this early period. In addition, this timeline was extended to include the more recent paradigm shifting discoveries of the ammonia-oxidizing archaea and complete ammonia oxidizers.</p></sec><sec id=\"sec2\"><title>Nitrification Is a Biotic Process</title><p>Up until the late 1870s, ammonium and nitrates held quite of bit of mystery in both agricultural and drinking water quality research. For instance, it was unknown how nitrates were replenished in unfertilized soils. Common hypotheses centered around abiotic chemical reactions, such as reactions of atmospheric nitrogen and oxygen, organic nitrogen with oxygen or ozone, or ammonia with ferric oxide (reviewed by <xref rid=\"ref55\" ref-type=\"bibr\">Warington, 1878a</xref>). In addition, it was Edmund Davy, an Irish medical doctor and professor of forensic medicine at the Royal College of Surgeons in Ireland, who first observed that ammonium concentrations decreased while nitrite (and he assumed nitrate) increased over time in sewage contaminated drinking water. Therefore, in order to rule out sewage contamination, Davy argued that drinking water should be tested for not only just ammonium but also nitrite and nitrate (<xref rid=\"ref10\" ref-type=\"bibr\">Davy, 1883</xref>). Notably, although Davy did not attempt to answer whether nitrification was a biotic process or not, he detailed how the process of nitrification: (1) is inhibited by high amounts of organic matter, (2) requires air or free oxygen, and (3) proceeds at an optimal temperature of between 21 and 27°C.</p><p>The first person to hypothesize that ammonia oxidation may in fact be a biological process was the French chemist/microbiologist Luis Pasteur, who in 1862 suggested that ammonia may be oxidized to nitrate in a similar manner as alcohol is oxidized to acetic acid (<xref rid=\"ref40\" ref-type=\"bibr\">Pasteur, 1862</xref>). A decade later, Alexander Müller, a German agricultural chemist, would put forward that ammonia oxidation must be performed by microorganisms. While investigating water quality from wells in Berlin, Müller noted that ammonium was stable in sterilized solutions in the laboratory but readily nitrified in natural waters (<xref rid=\"ref36\" ref-type=\"bibr\">Müller, 1875</xref>). Even with these observations, it would be ~25 years (1877) after Pasteur’s initial hypothesis until two French agricultural chemists working in Paris, Jean-Jacques Schloesing and Achille Müntz, demonstrated that the oxidation of ammonium in sewage and soils was indeed a microbially mediated process (<xref rid=\"ref47\" ref-type=\"bibr\">Schloesing and Müntz, 1877a</xref>,<xref rid=\"ref48\" ref-type=\"bibr\">b</xref>).</p><p>In order to demonstrate the biological nature of nitrification, Schloesing and Müntz slowly passaged liquid sewage through an artificial soil matrix column consisting of sterilized sand and powered chalk. With this experimental setup, they were able to make four key observations: (1) there was an initial lag phase, (2) as the ammonia concentration decreased the nitrate concentration increased in the filtrate, (3) nitrification irreversibly ceased when the column was exposed to chloroform or high heat, and (4) nitrification could be restarted by adding small amounts of soil washings (<xref rid=\"ref47\" ref-type=\"bibr\">Schloesing and Müntz, 1877a</xref>,<xref rid=\"ref48\" ref-type=\"bibr\">b</xref>). These results were independently confirmed by the English agricultural chemist Robert Warington who was investigating the nitrification ability of garden soil at the (still operational today) Rothamsted experimental station in Harpenden England. Warington was also able to demonstrate that soil added to dilute solutions of ammonium would produce nitrate, and that these nitrifying solutions could seed new nitrifying solutions – with this, nitrifier enrichment/cultivation was born (<xref rid=\"ref56\" ref-type=\"bibr\">Warington, 1878b</xref>). Together, these seminal studies kick-started the study of microbial nitrification.</p><p>Interestingly, one of the still remaining open questions in nitrification research became a topic of debate during these early years. Several researchers published observations very early on as to whether or not direct sunlight inhibited the process of nitrification, with varied results (<xref rid=\"ref56\" ref-type=\"bibr\">Warington, 1878b</xref>, <xref rid=\"ref57\" ref-type=\"bibr\">1879</xref>, <xref rid=\"ref58\" ref-type=\"bibr\">1884</xref>; <xref rid=\"ref10\" ref-type=\"bibr\">Davy, 1883</xref>; <xref rid=\"ref37\" ref-type=\"bibr\">Munro, 1886</xref>). However, the effect of 24-hour illumination on nitrification could not be studied, as the incandescent light bulb was still being developed.</p></sec><sec id=\"sec3\"><title>Nitrification Is a Two-Step Process</title><p>In 1879, Warington made the first observation that nitrification proceeds as a two-step process, involving the oxidation of ammonia to nitrite and the oxidation of nitrite to nitrate. Here, Warington described and propagated individual ammonia‐ and nitrite-oxidizing enrichment cultures. In addition, he observed that fully nitrifying cultures only built up nitrite as an intermediate if the rate of ammonia oxidation was sufficiently high (<xref rid=\"ref57\" ref-type=\"bibr\">Warington, 1879</xref>). The two-step process of nitrification would be later confirmed by the English chemist John Munro working at the College of Agriculture at Downton (<xref rid=\"ref37\" ref-type=\"bibr\">Munro, 1886</xref>). These observations were made well before the isolation of the first nitrifier in 1890. However, the hypothesis at the time was that the ammonia‐ and nitrite-oxidizing cultures represented different life phases or character traits of the single nitrifying microorganism (<xref rid=\"ref58\" ref-type=\"bibr\">Warington, 1884</xref>, <xref rid=\"ref59\" ref-type=\"bibr\">1891</xref>).</p></sec><sec id=\"sec4\"><title>Nitrifier Isolation Attempts</title><p>Once it was confirmed that nitrification was a microbially mediated process, the race was on to be the first researcher to isolate and characterize the microorganism(s) responsible. Over the course of the next ~15 years (1877-1890), several researchers made claims that they isolated cultures that had (or previously had) full nitrifying capabilities (the ability to oxidize ammonium to nitrate). At the time, it was still hotly debated whether microorganisms were pleomorphic – able to radically change their abilities or characteristics under different circumstances, sometimes irreversibly in short periods of time (<xref rid=\"ref65\" ref-type=\"bibr\">Winogradsky, 1936</xref>; <xref rid=\"ref12\" ref-type=\"bibr\">Doolittle, 2013</xref>). With the pleomorphic hypothesis in mind, some researchers observed that nitrifying microorganisms irreversibly lost their nitrifying capabilities through the process of isolation. These studies attempted to isolate single nitrifier colonies on solid growth medium containing high amounts of organic carbon. In a somewhat ironic twist, this exact approach (growth in or on medium containing high amounts of organic carbon) is now common practice in order to detect heterotrophic contaminants in pure nitrifier cultures. However, because each researcher isolated their own unique environmental cultures and some of these cultures had seemingly lost their nitrifying capabilities by the time of publication, independent conformational studies were difficult if not impossible.</p><p>As a notable exception, the German chemist Wilhelm Heraeus, working at the Institute of Hygiene in Berlin, published in 1886 that in addition to cultures, he freshly isolated from sites around Hanau Germany, several well-known bacterial cultures, including <italic>Bacillus anthracis</italic> and <italic>Bacillus ramosus</italic> had nitrifying capabilities. In his publication, Heraeus notes that some of these isolated cultures produced detectable but not quantifiable amounts of nitrite and nitrate when cultured in dilute urine (<xref rid=\"ref27\" ref-type=\"bibr\">Heraeus, 1886</xref>). As these <italic>Bacillus</italic> strains were more widely available to the scientific community, others could attempt to replicate the results. Indeed, the English agricultural chemist Percy Frankland attempted but could not independently verify Heraeus’s results. Frankland determined that Heraeus was most likely detecting small amounts of nitrate present in the urine, and that the <italic>Bacillus</italic> cultures were reducing this nitrate to nitrite. By inoculating the <italic>Bacillus</italic> cultures into ammonium solutions instead of dilute urine, Frankland was able to refute Heraeus’s claims as no nitrite or nitrate was produced (<xref rid=\"ref18\" ref-type=\"bibr\">Frankland, 1888</xref>). It would not be until 1890 that Percy and Grace Frankland, two English scientists working in Dundee Scotland would publish about the isolation of what is most likely the first pure nitrifier (ammonia-oxidizing) culture (<xref rid=\"ref19\" ref-type=\"bibr\">Frankland and Frankland, 1890</xref>). This breakthrough was made possible by the combined expertise of Grace as a bacteriologist and Percy as an agricultural chemist.</p></sec><sec id=\"sec5\"><title>Isolation of the First Nitrifiers</title><p>For years researchers (including the Frankland’s) failed to isolate actively nitrifying cultures using Robert Koch’s solid medium isolation techniques (<xref rid=\"ref30\" ref-type=\"bibr\">Koch, 1882</xref>). However, Warington, Winogradsky, and the Frankland’s were all able to establish and propogate nitrifying enrichment cultures (<xref rid=\"ref56\" ref-type=\"bibr\">Warington, 1878b</xref>; <xref rid=\"ref19\" ref-type=\"bibr\">Frankland and Frankland, 1890</xref>; <xref rid=\"ref63\" ref-type=\"bibr\">Winogradsky, 1890</xref>). Ultimately Frankland and Frankland were the first to isolate a pure nitrifier (ammonia-oxidizing bacteria) from their enrichment cultures. In order to achieve a pure culture, they employed a system of serial dilutions with very low inoculum over the period of several years (<xref rid=\"ref19\" ref-type=\"bibr\">Frankland and Frankland, 1890</xref>). This method of serial dilution to extinction is a tried and true method that is still used for the (painstakingly slow) isolation of nitrifiers today.</p><p>In 1890, both Winogradsky as well as Frankland and Frankland would both claim that they were the first to have a pure nitrifier culture (<xref rid=\"ref19\" ref-type=\"bibr\">Frankland and Frankland, 1890</xref>; <xref rid=\"ref63\" ref-type=\"bibr\">Winogradsky, 1890</xref>). However, at this time, Winogradsky’s pure culture was still a fully nitrifying culture, and he would later disclose through personal communications (~40 years later) that his nitrifying cultures were suitable for nitrification studies but not pure in the strictest sense of the word (<xref rid=\"ref25\" ref-type=\"bibr\">Hanks and Weintraub, 1936</xref>). With the more recent discovery of complete ammonia oxidizers in mind, it is tempting to speculate that Winogradsky did in fact have a pure culture of a complete ammonia oxidizer capable of fully nitrifying. However, based on his work in the years to come, this culture was much more likely a co-culture of ammonia‐ and nitrite-oxidizing bacteria. Winogradsky was able to separate the ammonia‐ and nitrite-oxidizing bacteria just a year later (<xref rid=\"ref64\" ref-type=\"bibr\">Winogradsky, 1891</xref>).</p><p>In contrast to other researchers before them, both the team of Frankland and Frankland as well as Winogradsky took many steps in order to ensure they had an actively nitrifying pure culture. Both noted that pure nitrifying cultures did not produce any growth on solid media, and that the nitrifying liquid medium remained clear throughout the entirety of nitrification. Additionally, they monitored and quantified the conversion of ammonium into nitrite and nitrate (<xref rid=\"ref19\" ref-type=\"bibr\">Frankland and Frankland, 1890</xref>; <xref rid=\"ref63\" ref-type=\"bibr\">Winogradsky, 1890</xref>, <xref rid=\"ref64\" ref-type=\"bibr\">1891</xref>).</p><p>Although Frankland and Frankland were confident that they had isolated the microorganism responsible for nitrification, the isolate only oxidized ammonium to nitrite when in its pure form (<xref rid=\"ref19\" ref-type=\"bibr\">Frankland and Frankland, 1890</xref>). Warington had observed a similar phenomenon years ago, noting that after several passages in the laboratory nitrifying cultures would often produce nitrite rather than fully nitrifying (<xref rid=\"ref57\" ref-type=\"bibr\">Warington, 1879</xref>, <xref rid=\"ref58\" ref-type=\"bibr\">1884</xref>). There were several hypotheses to explain this partial nitrification: (1) the oxidation of nitrite to nitrate was completed by a second uncultured microorganism, (2) the conditions for full ammonia oxidation were not met in the laboratory, or (3) part of the microorganism’s physiology present when in soil was lost rapidly during laboratory cultivation. In hindsight, Warington as well as Frankland and Frankland, were unknowingly separating ammonia‐ and nitrite-oxidizing bacteria through the use of different growth mediums and transfer techniques.</p><p>As is the case with the ammonia-oxidizing bacteria, determining which researcher(s) truly isolated the first nitrite-oxidizing bacteria, is not without uncertainty. Again, there were researchers that claimed to isolate nitrite-oxidizing bacteria that quickly lost their nitrite oxidation abilities once grown and isolated on or in organic growth medium (<xref rid=\"ref4\" ref-type=\"bibr\">Beijerinck, 1914</xref>), which were most likely not pure nitrite-oxidizing bacteria. In addition, some seemingly pure cultures were later discovered to be highly enriched cultures with only a few bacterial members (<xref rid=\"ref5\" ref-type=\"bibr\">Burri and Stutzer, 1895</xref>). But there are about 10 research groups, who between 1900 and 1960 claim to have purified nitrite-oxidizing bacteria, which did not produce noticeable growth when inoculated on or in growth medium containing high amounts of organic matter (reviewed by <xref rid=\"ref68\" ref-type=\"bibr\">Zavarzin and Legunkova, 1959</xref>; but missing <xref rid=\"ref20\" ref-type=\"bibr\">Fred and Davenport, 1921</xref>). As with the ammonia oxidizers, Winogradsky was the first to claim a pure nitrite-oxidizing culture (<xref rid=\"ref64\" ref-type=\"bibr\">Winogradsky, 1891</xref>). Unfortunately, unlike with the isolation of the first ammonia oxidizers, where there was a clear difference between theoretically pure cultures that produce nitrite versus those that produce nitrate from ammonium; here, all theoretically pure nitrite oxidizer cultures share the same reported physiology (nitrite consumption and nitrate production). Therefore, without preserved cultures, it is difficult if not impossible to say with certainty which cultures contained no heterotrophic contaminants.</p><p>Nitrite oxidizer isolation represents another close call for Robert Warington. After being the first to observe the two steps of nitrification (even if the significance of the observation was not immediately clear), he spent years attempting to isolate nitrite-oxidizing microorganisms through the serial dilution technique shown to be successful by Frankland and Frankland with ammonia oxidizers, but to no avail. He would end his scientific career before he was able to produce a pure nitrite-oxidizing isolate (<xref rid=\"ref59\" ref-type=\"bibr\">Warington, 1891</xref>).</p></sec><sec id=\"sec6\"><title>New Nitrifiers Discovered In The 21<sup>st</sup> Century</title><p>Since the inaugural era of nitrification research discussed so far, there has been a wealth of studies that have propelled the field of nitrification forward during the subsequent ~100 years. These advances include (but are not limited to) the (1) discovery of many phylogenetically and physiologically distinct ammonia‐ and nitrite-oxidizing bacteria, (2) a deeper understanding of nitrifier (eco)physiology/enzymology, and (3) extensive environmental surveys. These discoveries, isolations, and characterizations have previously been thoroughly reviewed throughout the years (<xref rid=\"ref61\" ref-type=\"bibr\">Watson et al., 1981</xref>, <xref rid=\"ref60\" ref-type=\"bibr\">1989</xref>; <xref rid=\"ref43\" ref-type=\"bibr\">Prosser, 1989</xref>; <xref rid=\"ref33\" ref-type=\"bibr\">Koops and Pommerening-Röser, 2001</xref>; <xref rid=\"ref35\" ref-type=\"bibr\">Kowalchuk and Stephen, 2001</xref>; <xref rid=\"ref34\" ref-type=\"bibr\">Koops et al., 2006</xref>; <xref rid=\"ref3\" ref-type=\"bibr\">Arp et al., 2007</xref>; <xref rid=\"ref8\" ref-type=\"bibr\">Daims et al., 2011</xref>; <xref rid=\"ref49\" ref-type=\"bibr\">Stahl and de la Torré, 2012</xref>).</p><p>Even with this immense increase in the diversity of known nitrifiers, up until just over 15 years ago, it was widely accepted that (1) there was a complete division of labor in nitrification and (2) nitrification was carried out solely by bacteria. Then, over the course of a 10-year period both of these century-old paradigms were shattered with the discovery and isolation of ammonia-oxidizing archaea and complete ammonia oxidizers. Just as with the discovery and isolation of the ammonia‐ and nitrite-oxidizing bacteria, these discoveries too, took a village.</p><sec id=\"sec7\"><title>Ammonia-Oxidizing Archaea</title><p>In 2004, two independent research groups identified putative ammonia monooxygenase genes (the main functional genes used to identify ammonia oxidizers) in genomic sequence material belonging to a <italic>Crenarchaeota</italic> microorganism (<xref rid=\"ref50\" ref-type=\"bibr\">Treusch et al., 2004</xref>; <xref rid=\"ref53\" ref-type=\"bibr\">Venter et al., 2004</xref>).</p><p>Almost immediately, these findings were followed key genomic comparisons (<xref rid=\"ref45\" ref-type=\"bibr\">Schleper et al., 2005</xref>) and environmental survey studies (<xref rid=\"ref17\" ref-type=\"bibr\">Francis et al., 2005</xref>). Amazingly, the first isolated ammonia-oxidizing archaea culture, <italic>Nitrosopumilus maritimus</italic>, was published within the next year (<xref rid=\"ref32\" ref-type=\"bibr\">Könneke et al., 2005</xref>), and the first genome of an ammonia-oxidizing archaea (<italic>Candidatus Cenarchaeum symbiosum</italic>) was published soon after (<xref rid=\"ref23\" ref-type=\"bibr\">Hallam et al., 2006a</xref>,<xref rid=\"ref24\" ref-type=\"bibr\">b</xref>). Since these works, there have been hundreds of studies focusing on the diversity, physiology, and environmental distribution of ammonia-oxidizing archaea, highlighting to what extent they have changed the field of nitrification in such a short time (reviewed by <xref rid=\"ref14\" ref-type=\"bibr\">Erguder et al., 2009</xref>; <xref rid=\"ref46\" ref-type=\"bibr\">Schleper and Nicol, 2010</xref>; <xref rid=\"ref51\" ref-type=\"bibr\">Urakawa et al., 2011</xref>; <xref rid=\"ref26\" ref-type=\"bibr\">Hatzenpichler, 2012</xref>; <xref rid=\"ref44\" ref-type=\"bibr\">Prosser and Nicol, 2012</xref>; <xref rid=\"ref49\" ref-type=\"bibr\">Stahl and de la Torré, 2012</xref>; <xref rid=\"ref2\" ref-type=\"bibr\">Alves et al., 2018</xref>).</p></sec><sec id=\"sec8\"><title>Complete Ammonia Oxidizers</title><p>Although a complete ammonia oxidizer had never been described, their existence was by no means a new hypothesis. The whole field of nitrification began with the belief that nitrification was completed by one microorganism (a nitrifying ferment). In addition, a recent theoretical kinetic study hypothesized that complete ammonia oxidizers would be found in environments that select for slow growing but high yield microorganisms (<xref rid=\"ref6\" ref-type=\"bibr\">Costa et al., 2006</xref>). In 2015, just 10 years after the discovery of the ammonia-oxidizing archaea, the existence of complete ammonia oxidizers was published in parallel by two research groups. Both studies yielded metagenomic evidence for, and enrichment cultures of, a complete ammonia oxidizer (<xref rid=\"ref7\" ref-type=\"bibr\">Daims et al., 2015</xref>; <xref rid=\"ref52\" ref-type=\"bibr\">van Kessel et al., 2015</xref>). Within a year, two additional research groups published evidence supporting these findings (<xref rid=\"ref42\" ref-type=\"bibr\">Pinto et al., 2015</xref>; <xref rid=\"ref38\" ref-type=\"bibr\">Palomo et al., 2016</xref>). The isolation of <italic>Nitrospira inopinata</italic>, the first complete ammonia oxidizer brought into pure culture, followed shortly thereafter (<xref rid=\"ref7\" ref-type=\"bibr\">Daims et al., 2015</xref>; <xref rid=\"ref29\" ref-type=\"bibr\">Kits et al., 2017</xref>). In the same manner as the discovery of the ammonia-oxidizing archaea before them, studies focusing on the diversity, physiology, and environmental distribution of complete ammonia oxidizers are now increasing at a rapid pace (reviewed by <xref rid=\"ref9\" ref-type=\"bibr\">Daims et al., 2016</xref>; <xref rid=\"ref39\" ref-type=\"bibr\">Palomo et al., 2018</xref>; <xref rid=\"ref66\" ref-type=\"bibr\">Xia et al., 2018</xref>; <xref rid=\"ref31\" ref-type=\"bibr\">Koch et al., 2019</xref>). It took about 150 years, but the field of nitrification has now come full circle – a fully nitrifying microorganism with the ability to oxidize ammonia all the way to nitrate.</p></sec></sec><sec id=\"sec9\"><title>In Summary</title><p>While Winogradsky has certainly earned his title of “the founder of microbial ecology,” the breakthroughs illustrated here highlight the work of many of his often-overlooked contemporaries. Schloeing, Muntz, Warington, Frankland, and Frankland among others, all contributed essential observations and discoveries that now form the foundation of nitrification-related research. With the surprising discoveries of new types of nitrifiers within just the past 15 years, the future of nitrification research looks bright. It will be exciting to see whether there are even more fundamentally different types of nitrifiers including nitrite-oxidizing or complete ammonia-oxidizing archaea out there waiting to be discovered and isolated.</p></sec><sec id=\"sec10\"><title>Author Contributions</title><p>The author confirms being the sole contributor of this work and has approved it for publication.</p><sec id=\"sec11\" sec-type=\"coi\"><title>Conflict of Interest</title><p>The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></sec></body><back><ack><p>I would like to thank Michael Wagner, Holger Daims, Petra Pjevac, Andrew Giguere, Anna Aplenc, and Heather Beck for fruitful discussions and helpful comments on the manuscript.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> CS was supported by the Wittgenstein Award of the Austrian Science Fund presented to Michael Wagner, the Austrian Science Fund (FWF, project P30570-B29), and the Comammox Research Platform of the University of Vienna.</p></fn></fn-group><ref-list><title>References</title><ref id=\"ref1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ackert</surname><given-names>L. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Med (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Med (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Med.</journal-id><journal-title-group><journal-title>Frontiers in Medicine</journal-title></journal-title-group><issn pub-type=\"epub\">2296-858X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850910</article-id><article-id pub-id-type=\"pmc\">PMC7431686</article-id><article-id pub-id-type=\"doi\">10.3389/fmed.2020.00417</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Medicine</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title><italic>H. pylori</italic> Eradication Treatment Causes Alterations in the Gut Microbiota and Blood Lipid Levels</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Martín-Núñez</surname><given-names>Gracia M.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/931505/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Cornejo-Pareja</surname><given-names>Isabel</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/549627/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Roca-Rodríguez</surname><given-names>M. del Mar</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Clemente-Postigo</surname><given-names>Mercedes</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/650164/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Cardona</surname><given-names>Fernando</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/87452/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Fernández-García</surname><given-names>José C.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/932603/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Moreno-Indias</surname><given-names>Isabel</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/152769/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Tinahones</surname><given-names>Francisco J.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/711845/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Unidad de Endocrinología y Nutrición, Hospital Universitario Virgen de la Victoria</institution>, <addr-line>Málaga</addr-line>, <country>Spain</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y la Nutrición (CIBEROBN), Instituto de Salud Carlos III</institution>, <addr-line>Madrid</addr-line>, <country>Spain</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Departamento de Endocrinología y Nutrición, Hospital Universitario Puerta del Mar</institution>, <addr-line>Cádiz</addr-line>, <country>Spain</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Departamento de Biología Celular, Fisiología e Inmunología, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Universidad de Córdoba, Hospital Universitario Reina Sofía</institution>, <addr-line>Córdoba</addr-line>, <country>Spain</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Giovanni Tarantino, University of Naples Federico II, Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Mitsushige Sugimoto, Shiga University of Medical Science, Japan; Timothy Powell, University of Oxford, United Kingdom</p></fn><corresp id=\"c001\">*Correspondence: Gracia M. Martín-Núñez <email>graciamaria_mn@hotmail.com</email></corresp><corresp id=\"c002\">Isabel Moreno-Indias <email>isabel_moreno_indias@hotmail.com</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Translational Medicine, a section of the journal Frontiers in Medicine</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†These authors have contributed equally to this work</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>7</volume><elocation-id>417</elocation-id><history><date date-type=\"received\"><day>16</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>30</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Martín-Núñez, Cornejo-Pareja, Roca-Rodríguez, Clemente-Postigo, Cardona, Fernández-García, Moreno-Indias and Tinahones.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Martín-Núñez, Cornejo-Pareja, Roca-Rodríguez, Clemente-Postigo, Cardona, Fernández-García, Moreno-Indias and Tinahones</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p><bold>Background:</bold> The gut microbiome plays an important role in the lipid metabolism. Antibiotic treatment causes changes in the intestinal microbiota. Our objective was to explore the relationship between changes in the intestinal microbiota and the level of plasma high density lipoprotein cholesterol (HDL) and low density lipoprotein cholesterol (LDL).</p><p><bold>Methods:</bold> Prospective case-control study with <italic>Helicobacter pylori</italic>-positive patients undergoing eradication therapy with omeprazole, clarithromycin, and amoxicillin. Stool and blood samples were obtained from 20 controls (<italic>H. pylori</italic> negative) and 40 patients before and 2 months after antibiotic treatment. Gut microbiota was determined through 16S rRNA amplicon sequencing (Illumina MiSeq).</p><p><bold>Results:</bold> Eradication treatment for <italic>H. pylori</italic> increased the HDL levels, and caused changes in gut microbiota profiles. An unfavorable lipid profiles (high LDL and low HDL levels) was associated with a low microbial richness and an increase of the Bacteroidetes phylum. <italic>Prevotella copri, Lachonobacterium</italic>, and <italic>Delsufovibrio</italic> were positively associated with HDL while <italic>Rikenellaceae</italic> was negatively associated with HDL after completing antibiotic treatment.</p><p><bold>Conclusions:</bold>\n<italic>Helicobacter pylori</italic> eradication treatment could improve lipid metabolism in relation with an increase in the HDL. Changes in the abundance of specific bacteria, such as <italic>P. copri, Lachonobacterium, Delsufovibrio</italic>, and <italic>Rikenellaceae</italic> could be associated with change in the plasma HDL levels.</p></abstract><kwd-group><kwd>LDL</kwd><kwd>HDL</kwd><kwd>gut microbiota</kwd><kwd>antibiotic</kwd><kwd><italic>H. pylori</italic></kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Instituto de Salud Carlos III<named-content content-type=\"fundref-id\">10.13039/501100004587</named-content></funding-source><award-id rid=\"cn001\">CM 17/00169</award-id><award-id rid=\"cn001\">CP16/00163</award-id><award-id rid=\"cn001\">PI14/00082</award-id><award-id rid=\"cn001\">PI15/01114</award-id><award-id rid=\"cn001\">PI18/01160</award-id></award-group><award-group><funding-source id=\"cn002\">Centro de Investigación Biomédica en Red-Fisiopatología de la Obesidad y Nutrición<named-content content-type=\"fundref-id\">10.13039/501100012514</named-content></funding-source><award-id rid=\"cn002\">CB06/03/0018</award-id></award-group><award-group><funding-source id=\"cn003\">Ministerio de Ciencia e Innovación<named-content content-type=\"fundref-id\">10.13039/501100004837</named-content></funding-source><award-id rid=\"cn003\">FJCI-2017-34349</award-id></award-group></funding-group><counts><fig-count count=\"2\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"37\"/><page-count count=\"7\"/><word-count count=\"5225\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Increasing evidence has demonstrated that the intestinal microbiota is critical for the development of diseases associated with altered lipid metabolism (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>) including variation in the level of blood lipids (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). The microbiome affects ability to metabolize lipids by the host taking part in the absorption, storage, and energy resulted from the diet (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>), because of its ability to convert bile acids and produce short-chain fatty acids (SCFAs) within the intestinal lumen (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B5\" ref-type=\"bibr\">5</xref>).</p><p>Triglycerides and high density lipoproteins cholesterol (HDL) levels have been associated to the diversity and amount of Proteobacteria and Bacteroidetes members (including <italic>Christensenellaceae, Pasteurellaceae</italic>, and genus <italic>Butyricimonas</italic>) (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). HDL function is to transport cholesterol to the liver from peripheral tissues for its subsequent excretion as biliary sales, and to endocrine organs for the synthesis of steroid hormones. The concentration of serum low-density lipoproteins cholesterol (LDL) is regulated, in part, by a cross-talk between the absorption of dietary and biliary cholesterol in the intestine, and the biosynthesis of cholesterol in the liver (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). LDL and HDL levels are biomarkers associated with the CVD risk (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>).</p><p><italic>Helicobacter pylori</italic> is a Gram-negative bacterium that colonizes the gastric mucosa commonly causing a chronic infection. Also, it has been associated with extradigestive pathologies including coronary heart disease (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). <italic>H. pylori</italic> eradication through oral administration of antibiotic and a proton-pump inhibitor has been associated with alterations in the gut microbiota (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B9\" ref-type=\"bibr\">9</xref>). We have previously found that antibiotic therapy used for <italic>H. pylori</italic> eradication can alter the intestinal microbiota population, and more importantly, these changes were associated to glucose metabolism and GLP-1 (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B11\" ref-type=\"bibr\">11</xref>). However, there is scarce data in humans about the relationship between blood lipids and changes in the profile of gut microbiota after antibiotic therapy (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Thus, in the present study, our objective was to test the relationship between changes in the gut microbiota and blood lipids level in patients who received antibiotic treatment for <italic>H. pylori</italic> eradication.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Study Subjects and Design</title><p>Forty volunteers with positive <italic>H. pylori</italic> antigen in faeces tested by immunochromatography were derived from the Microbiology Unit of the Virgen de la Victoria Hospital (Málaga, Spain). Sample size was calculated taking into account a reduction in richness (according to Chao1 index) of 16% after the antibiotic therapy (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>). The inclusion criteria were: (1) adulthood (18–65 years old), and (2) primary infection with <italic>H. pylori</italic>. Besides, a group of non-infected volunteers (20 individuals) was included as control. Exclusion criteria were (1) diabetes (type 1 or 2); (2) prior H. <italic>pylori</italic> documented treatment; (3) antibiotherapy 3 months before recruitment; and (4) without informed consent.</p><p>The study included two visits, before and 60 days post-treatment (20 mg omeprazole, 500 mg clarithromycin, and 1000 mg amoxicillin, twice daily during 10 days) for patients, while a unique visit was assessed in the case of the control volunteers. Visitations comprised a physical exploration, a fasting blood sample, and an oral glucose tolerance test (OGTT) with 75 g glucose and measurements at 30, 60, and 120 min. Moreover, samples of stool were obtained in every visit and immediately frozen at −80°C. The investigation protocol was conducted consonantly with the Declaration of Helsinki and conveniently approved by the Medical Ethics Committee at Virgen de la Victoria University Hospital. Volunteers were supplied with a written informed consent, as well as they were advised of the study characteristics.</p></sec><sec><title>Anthropometric, Biochemical, and Dietetics Measurements</title><p>Anthropometric measurements (height, weight, and waist and hip circumferences) were recorded (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). An enzymatic method was used for the measurement of triglycerides (mmol/L), total cholesterol (mg/dl), and HDL (mg/dl) (Randox Laboratories Ltd.), while C-reactive protein (CRP) was measured with a Dimension autoanalyzer (Dade Behring Inc.). LDL (mg/dl) was determined through the Friedewald formula. Food intake was evaluated with 7/24-h dietary recalls. The dietary variables (fats, fiber, proteins, etc.) were determined using DIAL nutrition software and the Diet Balancer software (Cardinal Health Systems Inc.).</p></sec><sec><title>Gut Microbiota Analysis</title><p>The determination of the gut microbiota was assessed as was described previously (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). In brief, the QIAamp DNA Stool Mini Kit was used for the extraction of DNA from fecal samples, and posteriorly the DNA concentrations were determined by Qubit® Fluorometric (Thermo Fisher Scientific). The 16S rRNA V3-V4 amplicon was analyzed with the universal primers reported by Klindworth et al. (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). The amplicon size ~460 bp was verified with a bioanalyzer (Agilent 2100, USA). AMPure XP beads (Beckman Coulter Genomic, CA, USA) were used to purify amplicon products. Samples were multiplexed with Nextera XT Index Kit (Illumina, CA, USA). Illumina MiSeq platform (Illumina, San Diego, USA) was used for the paired-end sequencing of amplicons.</p><p>Quantitative Insights into Microbial Ecology (Qiime) tool (version 1.9.1, open source software) was used for the analysis of the merged paired-end reads. The operational taxonomic units (OTUs) were generated at 97% similarity and alignment by UCLUST consensus using the Greengenes 16S rRNA gene database. Alpha diversity (Chao1 and Shannon indixes, as well as the observed species) was also assessed with Qiime. Alpha diversity was evaluated with the rarefaction workflow, with a threshold of 34,385 sequences. Rarefied data were used for the downstream analysis. OTUs in less than five different samples were excluded. Raw data is sored at the public repository SRA database (NCBI) with the BioProject PRJNA517270.</p></sec><sec><title>Statistical Analysis</title><p>SPSS 22.0 (SPSS Inc., Chicago, IL, USA) and QIIME (version 1.9.1; open source software) were used for the statistical analysis. The data were expressed as mean ± standard deviation. Statistical comparisons between the means for independent samples and paired samples (before and after antibiotic eradication treatment) was performed using the Student's <italic>t</italic>-test. Mann-Whitney and Wilcoxon signed-rank tests were used for the evaluation of non-parametric variables. Correlation analysis between analytical, clinical, and microbial populations variables was analyzed using the Spearman bivariate correlations test. Linear regression models were applied to identify bacterial changes as independent predictors of the selected variables (HDL/LDL ratio and HDL–). A <italic>p</italic> < 0.05 treshold was established for the statistical significance.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>Lipid Metabolism</title><p>The anthropometric and clinical variables of the patients and control subjects have been previously described (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Anthropometric variables as BMI and waist circumference or biochemical parameters as triglycerides and cholesterol level did not reach statistical differences between groups. Focusing on lipid metabolism, HDL levels increased significantly after <italic>H. pylori</italic> eradication with antibiotic therapy (<italic>p</italic> = 0.021), while LDL levels were significantly lower in controls than in <italic>H. pylori</italic>-infected subjects (<italic>p</italic> = 0.036; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). In addition, HDL/LDL ratio was significantly lower in patients pre- and post-<italic>H. pylori</italic> eradication than in control subjects (<italic>p</italic> = 0.007 and <italic>p</italic> = 0.03, respectively; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). HDL/LDL ratio and HDL were associated with BMI in a regression model (<italic>R</italic><sup>2</sup> = 0.079, β = −0.281, <italic>p</italic> = 0.005; <italic>R</italic><sup>2</sup> = 0.93, β = −0.305, <italic>p</italic> = 0.003, respectively). No statistically differences in the dietary intake energy, micro or macronutrients and dietary fiber between patients and controls (<italic>p</italic> > 0.05) (data not shown).</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Characteristics of the study groups.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Variables</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Pre-<italic>H. pylori</italic> eradication (<italic>n</italic> = 40)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Post-<italic>H. pylori</italic> eradication (<italic>n</italic> = 40)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Control group (<italic>n</italic> = 20)</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Age (years)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">46.95 ± 12.78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">46.95 ± 12.78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">43.86 ± 12.63</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Men/women (<italic>n</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16/24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16/24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9/13</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BMI (kg/m<sup>2</sup>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.92 ± 4.30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.91 ± 4.40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25.89 ± 4.54</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Waist (cm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92.10 ± 12.06</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91.27 ± 11.73</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">89.8 ± 13.23</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HDL (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">52.97 ± 12.9<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">55.36 ± 16.36<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">57 ± 15.8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">LDL (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">121.45 ± 35.8<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">117.96 ± 33.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">102.05 ± 34<sup>a</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HDL/LDL</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.47 ± 0.17<sup>b</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.51 ± 0.22<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.62 ± 0.25<sup>b, a</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Triglycerides (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">97.2 ± 39.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">93.5 ± 36.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">89.70 ± 41.78</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cholesterol (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">194.22 ± 40.84</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">191.34 ± 37.15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">177.05 ± 39.5</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DBP (mmHg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">77.75 ± 9.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80.50 ± 11.37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">75.95 ± 10</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SBP (mmHg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">123.84 ± 16.62</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">125.42 ± 21.36</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">120.3 ± 13.35</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CRP (mg/L)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.07 ± 2.44</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.56 ± 2.11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.14 ± 2.92</td></tr></tbody></table><table-wrap-foot><p><italic>All values are means ± standard deviations. Wilcoxon's test was used to compare patients before and after H. pylori eradication. The Mann-Whitney U-test or Student's t-test were used to compare independent group. Equal letter indicates statistical signiticative differences in the mean between those 2 groups, p < 0.05. BMI, body mass index; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TGs, triglycerides, TC, total cholesterol; DBP, diastolic blood pressure; SBP, systolic blood pressure; CRP, C-reactive protein</italic>.</p></table-wrap-foot></table-wrap></sec><sec><title>Gut Microbiome and Lipids Metabolism</title><p>Our <italic>H. pylori</italic>-positive patients study model who received antibiotics for a limited period of time has allowed us to evaluate changes in the microbiota, HDL and LDL, avoiding the confounding factors derived from the presence of other diseases, or metabolic alterations.</p><p><italic>Helicobacter pylori</italic> infection and its eradication with antibiotic affected alpha diversity (Chao 1 and Shannon indexes) and this data has been previously described by our group (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Control subjects showed a greatest diversity and richness, with statistically significant differences, compared to the <italic>H. pylori</italic> patients (pre- and post-eradication treatment) (<italic>p</italic> < 0.05) (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Chao1 index correlated positively with the HDL/LDL ratio (<italic>r</italic> = 0.258, <italic>p</italic> = 0.04).</p><p>According to the abundance of each identified OTU, the most abundant bacterial phyla were Firmicutes and Bacteroidetes, while Actinobacteria, Proteobacteria, and Verrucomicrobia contributed with smaller proportions (1–5%). Changes in the microbiome between these subjects has been described in detail by our group in a previous work (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). The phylum Bacteroidetes, that was greater in patients (58.72 ± 13.62 and 63.50 ± 10.30 vs. 45.89 ± 13.57), showed negative correlations with the HDL/LDL ratio (<italic>r</italic> = −0.237, <italic>p</italic> = 0.021), while the phylum Firmicutes, which was greater in controls (45.68 ± 15.61 vs. 35.29 ± 11.82 and 32.06 ± 10.29, <italic>p</italic> < 0.05), showed positive correlations with the HDL/LDL ratio (<italic>r</italic> = 0.230, <italic>p</italic> = 0.025) in the studied groups. In the regression analysis, Bacteroidetes and Firmicutes predicted changes in the HDL/LDL ratio (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Bacteroidetes and Firmicutes in the prediction of HDL/LDL ratio in a linear regression model including the 3 groups studied. Asterisk: regression model adjusted by age, sex, and BMI.</p></caption><graphic xlink:href=\"fmed-07-00417-g0001\"/></fig><p>Genera and species of the phyla Bacteroidetes and Firmicutes showed association with the HDL/LDL ratio (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). <italic>Bacteroides coprophilus, Eubacterium</italic>, and <italic>E. biforme</italic> predicted changes in the HDL/LDL ratio, in a linear regression model adjusted for age, sex, and BMI (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). The presence of these bacteria was higher in subjects with <italic>H. pylori</italic> infection compared to controls (<italic>p</italic> < 0.05, data not shown). The associations shown in the <xref rid=\"T2\" ref-type=\"table\">Table 2</xref> were found in patients and controls.</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Association between intestinal bacteria and HDL/LDL ratio.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Genus/Species</bold></th><th valign=\"top\" align=\"center\" colspan=\"3\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Model 1</bold></th><th valign=\"top\" align=\"center\" colspan=\"3\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Model 2</bold></th></tr><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>R</italic><sup><bold>2</bold></sup></bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>β</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>p</italic></bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>R</italic><sup><bold>2</bold></sup></bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>β</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>P</italic></bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>B. coprophilus</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.049</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.243</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.018</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.148</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.233</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.018</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Eubacterium</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.043</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.231</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.025</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.148</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.235</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.018</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>E. biforme</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.045</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.236</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.021</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.149</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.237</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.017</td></tr></tbody></table><table-wrap-foot><p><italic>Dependent variable: HDL/LDL ratio. Model 1: linear univariate regression model. Model 2: linear multivariate regression model adjusted by age, sex, and BMI</italic>.</p></table-wrap-foot></table-wrap><p>We evaluated the impact of antibiotic eradication therapy on the abundance of identified OTU, LDL, and HDL level. Significant correlations between changes in the number of particular bacteria and HDL were found after <italic>H. pylori</italic> eradication (<italic>Prevotella copri</italic>: <italic>r</italic> = 0.34, <italic>p</italic> = 0.037; <italic>Prevotella stercorea</italic>: <italic>r</italic> = −0.34, <italic>p</italic> = 0.04; <italic>Lachnobacterium r</italic> = 0.36 <italic>p</italic> = 0.028; <italic>Delsufovibrio r</italic> = 0.48, <italic>p</italic> = 0.003). Changes in the abundance of <italic>Bacteroides</italic> were associated with changes in HDL/LDL ratio after <italic>H. pylori</italic> eradication (<italic>r</italic> = −0.364, <italic>p</italic> = 0.029). In multivariate regression analysis, changes in <italic>Delsufovibrio</italic> (<italic>R</italic><sup>2</sup> = 0.130, β = 0.39, <italic>p</italic> = 0.017, and <italic>R</italic><sup>2</sup> = 0.082, β = 0.44, <italic>p</italic> = 0.015, after including age, sex, and BMI) and <italic>Rikenellaceae</italic> (<italic>R</italic><sup>2</sup> = 0.098, β = −0.353, <italic>p</italic> = 0.035, and <italic>R</italic><sup>2</sup> = 0.022, β = −0.350, <italic>p</italic> = 0.045, after including age, sex, and BMI) predicted the proportion of changes in the HDL level, in patients after eradication treatment (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>).</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Changes in <italic>Desulfovibrio</italic> and <italic>Rikenellaceae</italic> in the prediction of modifications in HDL levels after <italic>H pylori</italic> eradication treatment. Asterisk: linear regression model adjusted by age, sex, and BMI.</p></caption><graphic xlink:href=\"fmed-07-00417-g0002\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>There are no previous studies that evaluate changes in microbiota related to lipid metabolism in otherwise healthy subjects pre-post-<italic>H. pylori</italic> eradication. Alterations in blood cholesterol levels (high LDL and low HDL) are major risk factors for metabolic syndrome and cardiovascular disease (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). Antibiotic treatment for <italic>H. pylori</italic> eradication increased the HDL, reaching values similar to controls, in agreement with previous studies (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>), and both eradication and infection produced changes in the microbiota, data described in detail in our previous study (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). More interestingly, this is the first time that variations in blood lipid levels have been related to specific bacteria from the microbiome independent of age, sex, and BMI resulting <italic>H. pylori</italic> infection and eradication, suggesting that gut microbiota may affect specific aspects of lipid metabolism. Further, many of the identified taxa related to lipid metabolism are novel findings.</p><p>The use of broad spectrum antibiotics have been widely established to cause community-wide microbiota perturbations (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). In our study, <italic>H. pylori</italic> infection and its eradication with amoxicillin and clarithromycin for 10 days induced a dysbiosis affecting microbial diversity, something that has been associated to metabolic functions of intestinal bacteria. Low microbial functional richness has been associated to metabolic disease, including levels of fasting triglyceride, LDL and a raise in inflammation markers (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). In this sense, our study associated unfavorable lipid profiles (high LDL and low HDL level) with a low microbial richness in metabolically healthy subjects. In addition, this unfavorable lipid profile (lower HDL/LDL ratio) was associated with the phylum Bacteroidetes. As opposed, Firmicutes was associated with a favorable lipid profile (higher HDL/LDL ratio). The association between blood lipids and gut microbiome has been previously reported (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B21\" ref-type=\"bibr\">21</xref>). Firmicutes and Bacteroidetes are the two main bacteria phyla and they are implicated in the homeostasis of the host and fat accumulation. Interestingly, in our study, for the first time, Bacteroidetes and Firmicutes predicted HDL and LDL levels, regardless of age, sex, and BMI.</p><p>The raise in Bacteroidetes and the disminution in Firmicutes associated to the <italic>H. pylori</italic> infection and antibiotic-treatment could affect the production of key metabolites for the host, including SCFAs, bile acids, and lipopolysaccharide (LPS). In fact, acetate and propionate are produced mainly by members of Bacteroidetes while butyrate is typically produced by members of Firmicutes (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). Alterations in the microbiota as well as in their metabolites influence key processes as energy harvesting, the activation of the immune system, modulation of the chronic inflammation through the modification of the intestinal barrier permeability and perturbation of the reverse cholesterol transport, triggering in a higher susceptibility for some metabolic diseases (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>).</p><p>The impact of the antibiotherapy, amoxicillin, and clarithromycin, on lipid metabolism as a result of the disturbance on the microbiota is an area of interest with limited knowledge at present. In this sense, we show a negative association between changes in the abundance of <italic>P. stercorea</italic> and HDL level and a positive association between changes in the abundance of <italic>P. copri, Lachonobacterium, Delsufovibrio</italic>, and HDL level at 2 months after completing antibiotic treatment.</p><p><italic>Lachnospiraceae</italic> family has been specially associated with LDL (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>) and development of metabolic disorders (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). Our study associated <italic>Lachnospiraceae/Lachonobacterium</italic> with HDL. <italic>Lachnospiraceae</italic> family is known by its capacity to metabolize complex polysaccharides to SCFAs, as butyrate, acetate, and propionate (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Butyrate controls gene expression through epigenetic control. Buryrate is a key energy source for the colonic mucosa (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). Acetate is implicated in the <italic>de novo</italic> hepatic lipogenesis through acetyl-coA and fatty acid synthase (FAS), while propionate balances lipogenesis (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). Moreover, acetate is needed for the biosynthesis of cholesterol in liver. Thus, changes in the acetate/propionate ratio could play a crucial role in the regulation of lipid and cholesterol metabolism (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>).</p><p>More interestingly is that alterations in <italic>Delsufovibrio</italic>, from the Proteobacteria phylum, and <italic>Rikenellaceae</italic>, from the Bacteroidetes phylum, predicted changes in HDL levels at 2 months after completing antibiotic treatment. To date, lower abundance of <italic>Desulfovibrio</italic> has been associated with obesity, blood pressure, insulin, and LDL (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). <italic>Desulfovibrio</italic> also has been inversely correlated to BMI (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). We showed, for the first time, that <italic>Delsufovibrio</italic> positively contributed to the HDL level independently of BMI, suggesting that this bacterium affects blood lipids partly. Bacteria such as <italic>Delsufovibrio</italic> and <italic>P. copri</italic> are producers of lipopolysaccharide (LPS). LPS is a ligand for toll-like receptor 4 (TLR4), which activates innate immune system, something that could trigger in a pro-inflammatory status (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). The inflammatory response to microbial stimulus may induce metabolic modifications. Low grade systemic inflammation associated with bacterial LPS-induced endotoxaemia as well as a gut permeability increase have been associated with elevated plasma HDL (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). This finding could be controversial, as obesity, metabolic syndrome and type-2 diabetes are characterized by and impaired barrier function and local systemic inflammation (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>), and they are characterized by a low HDL. On the other hand, products secreted by Desulfovibrio can up-regulate CD36 expression (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>). CD36 is a critical regulator of lipid absorption, which has been positively correlated with HDL (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). All of these works would be in accordance with our data, and could help explain the results obtained in this study. In addition, we have reported an inverse relationship between <italic>Rikenellaceae</italic>, glucose (AUC) (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>) and HDL levels at 2 months antibiotic treatment. <italic>Rikenellaceae</italic> is able to use the environmental glucose for acetate production (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). Acetic acid stimulates “<italic>de novo</italic>” lipogenesis and cholesterogenesis in the liver (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). The increase in the production of SCFAs as well as the elimination of glucose from the environment by this bacterium could explain part of the observed results. In addition, tudies have been showed a possible role of <italic>Clostridiales</italic> (including <italic>Lachonobacterium</italic>) and <italic>Bacteroidales</italic> (including <italic>Rikenellaceae, P. copri, and P. stercorea</italic>) in the metabolism of lipids, through the metabolic pathway of bile acids (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>). In fact, secondary bile acids from the bacteria metabolism are absorbed being able to systemically modulate lipid and glucose metabolisms through nuclear or G protein-coupled receptors (GPCRs), such as FXR or TGR5 (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>).</p><p>The current study, although it has some strengths, there are also a few limitations to be considered. The targeted sequencing of the16S rRNA, although it permits know who is who, has the impediment to identify specific species and strains as well as gives little information on bacterial functions. Moreover, although previous sample size calculations were performed to ensure a real approach, sample size could be augmented. The lack of a group of subjects without an <italic>H. pylori</italic> infection exposed to the eradication treatment could be indicated as another limitation, although it was not performed because of ethical reasons. This group could have provided more detailed data on the role of antibiotic treatment. Lastly, the follow-up period was based on a visit at 2 months the administration of the antibiotic treatment. The introduction of new time-points after 2 months could permit us know more about the dynamics suffered after eradication treatment.</p><p>In summary, antibiotic eradication treatment for <italic>H. pylori</italic> could increase HDL levels and affected the gut microbiota composition. The results obtained indicate that intestinal microbiome, specifically bacteria such as <italic>P. copri, Lachonobacterium</italic> and <italic>Delsufovibrio, Rikenellaceae</italic> could plays a role in lipid metabolism.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The datasets generated for this study can be found in the SRA database public repository from NCBI within the BioProject accession number PRJNA517270.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by Medical Ethics Committee at Virgen de la Victoria University Hospital and conducted in accordance with the Declaration of Helsinki. Written informed consent was provided by all participants, who also were verbally informed of the characteristics on the study. The patients/participants provided their written informed consent to participate in this study.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>GM-N was person in charge of the metagenomic laboratory analysis, statistical analysis and interpretation of data, as well as the drafting, and reviewing of the manuscript. IC-P was person in charge of the recruitment of the patients and their follow-up and contributed to the design of the study and the revision of the manuscript. MR-R and JF-G also participated in the recruitment and follow-up of the patients, as well as reviewed the manuscript. MC-P performed laboratory analysis and participated in the revision of the manuscript. FC participated in the study design and reviewed the manuscript. IM-I assessed the bioinformatic analysis and critical revision of the manuscript. FT contributed to the study concept and design, interpretation of data, reviewed, and critically revised the article. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> GM-N was supported by a Juan de la Cierva, Formación contract (FJCI-2017-34349) from the Spanish Ministry of Science (Spain). IC-P was now the recipient of a postdoctoral grant (Juan Rodés JR19/00054) from the Instituto de Salud Carlos III (ISCIII) and co-funded by Fondo Europeo de Desarrollo Regional-FEDER. MC-P was supported by a Juan de la Cierva, Formación contract (FJCI-2017-32194) from the Spanish Ministry of Science (Spain). FC was supported by a Nicolas Monardes contract (C-0032-2016) from Servicio Andaluz de Salud (SAS). JF-G was supported by a research contract from SAS (B-0003-2017). IM-I was supported by a MS type I contract (CP16/00163) from the ISCIII and co-funded by Fondo Europeo de Desarrollo Regional-FEDER. 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] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Psychol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Psychol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Psychol.</journal-id><journal-title-group><journal-title>Frontiers in Psychology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-1078</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849128</article-id><article-id pub-id-type=\"pmc\">PMC7431687</article-id><article-id pub-id-type=\"doi\">10.3389/fpsyg.2020.01919</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Psychology</subject><subj-group><subject>Brief Research Report</subject></subj-group></subj-group></article-categories><title-group><article-title>Understanding When Similarity-Induced Affective Attraction Predicts Willingness to Affiliate: An Attitude Strength Perspective</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Philipp-Muller</surname><given-names>Aviva</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/827080/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wallace</surname><given-names>Laura E.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Sawicki</surname><given-names>Vanessa</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/764603/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Patton</surname><given-names>Kathleen M.</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wegener</surname><given-names>Duane T.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1045107/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Psychology, The Ohio State University</institution>, <addr-line>Columbus, OH</addr-line>, <country>United States</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Psychology, The Ohio State University</institution>, <addr-line>Marion, OH</addr-line>, <country>United States</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Hanover Research</institution>, <addr-line>Arlington, VA</addr-line>, <country>United States</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Matthew Montoya, Murdoch University, Australia</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Johannes Ullrich, University of Zurich, Switzerland; Estelle Michinov, University of Rennes 2 - Upper Brittany, France</p></fn><corresp id=\"c001\">*Correspondence: Aviva Philipp-Muller, <email>philipp-muller.1@osu.edu</email>; <email>aviva.philipp.muller@gmail.com</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Personality and Social Psychology, a section of the journal Frontiers in Psychology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1919</elocation-id><history><date date-type=\"received\"><day>31</day><month>1</month><year>2020</year></date><date date-type=\"accepted\"><day>13</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Philipp-Muller, Wallace, Sawicki, Patton and Wegener.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Philipp-Muller, Wallace, Sawicki, Patton and Wegener</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Individuals reliably feel more attracted to those with whom they share similar attitudes. However, this affective liking does not always predict affiliative behavior, such as pursuing a friendship. The present research examined factors that influence the extent to which similarity-based affective attraction increases willingness to affiliate (i.e., behavioral attraction) – one potential step toward engaging in affiliative behavior. Research on attitude strength has identified attitude properties, such as confidence, that predict when an attitude is likely to impact relevant outcomes. We propose that when one’s attitudes possess these attitude strength-related properties, affective attraction to those who share that attitude will be more likely to spark willingness to affiliate. Across four studies on a variety of topics, participants (<italic>N</italic> = 428) reported their attitudes and various attitude properties regarding a topic. They were introduced to a target and learned the target’s stance on the issue. Participants reported their affective attraction and willingness to pursue friendship with the target. Consistent with past research, attitude similarity predicted affective attraction. More importantly, the relation between affective attraction and willingness to affiliate with the target was moderated by the attitude strength-related properties. A mini meta-analysis found this effect to be consistent across the four studies.</p></abstract><kwd-group><kwd>attraction</kwd><kwd>attitude similarity</kwd><kwd>attitude strength</kwd><kwd>affiliation</kwd><kwd>confidence</kwd><kwd>morality</kwd></kwd-group><counts><fig-count count=\"3\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"34\"/><page-count count=\"7\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>In the film <italic>Serendipity</italic>, Jonathan Trager and Sara Thomas, the film’s romantic leads, meet while shopping when they both reach for the same pair of gloves. This shared interest sparks an attraction between the two that ultimately leads them to pursue a long-term relationship. Indeed, inter-personal similarity is considered a cornerstone of many relationships (<xref rid=\"B7\" ref-type=\"bibr\">Byrne, 1961</xref>). Yet often people do not act upon their attraction to similar others. Why does similarity-based attraction sometimes blossom into a willingness to form relationships while, at other times, it is nothing more than an ephemeral moment that is never further pursued? Could the strength of those initial shared attitudes or interests perhaps play a role in determining when people act upon their attraction?</p><sec id=\"S1.SS1\"><title>Attitude Similarity and Attraction</title><p>Typically, when individuals encounter a target with similar attitudes, they feel affective attraction for the similar other (<xref rid=\"B7\" ref-type=\"bibr\">Byrne, 1961</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Byrne et al., 1971</xref>). When individuals share a high degree of similarity, their interactions are smoother and more enjoyable (<xref rid=\"B6\" ref-type=\"bibr\">Burleson and Denton, 1992</xref>). These similarity-attraction effects seem to be primarily driven by perceived similarity, rather than actual similarity (<xref rid=\"B18\" ref-type=\"bibr\">Montoya et al., 2008</xref>).</p></sec><sec id=\"S1.SS2\"><title>Antecedents to Attitude Strength</title><p>Some features of attitudes seem to impact the size of this similarity-attraction effect. Research on attitude strength has identified many attitude properties that predict attitude-behavior consistency, dubbed <italic>attitude strength-related properties</italic>. These properties include confidence in one’s attitude (<xref rid=\"B33\" ref-type=\"bibr\">Wegener et al., 1995</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Petty et al., 1997</xref>), perceptions that an attitude is based in moral convictions (<xref rid=\"B26\" ref-type=\"bibr\">Skitka and Bauman, 2008</xref>), perceptions that an attitude stems from core values (<xref rid=\"B22\" ref-type=\"bibr\">Pomerantz et al., 1995</xref>), one-sidedness of an attitude (<xref rid=\"B29\" ref-type=\"bibr\">van Harreveld et al., 2015</xref>), personal importance of the attitude (<xref rid=\"B5\" ref-type=\"bibr\">Boninger et al., 1995</xref>), and the amount of knowledge associated with that attitude (<xref rid=\"B13\" ref-type=\"bibr\">Kallgren and Wood, 1986</xref>), among others.</p><p>Previous work has suggested that attitude strength antecedents can play an important role in determining affective attraction to similar others. For example, when attitudes are based on equally high levels of knowledge, similarity with relatively univalent (rather than ambivalent) attitudes better predict liking and desire to talk with an attitudinally similar other (see <xref rid=\"B32\" ref-type=\"bibr\">Wallace et al., 2020</xref>). Similarly, when perceivers focus on forming an impression of a target, similarity of attitudes held with confidence vs. doubt better predict affective attraction (<xref rid=\"B25\" ref-type=\"bibr\">Sawicki and Wegener, 2018</xref>). A recent meta-analysis found that attitudes more central or important (vs. peripheral or unimportant) to participants’ identities produced larger similarity-attraction effect sizes (<xref rid=\"B16\" ref-type=\"bibr\">Montoya and Horton, 2013</xref>). Thus, attitude strength antecedents seem to increase affective attraction to similar others.</p></sec><sec id=\"S1.SS3\"><title>Attraction and Willingness to Affiliate</title><p>Individuals who are affectively attracted to a given target often express a willingness to affiliate (otherwise referred to as behavioral attraction; <xref rid=\"B17\" ref-type=\"bibr\">Montoya and Horton, 2014</xref>), which could represent a step toward engaging in affiliative behavior (cf. <xref rid=\"B11\" ref-type=\"bibr\">Gibbons et al., 1998</xref>). Often, however, affective attraction does not result in willingness to affiliate. As attraction is composed of affective and behavioral components (<xref rid=\"B17\" ref-type=\"bibr\">Montoya and Horton, 2014</xref>), when feelings of attraction do not produce a willingness to affiliate, there is a curious disconnect between the affective and behavioral facets of attraction. For example, though similarity on a variety of attitudes predicts affective attraction, only similarity on attitudes directly relevant to the interaction context (e.g., attitudes regarding school when choosing who to sit next to in class) predict behavioral attraction (<xref rid=\"B15\" ref-type=\"bibr\">Michinov and Monteil, 2002</xref>). What factors might encourage or prevent individuals who are affectively attracted to one another from being willing to affiliate? The obstacles impeding behavioral attraction have been under-studied in the relationships literature (cf. <xref rid=\"B17\" ref-type=\"bibr\">Montoya and Horton, 2014</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Montoya et al., 2018</xref>), and it therefore seems important to gain a better understanding of when and why the link between affective attraction and willingness to affiliate is strengthened or broken.</p></sec><sec id=\"S1.SS4\"><title>Potential Role of Attitude Strength</title><p>Whereas previous work has examined the moderating influence of antecedents to attitude strength on the link between attitudes and affective attraction, it remains an open question whether feelings of attraction to similar others might be more predictive of willingness to affiliate when the similarity-based attraction is rooted in strong attitudes. One reason this may occur is that increased strength of the attitude on which the affective attraction is based may strengthen the affective attraction, making it particularly influential in determining behavioral willingness.</p></sec><sec id=\"S1.SS5\"><title>Present Research</title><p>In the present work, we examined the impact of attitude strength-related properties on the link between affective attraction and willingness to affiliate by examining the moderating role of various antecedents to attitude strength (across the various studies, we measured the moral basis, values basis, importance, confidence, ambivalence, and subjective knowledge associated with the attitude). We report a mini-meta-analysis (<xref rid=\"B10\" ref-type=\"bibr\">Fabrigar and Wegener, 2016</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Goh et al., 2016</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Wallace et al., 2020</xref>) of four studies<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup> conceptually related to the same hypotheses. Though there were variations in topic and attitude properties measured, the study design was quite similar across studies. These studies were not all designed to test the current hypothesis but were rather selected for the present meta-analysis because they included key measures of interest, and several of them would be underpowered to detect our central effects if examined individually. We therefore combined the studies into a meta-analysis. Conducting a meta-analysis allowed us to examine whether there was consistent evidence in the direction of the hypothesized effect (<xref rid=\"B12\" ref-type=\"bibr\">Goh et al., 2016</xref>). Each study first measured participants’ attitudes and various attitude strength-related properties. Then participants encountered a target who held a particular position on a topical issue. Finally, we measured participants’ affective attraction toward the target and willingness to pursue a friendship with the target (i.e., their behavioral attraction).</p><p>Generally, attitude strength-related properties are considered distinct constructs (<xref rid=\"B31\" ref-type=\"bibr\">Visser et al., 2006</xref>), with some properties relating more than others (i.e., some reflecting an attitudes embeddedness and others reflecting its internal consistency; <xref rid=\"B21\" ref-type=\"bibr\">Philipp-Muller et al., 2020</xref>). However, subjective strength-related properties can also predict the same outcomes in similar ways (<xref rid=\"B4\" ref-type=\"bibr\">Bassili, 1996</xref>). In the present work, there were no unique effects of either category of features. Thus, we combined all strength-related properties for analysis. Because the method was comparable across studies, we present combined results for efficiency’s sake. Separate results for each study are consistent with this combined analysis and are available in <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Material</xref>.</p></sec><sec id=\"S1.SS6\"><title>Hypothesis</title><p>We predicted that when people’s attitudes are high in these strength-related properties (e.g., high in confidence), their affective attraction to others with that same attitude will be more predictive of their willingness to engage in affiliative behavior. Likewise, when people’s attitudes are low in these properties, their affective attraction to others with that same attitude will be less predictive of willingness to affiliate. More concretely, imagine that Beatrice loves composting and sees this love of composting as an important part of who she is (i.e., based in her moral beliefs, following from her values, and she holds the view with certainty). Any attraction Beatrice might feel toward Bartholomew should be more likely to lead to a willingness to form a friendship, compared to someone whose composting attitude is not an important part of who they are.</p></sec></sec><sec id=\"S2\"><title>Methods</title><p>In all studies, prior to data collection, we obtained ethics approval from our respective institutional review boards to conduct this study.</p><sec id=\"S2.SS1\"><title>Participants</title><p>Four hundred eighty individuals (Study 1: <italic>N</italic> = 144; Study 2: <italic>N</italic> = 124; Study 3: <italic>N</italic> = 63; Study 4: <italic>N</italic> = 149) participated in exchange for undergraduate-level course credit (participants in Studies 2–4 were introductory psychology students, and Study 1 consisted of introductory marketing students). Study samples were comparable in gender and age composition (aggregate: <italic>M</italic><sub>age</sub>: 20.33, <italic>SD</italic><sub>age</sub>: 1.26, 213 males, 202 females; see <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Material</xref> for individual study sample details)<sup><xref ref-type=\"fn\" rid=\"footnote2\">2</xref></sup>. Participants were excluded from analysis (Study 1: <italic>N</italic> = 23; Study 2: <italic>N</italic> = 22; Study 4: <italic>N</italic> = 7) if they either failed to identify the target’s position (Studies 1 and 2), or selected “no” (on a dichotomous scale) when asked “did you answer the questions attentively and thoughtfully today?” (for the complete wording of the checks used in Studies 1, 2, and 4, see <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Material</xref>). The final aggregate sample was 428.</p><p>We calculated the effect size of the interaction between strength-related properties and affective attraction that this meta-analysis would be able to detect with 80% power (see <xref rid=\"B28\" ref-type=\"bibr\">Valentine et al., 2010</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Quintana, 2017</xref>). We entered <italic>N</italic> = 107 as our average <italic>N</italic> per study and four as the number of studies. The meta-analysis would have 80% power to detect an effect of <italic>d</italic> = 0.222, 0.272, and.387, for low, medium, and high heterogeneity of effect sizes, respectively. Because we ultimately observed low heterogeneity across studies for this central effect, the value assuming low heterogeneity of effects might be the most informative. In comparison, sensitivity analyses revealed that, although Studies 1, 2, and 4 have large enough samples to detect a small to moderate effect (Cohen’s <italic>d</italic> = 0.36, 0.39, and.34, respectively) with 80% power, Study 3 could only detect a moderate effect (Cohen’s <italic>d</italic> = 0.50) with 80% power. As we expect the interaction effect of interest to be relatively small, we focus on the combined effects.</p></sec><sec id=\"S2.SS2\"><title>Procedure</title><p>Prior to commencing each study, participants provided informed consent to participate using online consent forms. Participants reported their attitudes toward the relevant topic (Studies 1, 2, and 4: a junk food tax; Study 3: mandatory drug testing for welfare recipients) as well as various attitude properties (Studies 1–4). We chose these topics because we expected that participants’ attitude positions and attitude properties would vary across participants. Participants next read about a target who held a strong position on the topical issue. In Studies 1 and 2, the target opposed the junk food tax; in Study 3, the target supported mandatory drug testing for welfare recipients; and in Study 4, the target supported a junk food tax (see <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Material</xref> for complete message wordings). Participants then completed an attention check [i.e., they reported what they perceived the target’s position to be (Studies 1 and 2 only) or reported the extent to which they took the study seriously (Studies 1, 2, and 4)]. By presenting participants with a target who held one particular stance on a topical issue, attitude similarity varied naturalistically as individuals’ own attitudes toward these issues varied. Next, participants reported the extent to which they felt affectively attracted to the target and the extent to which they were willing to pursue a friendship with the target. These questions were followed by additional exploratory measures that are listed, in full, in <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Material</xref>, along with a complete list of item and anchor wordings, which varied slightly by study.</p></sec><sec id=\"S2.SS3\"><title>Measures</title><sec id=\"S2.SS3.SSS1\"><title>Attitude</title><p>Participants reported their attitudes on three 9-point scales with “bad,” “harmful,” and “unfavorable” anchoring the low end and “good,” “beneficial,” and “favorable” anchoring the positive end. Because the responses to the attitude items were highly correlated, they were averaged to create a composite attitude score (internal reliability: Study 1: α = 0.79; Study 2: α = 0.86; Study 3: α = 0.98; Study 4: α = 0.85).</p></sec><sec id=\"S2.SS3.SSS2\"><title>Antecedents to Attitude Strength</title><p>In Studies 1 and 2, all measures of attitude features were anchored by “strongly disagree” and “strongly agree.” For a summary of which strength-related properties were measured in each study, see <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Summary of all attitude strength-related features measured in each study.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Study</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Moral basis</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Values basis</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Confidence</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Importance</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Ambivalence</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Knowledge</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">X</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">x</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">x</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">X</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">x</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">x</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">x</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">x</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">x</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">x</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">x</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">x</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">x</td></tr></tbody></table></table-wrap><sec id=\"S2.SS3.SSS2.Px1\"><title>Studies 1 and 2</title><p>Participants reported the extent to which their attitudes were based in their moral beliefs and convictions on three 7-point scales (e.g., “I feel that my attitude on the junk food tax is based on strong moral principles”).</p></sec><sec id=\"S2.SS3.SSS2.Px2\"><title>Studies 1–3</title><p>Participants reported the extent to which their attitudes were based in their values on three 7-point scales (e.g., “My attitude on the junk food tax is based on my core values”). Participants also rated the extent to which they felt confident in their attitude on three 7-point scales (e.g., “I am confident in my attitude toward the junk food tax”).</p></sec><sec id=\"S2.SS3.SSS2.Px3\"><title>Study 3</title><p>Participants also reported how personally important their attitude was to them on a single item (“Mandatory drug testing for welfare recipients is…”), anchored with “not important to me” and “very important to me.”</p></sec><sec id=\"S2.SS3.SSS2.Px4\"><title>Studies 3 and 4</title><p>Participants rated the extent to which they felt conflicted in their attitude on three 11-point scales (e.g., “How mixed are your thoughts and feelings about taxing junk food,” with “I feel completely one-sided reactions” anchoring the low end and “I feel completely mixed reactions” anchoring the high end of the scale). Participants also reported how knowledgeable they were about the topic on three 7-point scales (e.g., “How much knowledge do you have about taxing junk food,” with “Very little knowledge” anchoring the low end and “A lot of knowledge” anchoring the high end of the scale). In Study 3, values basis, confidence, and knowledge were each measured with a single item.</p><p>These attitude properties were highly related and were averaged to make a composite antecedent to attitude strength variable (Study 1: α = 0.88; Study 2: α = 0.87; Study 3: α = 0.84; Study 4: α = 0.71).</p></sec></sec></sec><sec id=\"S2.SS4\"><title>Attention Check</title><p>In Studies 1 and 2, participants were asked what Keith’s position was on the junk food tax. They could choose one of three options: “Really hates the junk food tax,” “Neutral,” and “Really likes the junk food tax.”</p></sec><sec id=\"S2.SS5\"><title>Affective Attraction</title><p>Measures of both affective attraction and willingness to affiliate were adapted from the Interpersonal Judgment Scale (<xref rid=\"B8\" ref-type=\"bibr\">Byrne, 1971</xref>), though in the present work, we differentiate between items that assess affective attraction from items that measure willingness to affiliate. Participants reported how they felt about the target on two 7-point scales (e.g., “How much do you like Keith Brown?”), with “not at all” and “very much” anchoring the low and high ends of the scale, respectively (internal reliability: Study 1: α = 0.89; Study 2: α = 0.72). In Studies 3 and 4, affective attraction was measured with a single item.</p></sec><sec id=\"S2.SS6\"><title>Affiliative Behavioral Willingness</title><p>Participants reported the extent to which they would be willing to pursue a friendship with the target on two additional 7-point scales (e.g., “Would you want to be Keith Brown’s friend?”), with “not at all” and “very much” anchoring the low and high ends of the scale, respectively. Because the responses were highly correlated, the responses were averaged to create a composite affiliative willingness score (internal reliability: Study 1: α = 0.84; Study 2: α = 0.80). In Studies 3 and 4, affiliative behavioral willingness was measured with a single item.</p></sec></sec><sec id=\"S3\"><title>Results</title><p>To see the results broken down by study, consult <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Material</xref>. We report here the results of a meta-analysis combining data from all four studies. For all analyses, we obtained effect sizes by calculating the partial correlation for the relevant term (<xref rid=\"B3\" ref-type=\"bibr\">Aloe and Thompson, 2013</xref>). We conducted random effects meta-analyses using the metafor package in R (<xref rid=\"B30\" ref-type=\"bibr\">Viechtbauer, 2010</xref>).</p><sec id=\"S3.SS1\"><title>Attitude Similarity and Attraction</title><p>We first meta-analyzed the extent to which initial attitudes toward the topical issue (e.g., a junk food tax) predicted affective attraction. Across the four studies, we found that, indeed, the more similar the participants’ attitudes were to the target, the more they liked the target, <italic>r</italic> = 0.46, <italic>p</italic> < 0.001, 95% CI: [0.25, 0.67] (see <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). There was, however, significant heterogeneity of effect sizes across studies, <italic>Q</italic>(3) = 24.54, <italic>p</italic> < 0.001. We provide a more extensive discussion of effect sizes heterogeneity in “General Discussion.”</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>A forest plot of the meta-analyzed effect of attitude similarity on affective attraction.</p></caption><graphic xlink:href=\"fpsyg-11-01919-g001\"/></fig></sec><sec id=\"S3.SS2\"><title>Moderation of the Link Between Affective Attraction and Willingness to Affiliate</title><p>We subjected the data to a regression analysis examining the impact of affective attraction, the attitude strength-related properties, and their two-way interaction on affiliative willingness. We meta-analyzed the two-way attraction by attitude properties interaction, <italic>r</italic> = 0.19, <italic>p</italic> < 0.001, 95% CI: [0.09, 0.29], finding it to be significant (see <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). There was no significant heterogeneity of effect sizes across studies, <italic>Q</italic>(3) = 3.52, <italic>p</italic> = 0.32. We also meta-analyzed the simple slopes at low and high levels of the attitude strength-related properties. When attitudes were relatively low in strength-related properties (-1 SD), affective attraction did not significantly predict affiliative willingness, <italic>r</italic> = 0.16, <italic>p</italic> = 0.08, 95% CI: [-0.02, 0.35]. There was significant heterogeneity of effect sizes across studies, <italic>Q</italic>(3) = 10.33, <italic>p</italic> = 0.02. However, when attitudes were relatively high in strength-related properties (+ 1 SD), there was a stronger relation between affective attraction and affiliative willingness, <italic>r</italic> = 0.46, <italic>p</italic> < 0.001, 95% CI: [0.30, 0.62]. There was again significant heterogeneity of effect sizes across studies, <italic>Q</italic>(3) = 14.39, <italic>p</italic> = 0.002.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>A forest plot of the meta-analyzed two-way interaction between affective attraction and attitude strength on affiliative willingness.</p></caption><graphic xlink:href=\"fpsyg-11-01919-g002\"/></fig></sec><sec id=\"S3.SS3\"><title>Moderation of the Attitude Similarity-Attraction Effect</title><p>We also examined whether we replicated previous findings that the antecedents to attitude strength moderated the attitude similarity-affective attraction effect (<xref rid=\"B16\" ref-type=\"bibr\">Montoya and Horton, 2013</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Sawicki and Wegener, 2018</xref>). Consistent with previous work, we found evidence for an attitude-by-strength-related properties interaction (see <xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>), <italic>r</italic> = 0.13, <italic>p</italic> = 0.01, 95% CI: [0.04, 0.23]. There was no significant heterogeneity of effect sizes across studies, <italic>Q</italic>(3) = 1.97, <italic>p</italic> = 0.58. Simple slopes revealed that though there was an effect of attitude on attraction when attitudes were relatively low in the attitude-strength properties (-1 SD), <italic>r</italic> = 0.22, <italic>p</italic> = 0.001, 95% CI: [0.09, 0.35]. There was an even stronger impact of attitudes when they were relatively high in the strength-related properties (+ 1 SD), <italic>r</italic> = 0.44, <italic>p</italic> < 0.001, 95% CI: [0.28, 0.61]. Though there was no significant heterogeneity of effect sizes when attitudes were relatively low in the strength-related properties, <italic>Q</italic>(3) = 6.03, <italic>p</italic> = 0.11, there was significant heterogeneity of effect sizes when attitudes were relatively high in the strength-related properties, <italic>Q</italic>(3) = 12.23, <italic>p</italic> = 0.01.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>A forest plot of the meta-analyzed two-way interaction between attitude and attitude strength on affective attraction.</p></caption><graphic xlink:href=\"fpsyg-11-01919-g003\"/></fig></sec></sec><sec id=\"S4\"><title>Discussion</title><p>This research provided meta-analytic evidence (across four studies) that attitude strength might not only influence the link between attitudes and attraction but also between affective attraction and willingness to initiate a friendship (i.e., behavioral attraction). The patterns for the attitude strength-related properties followed previous work demonstrating stronger influences of attitudes associated with high levels of various attitude properties (e.g., <xref rid=\"B21\" ref-type=\"bibr\">Philipp-Muller et al., 2020</xref>). We also replicated two previously established findings: that similarity breeds attraction and that attitude strength-related properties moderate this relation (e.g., <xref rid=\"B16\" ref-type=\"bibr\">Montoya and Horton, 2013</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Sawicki and Wegener, 2018</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Wallace et al., 2020</xref>).</p><p>These findings suggest that a willingness to pursue a relationship can be subtly increased by strengthening the attitudes on which that attraction is based. Also, those with dispositionally strong attitudes (e.g., those who are generally confident in their attitudes) might be more willing to pursue relationships with those who share their perspectives. This possibility could shed light on one mechanism by which individuals seem willing to engage in inappropriate affiliative behaviors, such as extramarital affairs or inappropriate workplace relationships. Those with attitudes high in confidence (or attitudes that are strong for another reason) may feel more willing to act on their attraction, even in instances where that would be inappropriate or harmful.</p><sec id=\"S4.SS1\"><title>Limitations</title><p>One limitation of the present work is that it examined a limited set of attitude features. Thus, it is possible that other attitude features not measured in the present work (e.g., centrality) would have different effects than the features measured. Previous work has found that many of the most commonly examined subjective attitude features fall onto one of two major factors when examined factor analytically: namely, an attitude’s embeddedness in one’s identity or an attitude’s internal consistency (<xref rid=\"B21\" ref-type=\"bibr\">Philipp-Muller et al., 2020</xref>). Attitude centrality, for example, consistently indicates an attitude’s embeddedness and loads with an attitude’s personal importance and its basis in morals and values. Thus, we would predict centrality to increase the link between affective attraction and willingness to affiliate, much as the strength-related properties did in the present work. We therefore consider the specific attitude features measured in the present work to be surrogates for the attitude strength-related properties. It also remains to be seen whether “structural” indicators of strength, such as objective knowledge, might play a similar role to the current subjective measures.</p><p>Another limitation is that we did not directly manipulate attitude similarity in any one study. Instead, participants encountered a target that held one particular position whose divergence from participants’ own attitudes determined the degree of attitude similarity. However, we did include multiple studies where the target held positions on both sides of an issue (i.e., in two studies, the target opposed a junk food tax, and in one study, the target supported the tax). Inclusion of a study that reverses the position of the target provides some indication that the effects are due to attitudes that vary in similarity to the position taken by the target, rather than one particular attitude position.</p><p>An additional potential limitation concerns the heterogeneity observed for some effects tested. Though effect sizes were heterogeneous across studies, the variability in our effects is not particularly striking, given how differences in samples, topics, changes to materials (<xref rid=\"B14\" ref-type=\"bibr\">Kenny and Judd, 2019</xref>), and even sampling error (<xref rid=\"B27\" ref-type=\"bibr\">Stanley and Spence, 2014</xref>) can introduce variability in effect sizes. Thus, we do not view heterogeneity of effects in this context as particularly concerning.</p></sec></sec><sec id=\"S5\"><title>Future Directions</title><p>The link between behavioral willingness and enacted behavior is not always strong (<xref rid=\"B19\" ref-type=\"bibr\">Montoya et al., 2018</xref>). The present work helped provide one piece of the attraction puzzle by demonstrating when we should expect affective attraction to impact willingness to affiliate.</p><sec id=\"S5.SS1\"><title>A Broader Model of Attraction and Affiliation</title><p>In order to better contextualize the current research, we end the article by discussing a broader view of when and how affective attraction might lead to affiliative behavior. We propose three paths by which affective attraction might result in affiliative behavior, informed by extant models. The path most related to the present work is that affective attraction might enhance willingness to affiliate (i.e., behavioral attraction) and that willingness to affiliate might enhance affiliative behavior. Additional routes unexplored in the present work include the impact of affective attraction on behavioral intentions to affiliate (e.g., <xref rid=\"B2\" ref-type=\"bibr\">Albarracín et al., 2001</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Ajzen and Fishbein, 2005</xref>) and the direct effect of attraction on enacted behavior (cf. <xref rid=\"B2\" ref-type=\"bibr\">Albarracín et al., 2001</xref>). Different contexts might make different routes more viable. For example, in risky situations, willingness to affiliate might predict behavior better than behavioral intentions (cf. <xref rid=\"B11\" ref-type=\"bibr\">Gibbons et al., 1998</xref>). Thus, affective attraction and behavioral attraction might have relatively equal impact on affiliative behavior (see <xref rid=\"B19\" ref-type=\"bibr\">Montoya et al., 2018</xref>). We only tested moderation of one of these routes by attitude properties, but we suspect that stronger attitudes might predict enhanced affiliative behavior through a number of different routes.</p><p>There might also be other moderators of the link between willingness to affiliate and enacted affiliation, such as the content of the attitudes upon which attraction is built. Attitudes related to activities that can be enacted together (e.g., shared hobbies) might be more likely to result in friendship pursuit than attitudes about more abstract matters (<xref rid=\"B34\" ref-type=\"bibr\">Werner and Parmelee, 1979</xref>). Another potential moderator is experience with relationship initiation. As experience with a risky situation increases, behavioral willingness (compared to more deliberative behavioral intentions) becomes a less proximal predictor of enacted behavior (<xref rid=\"B23\" ref-type=\"bibr\">Pomery et al., 2009</xref>). Thus, for those with extended experience initiating relationships being willing to affiliate might not be as robust a predictor of actual affiliation compared to relationship novices. Of course, additional data will be required to empirically test the various possibilities, but the present data offer evidence for one aspect of this broader approach. That is, when liking is based on a shared attitude that is relatively strong, this liking will better predict willingness to affiliate with the other person. Thus, perhaps when two strangers alight upon a common attitude, it is the strength of that attitude – and not just the degree of liking – that determines whether a relationship will bloom.</p></sec></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>The datasets generated for this study are available on request to the corresponding author.</p></sec><sec id=\"S7\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by The Ohio State University Office of Responsible Research Practices. The participants provided their written informed consent to participate in this study.</p></sec><sec id=\"S8\"><title>Author Contributions</title><p>AP-M, LW, and DW developed the project concept. AP-M, KP, and VS performed the data collection. AP-M performed the data analyses and drafted the manuscript. LW and DW provided integral revisions to the initial submission. All authors contributed to study design and revising the manuscript.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This research was supported by the Social Sciences and Humanities Research Council of Canada and the National Science Foundation Graduate Research Fellowship Program (DGE-1343012).</p></fn></fn-group><ack><p>We would like to thank the members of the Attitudes and Persuasion Lab and the Group for Attitudes and Persuasion at Ohio State University for their helpful insight and ideas.</p></ack><fn-group><fn id=\"footnote1\"><label>1</label><p>Results of Study 4 were included in a previously published article (<xref rid=\"B32\" ref-type=\"bibr\">Wallace et al., 2020</xref>). However, the particular analyses presented in this work were not previously examined or published.</p></fn><fn id=\"footnote2\"><label>2</label><p>These demographic variables were not collected in Study 3.</p></fn></fn-group><sec id=\"S11\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fpsyg.2020.01919/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fpsyg.2020.01919/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"TS1\"><media xlink:href=\"Table_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Ajzen</surname><given-names>I.</given-names></name><name><surname>Fishbein</surname><given-names>M.</given-names></name></person-group> (<year>2005</year>). “<article-title>The influence of attitudes on behavior</article-title>,” in <source><italic>The Handbook of Attitudes</italic></source>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>Albarracín</surname><given-names>D.</given-names></name><name><surname>Johnson</surname><given-names>B. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Pharmacol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Pharmacol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Pharmacol.</journal-id><journal-title-group><journal-title>Frontiers in Pharmacology</journal-title></journal-title-group><issn pub-type=\"epub\">1663-9812</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32848799</article-id><article-id pub-id-type=\"pmc\">PMC7431688</article-id><article-id pub-id-type=\"doi\">10.3389/fphar.2020.01209</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Pharmacology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Vitamin K2 Can Rescue the Dexamethasone-Induced Downregulation of Osteoblast Autophagy and Mitophagy Thereby Restoring Osteoblast Function <italic>In Vitro</italic> and <italic>In Vivo</italic>\n</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Liang</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/1049551\"/></contrib><contrib contrib-type=\"author\"><name><surname>Shi</surname><given-names>Xiang</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Weng</surname><given-names>She-Ji</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Xie</surname><given-names>Jun</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Tang</surname><given-names>Jia-Hao</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yan</surname><given-names>De-Yi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Bing-Zhang</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Xie</surname><given-names>Zhong-Jie</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wu</surname><given-names>Zong-Yi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yang</surname><given-names>Lei</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><xref ref-type=\"author-notes\" rid=\"fn001\">\n<sup>*</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/947392\"/></contrib></contrib-group><aff id=\"aff1\">\n<sup>1</sup>\n<institution>Department of Orthopedic Surgery, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University</institution>, <addr-line>Wenzhou</addr-line>, <country>China</country>\n</aff><aff id=\"aff2\">\n<sup>2</sup>\n<institution>Key Laboratory of Orthopedics of Zhejiang Province</institution>, <addr-line>Wenzhou</addr-line>, <country>China</country>\n</aff><aff id=\"aff3\">\n<sup>3</sup>\n<institution>School of Mental Health, Wenzhou Medical University</institution>, <addr-line>Wenzhou</addr-line>, <country>China</country>\n</aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Lina Ghibelli, University of Rome Tor Vergata, Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Luzia Teixeira Sousa, Federal University of Ceara, Brazil; Ewa Tomaszewska, University of Life Sciences of Lublin, Poland; Thorsten Schinke, University Medical Center Hamburg-Eppendorf, Germany</p></fn><corresp id=\"fn001\">*Correspondence: Lei Yang, <email xlink:href=\"mailto:cl18958749973@163.com\" xlink:type=\"simple\">cl18958749973@163.com</email>\n</corresp><fn fn-type=\"other\" id=\"fn002\"><p>This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1209</elocation-id><history><date date-type=\"received\"><day>06</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>24</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Chen, Shi, Weng, Xie, Tang, Yan, Wang, Xie, Wu and Yang</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Chen, Shi, Weng, Xie, Tang, Yan, Wang, Xie, Wu and Yang</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Chronic long-term glucocorticoids (GC) use is associated with glucocorticoid-induced osteoporosis (GIOP) by inhibiting the survival and impairing the functions of osteoblasts. Autophagy and mitophagy play key roles in osteoblast differentiation, mineralization and survival, and mounting evidence have implicated osteoblast autophagy and mitophagy as a novel mechanism in the pathogenesis of GIOP. Vitamin K2 (VK2) is an essential nutrient supplement that have been shown to exert protective effects against osteoporotic bone loss including GIOP. In this study, we showed that the glucocorticoid dexamethasone (Dex) deregulated osteoblast autophagy and mitophagy by downregulating the expression of autophagic and mitophagic markers LC3-II, PINK1, Parkin. This consequently led to inhibition of osteoblast differentiation and mineralization function <italic>in vitro</italic>. Interestingly, co-treatment with VK2 significantly attenuated the Dex-induced downregulation of LC3-II, PINK1, Parkin, thereby restoring autophagic and mitophagic processes and normal osteoblastic activity. In addition, using an established rat model of GIOP, we showed that VK2 administration can protect rats against the deleterious effects of Dex on bone by reinstating autophagic and mitophagic activities in bone tissues. Collectively, our results provide new insights into the role of osteoblast autophagy and mitophagy in GIOP. Additionally, the use of VK2 supplementation to augment osteoblast autophagy/mitophagy may significantly improve clinical outcomes of GIOP patients.</p></abstract><kwd-group><kwd>Vitamin K2</kwd><kwd>dexamethasone</kwd><kwd>osteoblast</kwd><kwd>mitophagy</kwd><kwd>glucocorticoid-induced osteoporosis (GIOP)</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Natural Science Foundation of China<named-content content-type=\"fundref-id\">10.13039/501100001809</named-content></funding-source><award-id rid=\"cn001\">81772348</award-id></award-group><award-group><funding-source id=\"cn002\">Science and Technology Department of Zhejiang Province<named-content content-type=\"fundref-id\">10.13039/501100008990</named-content></funding-source><award-id rid=\"cn002\">2016C37122</award-id></award-group></funding-group><counts><fig-count count=\"7\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"50\"/><page-count count=\"13\"/><word-count count=\"5704\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Glucocorticoids (GC), are widely used clinically for the treatment of allergic and autoimmune diseases and cancer owing to their anti-inflammatory and immunosuppressive properties (<xref rid=\"B32\" ref-type=\"bibr\">Rizzoli and Biver, 2015</xref>). However, chronic or prolonged GC treatment exhibits detrimental effects on the skeleton causing rapid bone loss, progressive decrease in bone mineral density leading to bone fragility, often termed glucocorticoid-induced osteoporosis (GIOP) (<xref rid=\"B36\" ref-type=\"bibr\">Seibel et al., 2013</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Hartmann et al., 2016</xref>). The molecular mechanism underlying GIOP is complex and has yet to be fully unraveled. This is because endogenous GC activities are necessary for the maintenance of bone homeostasis but excess GC impede bone formation by impairing osteoblast differentiation, mineralization function, and survival. The deleterious effects of GC on osteoblasts are now considered to play a major role in the development of GIOP (<xref rid=\"B43\" ref-type=\"bibr\">Weinstein et al., 1998</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Canalis et al., 2007</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Hartmann et al., 2016</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Rizzoli, 2017</xref>).</p><p>Autophagy is a highly conserved homeostatic process that plays an essential part in the regulation of osteoblast differentiation and mineralization function (<xref rid=\"B25\" ref-type=\"bibr\">Liu et al., 2013</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Nollet et al., 2014</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Han et al., 2018</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Lian et al., 2018</xref>). It has consistently been shown as a protective mechanism in maintaining osteoblast viability by allowing cells to survive various stresses (<xref rid=\"B41\" ref-type=\"bibr\">Todde et al., 2009</xref>; <xref rid=\"B45\" ref-type=\"bibr\">White and Lowe, 2009</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Gu et al., 2016</xref>; <xref rid=\"B46\" ref-type=\"bibr\">Yang et al., 2016</xref>; <xref rid=\"B50\" ref-type=\"bibr\">Zhu et al., 2019</xref>; <xref rid=\"B49\" ref-type=\"bibr\">Zhao et al., 2020</xref>). Furthermore, GC usage have been reported to inhibit osteoblast autophagy and this effect plays a pivotal role in the pathogenesis of GIOP (<xref rid=\"B12\" ref-type=\"bibr\">Han et al., 2018</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Lian et al., 2018</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Gremminger et al., 2019</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Wang et al., 2019</xref>). Recently a specialized form of autophagy that specifically degrades dysfunctional or redundant mitochondria, termed mitophagy, was found to contribute to the mineralization function of osteoblasts (<xref rid=\"B30\" ref-type=\"bibr\">Pei et al., 2018a</xref>). Under homeostatic conditions, PTEN induced putative kinase 1 (PINK1) translocates to the inner membrane of the mitochondria where it is rapidly degraded. When mitochondria are damaged (and becomes depolarized/loses membrane potential), PINK1 accumulates on the outer membrane where it phosphorylates ubiquitin and other mitochondrial outer membrane proteins, and facilitates the rapid recruitment of cytosolic Parkin, an E3 ubiquitin ligase, to the damaged mitochondria (<xref rid=\"B49\" ref-type=\"bibr\">Zhao et al., 2020</xref>). Multiple mitochondrial outer membrane proteins are ubiquitinated by Parkin resulting in the recruitment of ubiquitin-binding protein, p62/SQSTM1. The linkage of p62 to ubiquitin on the mitochondria and LC3-II (the lipidated form of LC3) on autophagosomes provides a physical attachment point for mitophagy (<xref rid=\"B8\" ref-type=\"bibr\">Geisler et al., 2010</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Palikaras et al., 2018</xref>), and disruption of either PINK1 or Parkin leads to the impaired mitophagy (<xref rid=\"B11\" ref-type=\"bibr\">Han et al., 2015</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Lazarou et al., 2015</xref>). Interestingly, impaired mitophagic function have been reported to negatively impact osteoblast differentiation and mineralization function <italic>in vitro</italic> (<xref rid=\"B38\" ref-type=\"bibr\">Shen et al., 2018b</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Jing et al., 2020</xref>) and the restoration of mitophagy helps alleviate steriod-induced bone loss <italic>in vivo</italic> (<xref rid=\"B48\" ref-type=\"bibr\">Zhang et al., 2020</xref>). Despite these encouraging reports, the precise role of autophagy and mitophagy in osteoblastic differentiation and function has not been thoroughly explored.</p><p>A more complete understanding of the effects of GC on osteoblast autophagy/mitophagy will be paramount in the exploration of new therapeutic strategy for the treatment of GIOP. Vitamin K2 (VK2) is a fat-soluble vitamin that have been used clinically to treat osteoporotic bone loss. Various reports have further demonstrated that dietary VK2 supplementation is associated with improved bone formation status, reduced risk of GIOP, and lowered the prevalence of osteoporotic fracture (<xref rid=\"B35\" ref-type=\"bibr\">Sasaki et al., 2005</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Iwamoto et al., 2010</xref>; <xref rid=\"B44\" ref-type=\"bibr\">Weng et al., 2019</xref>). Recent findings further indicate that the pro-osteoblastic effects of VK2 is mediated by enhanced autophagic activities (<xref rid=\"B23\" ref-type=\"bibr\">Li et al., 2019</xref>), but whether mitophagy is also associated with VK2’s beneficial effects in osteogenesis remains to be determined. In this study, we aim to investigate the potential beneficial effects of VK2 against the deleterious effects of GC (Dexamethasone; Dex) on osteoblasts particularly in terms of mitophagy and autophagy.</p></sec><sec sec-type=\"materials|methods\" id=\"s2\"><title>Materials and Methods</title><sec id=\"s2_1\"><title>Reagents, Antibodies, and Media</title><p>Vitamin K2 (VK2), Dexamethasone (Dex), and 3-Methyladenine (3-MA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against alpha-1 type I collagen (COL1A1), Runt-related transcription factor 2 (Runx2), and Osteocalcin (Ocn) were from Abcam (Cambridge, UK). The primary antibody against the mitochondrial import receptor subunit TOM20 was procured from Santa Cruz Biotechnology (Dallas, TX, USA). Primary antibodies against LC3-I/II, p62, PINK1, Parkin and GADPH were obtained from Cell Signaling Technology (Danver, MA, USA). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), and penicillin/streptomycin were purchased from Gibco BRL (Thermo Fisher Scientific; Waltham, MA, USA). Monodansylcadaverine (MDC) was acquired from Solarbio Science & Technology (Beijing, China). All other chemicals used were of analytical grade complying with tissue and cell culture standards.</p></sec><sec id=\"s2_2\"><title>Cell Culture</title><p>Primary rat osteoblasts were isolated <italic>via</italic> sequential trypsin-collagenase digestion of excised calvarial bone from 1-day-old neonatal Sprague Dawley (SD) rats as previously described (<xref rid=\"B2\" ref-type=\"bibr\">Bakker and Klein-Nulend, 2012</xref>). Primary rat calvarial osteoblasts were cultured in complete DMEM (1% (v/v) penicillin–streptomycin and 10% (v/v) FBS) humidified conditions of 5% CO<sub>2</sub> at 37°C. The medium was changed every other day and cells were passaged or used for downstream experiments upon reaching 80–90% confluence.</p></sec><sec id=\"s2_3\"><title>Cytotoxicity Assay</title><p>The effect of Dex and VK2 on the viability of osteoblasts was determined using the Cell Counting Kit-8 (CCK-8) assay (MedChemExpress LLC; Monmouth Junction, NJ, USA). Primary calvarial osteoblasts seeded in 96-well plate at density of 5×10<sup>3</sup> cells per well and divided into the following treatment groups: Dex (1, 5, 10, or 20 μM) for 12 and 24 h; VK2 (10<sup>−8</sup>, 10<sup>−7</sup>, 10<sup>−6</sup>, 10<sup>−5</sup>, or 10<sup>−4</sup> M) for 24 and 48 h; and pre-treatment with Dex (10 μM) for 24 h then treated with VK2 (10<sup>−6</sup> M) for further 48 h. Untreated cells were used as controls. At the end of the experimental period, 10 μl of CCK8 reagent was added to each well and for 2 further hours. The absorbance or optical density (OD) at wavelength of 450 nm were measured on a Multiskan Go Microplate Spectrophotometer (Thermo Fisher Scientific).</p></sec><sec id=\"s2_4\"><title>Osteoblast Differentiation and Mineralization Assays</title><p>Primary calvarial osteoblasts were seeded at a density of 5×10<sup>4</sup> cells per well in a 24-well plate. For the drug treatment, cells were pre-treated with 10 μM Dex for 24 h, then treated with 10<sup>−6</sup> M VK2 without or with 5 mM 3-MA for 48 h as indicated. After treatment, cells were cultured under osteogenic conditions (complete DMEM containing 20 μM ascorbic acid and 10 mM β-glycerophosphate) with osteogenic media replaced every other day. Total cellular proteins were extracted on the 7<sup>th</sup> day of osteogenesis for western blot analysis of proteins involved in osteoblast differentiation. Alkaline Phosphatase (ALP) activity was measured after 7 days of differentiation using the ALP Staining Kit (Beyotime Institute of Biotechnology; Jiangsu, China). For mineralization and bone nodule formation, cells were differentiated under osteogenic conditions for 21 days, fixed and then stained with Alizarin Red S (ARS) solution (Solarbio Science & Technology). The absorbance at 520 nm for ALP and at 570 nm for ARS staining was detected using a microplate reader.</p></sec><sec id=\"s2_5\"><title>Western Blot Analyses</title><p>Total cell protein was extracted from cultured cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) containing protease and phosphatase inhibitors (Sigma-Aldrich) for 30 min at 4°C. Cell lysates were cleared by centrifugation and protein concentration determined by the BCA Protein Assay Kit (Beyotime Institute of Biotechnology) as per manufacturer’s instruction. For each sample, 20 μg of extracted protein (diluted in SDS Sampling buffer and denatured by boiling for 5 min) were resolved on 10–15% SDS–polyacrylamide gel electrophoresis gel. Separated proteins then transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore; Burlington, MA, USA) overnight at 4°C. PVDF membranes were blocked with 5% skim milk diluted in Tris buffered saline with 0.1% Tween 20 (TBST) for 2 h at room temperature and then incubated with indicated primary antibodies for 12 h at 4°C. After extensive washes with TBST, membranes were incubated with corresponding HRP-conjugated secondary antibodies for 4 h at room temperature. Proteins were visualized by enhanced chemiluminescence and imaged on a ChemiDoc XRS+ (Bio-Rad; Hercules, CA, USA). Protein bands were quantified by densitometry analysis using Image Lab V3.0 software (Bio-Rad).</p></sec><sec id=\"s2_6\"><title>Immunofluorescence</title><p>After treatment period cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature and permeabilized with 0.5% (v/v) Triton X-100 in PBS for 20 min. Non-specific antibody binding was blocked with 1% (w/v) goat serum albumin for 1 h at room temperature and then incubated with indicated antibodies (the dilution for the antibodies is 1:200 respectively) in 0.2% BSA-PBS overnight at 4°C with gentle mechanical rocking. Cells were washed extensively and then incubated with fluorescence conjugated secondary antibodies (Alexa Fluor 488 or 546; Thermo Fisher Scientific) for 1 h at room temperature in the dark. Nuclei were stained with DAPI for 5 min at room temperature. Fluorescence images were captured on an Olympus BX53 fluorescence microscope (Olympus Life Science; Tokyo, Japan) and the level of expression was determined using integrated optical density (IOD) using Image-Pro Plus software (Media Cybernetics, Inc; Rockville, MD, USA). For MDC staining, primary calvarial osteoblasts cultured in 24-well plates and treated as indicated for 2 days were incubated with MDC reagent for 45 min at 37°C and fluorescence images captured as described earlier.</p></sec><sec id=\"s2_7\"><title>Transmission Electron Microscopy (TEM)</title><p>Treated primary calvarial osteoblasts were fixed in 2.5% glutaraldehyde for 12 h at 4°C, post‐fixed in 2% osmium tetroxide for 1 h (<xref rid=\"B7\" ref-type=\"bibr\">Florencio-Silva et al., 2017</xref>), and then stained with 2% uranyl acetate for 1 h at room temperature. Cells were dehydrated in cold graded ethanol series (10 min each of: 30, 50, 70, 80, 90, and 100% ethanol) and then washed three times with 100% acetone (20 min each time with gentle rocking). Cells were then embedded in araldite epoxy resin, semi-thin sections were cut and stained with toluidine blue. TEM images were captured on a Hitachi Field Emission Transmission Electron Microscope (Hitachi High-Technologies Corp; Tokyo, Japan).</p></sec><sec id=\"s2_8\"><title>Rat Model of Glucocorticoid-Induced Osteoporosis (GIOP)</title><p>All animal experiments were approved by the Animal Ethics Committee of The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, and conducted pursuant to the criteria outlined in the Guide for the Care and Use of Laboratory Animals (NIH, Bethesda, MD, USA). Forty 3-month-old SD male rats were purchased from Shanghai Laboratory Animal Center (SLACCAS; Shanghai, China) and housed in ventilated cages in groups of 5 under SPF conditions of 22–25°C with 12 h/day light duration. All animals had free access to tap water and standard rodent diet (containing 2.5% casein, 0.8% phosphorus, 1% calcium, 70–80% carbohydrates, and 5% fat) provided by Provimi Kliba, AG (Kaiseraugst, Switzerland). After one week of acclimatization, the rats were evenly and randomly divided into four groups (n = 10 per group): Sham (Vehicle) control group and three GIOP experimental groups. All rats in the GIOP groups received daily intraperitoneally injection of Dex (5mg/kg) for 4 weeks (Due to our results <italic>in vitro</italic> that high-dose and long-term use of Dex inhibited osteoblast mitophagy, we exerted the concentration of Dex of 5mg/kg/day to induce osteoblast mitophagy inhibiton <italic>in vivo</italic>). Rats in Control group received daily injections of PBS for the same period. After 4 weeks of Dex treatment, all rats in the GIOP groups were screened for osteoporotic bone loss by X-ray radiograph imaging and subsequently randomly assigned into the following treatment groups: Vehicle, VK2 (30 mg/kg), and VK2 + 3-MA (15 mg/kg). 3-MA is a widely acknowledged autophagy inhibitor. The aim of 3-MA administration is to block the pro-autophagic effects of VK2. VK2 and 3-MA were injected intraperitoneally daily for 8 weeks after which all rats were euthanized. Bilateral femurs were excised, cleaned of soft tissues and then fixed in 4% PFA prior to micro-computed tomography (CT) and histological assessment.</p></sec><sec id=\"s2_9\"><title>Micro-CT Analysis</title><p>Microstructural analysis of the distal femoral bones was carried out on a cabinet cone-beam micro-CT system and associated software (μCT 50, Scanco Medical; Brüttisellen, Switzerland). Images were acquired at a voltage of 70 kV, electric current of 200 μA and a spatial resolution of 14.8 mm in all directions. Three-dimensional reconstructed images were generated and the volume of interest (VOI) analyzed included the trabecular compartment 2 mm below the highest point of the growth plate to distal 100 CT slices. Quantitative bone parameters were assessed within the VOI included percentage bone volume to tissue volume (BV/TV), the mean trabecular thickness (Tb.Th, mm), the mean trabecular number (Tb.N, 1/mm), the mean trabecular separation (mean width of the medullary cavity between trabeculae, Tb.Sp, mm) using Evaluation V6.5-3 in micro-CT system and associated software.</p></sec><sec id=\"s2_10\"><title>Histology, Immunohistochemistry and Immunofluorescence Staining</title><p>For histological and immunohistochemical (IHC) assessments, fixed femoral bone tissues were decalcified in 10% EDTA for 3 weeks, dehydrated in graded ethanol series (70 to 100%), cleared in xylene, and paraffin-embedded with the long axis of the bone parallel to the base plane to preserve anatomical orientation. Longitudinal serial sections of 4 μm thick were cut and mounted on poly-lysine coated microscope slides and then subjected to hematoxylin and eosin (H&E), and Masson’s trichrome staining as per standard laboratory protocols. For IHC staining, 6 μm sections were prepared and incubated with primary antibodies against LC3-II, Parkin, and OCN (the dilution for the antibodies is 1:200 respectively) and the immuno-reactivities in the sections were detected using a horseradish peroxidase detection system in accordance with manufacturer’s protocol (Vector Laboratories; Burlingame, CA, USA). For immunofluorescence staining, 6 μm thick sections were deparaffinized, rehydrated and then stained with primary antibody against LC3-II (the dilution for the LC3-II is 1:200) overnight at 4°C and then with secondary fluorescence antibody for 1 hour at room temperature in the dark. Nuclei were counterstained with DAPI for 5 min. Tissue sections were imaged under an Olympus BX53 light/fluorescence microscope equipped with a Olympus DP71 digital color camera (Olympus Life Science). The level of expression was determined using integrated optical density (IOD) using Image-Pro Plus software (Media Cybernetics, Inc; Rockville, MD, USA).</p></sec><sec id=\"s2_11\"><title>Statistical Analyses</title><p>All statistical analyses were performed using the GraphPad Prism software (San Diego, CA, USA) and data presented herein are expressed as the mean ± standard error of mean (SEM) from at least three experimental repeats. Two tailed Student’s t-test was used to compare means between two groups and one-way ANOVA with Bonferroni or Dunnett corrections for multiple comparisons where appropriate. Differences were determined to be statistically significant when p < 0.05 unless otherwise stated.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec id=\"s3_1\"><title>Dexamethasone (Dex) Exhibits Dose- and Time-Dependent Effects on Osteoblast Autophagy and Mitophagy</title><p>We first assessed the biological effects of Dex on osteoblast viability using the CCK-8 assay. As shown in the <xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figure 1A</bold>\n</xref>, Dex exhibited an inhibitory effect to osteoblast viability in a dose-dependent manner at 12 and 24 h after treatment, with an IC50 of about 20 μM (p<0.01). Next, we used western blot to analyze the expression of proteins involved in autophagy and mitophagy in Dex-treated osteoblasts. To our surprise, we found that low concentrations of Dex treatment (1 and 5 μM) for 12 h upregulated the expression of autophagy and mitophagy related proteins LC3-II, PINK1 and Parkin (p<0.01). This was also associated with a concomitant reduction in the expression of p62 (<xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figures 1B, C</bold>\n</xref>). On the other hand, treatment with 10 μM Dex potently inhibited the expression of LC3-II, PINK1 and Parkin. At 24 h Dex treatment, the expression of LC3-II, PINK1, and Parkin were all markedly reduced (p<0.01) (<xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figures 1D, E</bold>\n</xref>). We further explored the inhibitory effect of Dex on autophagy by staining cells with MDC, a fluorescent marker that accumulates in autophagic vacuoles. As shown in <xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figure 1F</bold>\n</xref>, the number of MDC-stained autophagic vacuoles were dose-dependently reduced following treatment with Dex for 24 h (<xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figures 1F, G</bold>\n</xref>). Furthermore, extensive colocalization of TOM20, a mitochondrial outer membrane protein, with LC3-stained autophagosomes in untreated osteoblasts was observed indicative of active mitophagy (<xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figure 1H</bold>\n</xref>). Treatment with 10 μM Dex for 24 h significantly attenuated the expression of LC3 indicating the inhibition of autophagosome formation and consequently impairment of mitophagy.</p><fig id=\"f1\" position=\"float\"><label>Figure 1</label><caption><p>Dexamethasone (Dex) exhibits dose- and time-dependent effects on osteoblast autophagy and mitophagy. <bold>(A)</bold> Effects of different concentrations of Dexamethasone on the growth of osteoblasts after being treated for 12 h or 24 h, as measured by CCK8 assays; Western blotting results showed the change of mitophagy-related protein expression levels after 12 h <bold>(B)</bold> or 24 h <bold>(D)</bold> treatment; <bold>(C, E)</bold> Analysis of western blot results of <bold>(B, D)</bold>; <bold>(F, G)</bold> MDC is a marker for autolysosomes. MDC staining was used to confirm the alteration of the abundance of autophagic vacuoles in aforementioned group; <bold>(H)</bold> Representative images of immunofluorescence double staining of LC3 and Tom20 in osteoblasts. Data was expressed as mean ± SEM, n = 5; *p < 0.05, <sup>#</sup>p < 0.01 vs. control group.</p></caption><graphic xlink:href=\"fphar-11-01209-g001\"/></fig></sec><sec id=\"s3_2\"><title>Vitamin K2 (VK2) Activates Both Autophagy and Mitophagy in Osteoblasts</title><p>As with Dex, we first examined potential cytotoxic effects of VK2 using the CCK-8 assay. Treatment with increasing concentrations of VK2 (10<sup>−8</sup> to 10<sup>−4</sup> M) did not induce any deleterious effects on primary osteoblast viability at 24 and 48 h after treatment (<xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figure 2A</bold>\n</xref>). In fact, cell proliferation was elevated when cells were treated with 10<sup>−7</sup> to 10<sup>−5</sup> M at 24 h and with 10<sup>−8</sup> to 10<sup>−5</sup> M at 48 h. Having established that VK2 has no adverse effect cell viability/proliferation, we next examined whether VK2 can induced autophagy/mitophagy in treated primary osteoblasts. As shown in <xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figures 2B, C</bold>\n</xref>, treatment of primary osteoblasts with VK2 for 48 h upregulated the expression of LC3-II, PINK1 and Parkin. The expression of p62 was also accordingly decreased (p<0.01). The induction of autophagy by VK2 was also confirmed by immunofluorescence staining for LC3 autophagosomes with the most potent inductive effect at the concentration of 10<sup>−6</sup> M for 48 h (<xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figures 2D, E</bold>\n</xref>). This was further corroborated by TEM analysis showing increased formation of double-membraned vesicles in treated osteoblasts (<xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figure 2F</bold>\n</xref>). Collectively, VK2 treatment induces autophagy/mitophagy in primary calvarial osteoblasts and based on these results VK2 at concentration of 10<sup>−6</sup> M was used for subsequent experiments.</p><fig id=\"f2\" position=\"float\"><label>Figure 2</label><caption><p>Vitamin K2 (VK2) activates both autophagy and mitophagy in osteoblasts. <bold>(A)</bold> Effects of different concentrations of Vitamin K2 on the growth of osteoblasts after being treated for 24 h or 48 h, as measured by CCK8 assays; <bold>(B)</bold> Western blot results showed the change of mitophagy-related protein expression; <bold>(C)</bold> Analysis of the western blotting results in <bold>(B)</bold>; <bold>(D, E)</bold> Representative images and quantified analysis of immunofluorescence staining of LC3 in osteoblasts; <bold>(F)</bold> Mitophagosomes were detected by transmission electron microscopy (×10000) in osteoblasts. (Black arrow: mitophagosome). Data was expressed as mean ± SEM, n = 5; <sup>#</sup>p < 0.01 vs. control group.</p></caption><graphic xlink:href=\"fphar-11-01209-g002\"/></fig></sec><sec id=\"s3_3\"><title>VK2 Alleviates Inhibitory Effects of Dex on Osteoblast Autophagy and Mitophagy</title><p>Given the stimulatory effect of VK2 on osteoblast mitophagy, we next explored the possibility for VK2 in antagonizing the inhibitory effect of Dex against autophagic/mitophagic activities. To this end, primary osteoblasts were initially treated with 10 μM Dex for 24 h, then 10<sup>−6</sup> M VK2 without/with 5 mM 3-MA was added into the culture medium for another 48 h. The 3-MA is a widely acknowledged inhibitor of autophagy and thus used to reverse the pro-autophagic/mitophagic effects of VK2. As shown in <xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figure 3A</bold>\n</xref>, 10<sup>−6</sup> M VK2 can protect cells from the deleterious effects of 10 μM Dex on cell viability (p<0.01), whereas the addition of 3-MA partly attenuated the protective effects of VK2 treatment (p<0.01). Additionally, co-treatment of VK2 as indicated attenuated Dex-induced downregulation of LC3-II, PINK1, and Parkin protein expression (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figure 3B</bold>\n</xref>). Consistently, the upregulation of p62 following Dex treatment was also abrogated following co-treatment with VK2 treatment as well as the inhibition of autophagosome formation (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figures 3C–F</bold>\n</xref>). Immunofluorescence of co-localization of LC3 and TOM20 and TEM analysis further showed that treatment of cells with VK2 can alleviate the inhibitory effect of Dex on autophagosome formation mitophagy (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figures 3G, H</bold>\n</xref>). The co-treatment of 5 mM 3-MA potently blocked the upregulation of LC3-II, PINK1, and Parkin (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figure 3B</bold>\n</xref>), and subsequent induction of autophagy and mitophagy by VK2. Together, the above results indicate that VK2 can alleviate the inhibitory effect of Dex on osteoblast autophagy/mitophagy by preventing the downregulation of LC3-II, PINK1 and Parkin protein expressions.</p><fig id=\"f3\" position=\"float\"><label>Figure 3</label><caption><p>VK2 alleviates inhibitory effects of Dex on osteoblast autophagy and mitophagy. Primary osteoblasts were initially treated with 10 μM Dex for 24 h, then 10<sup>−6</sup> M VK2 without/with 5 mM 3-MA was added into the culture medium for another 48 h. <bold>(A)</bold> Effects of Vitamin K2 on the growth of osteoblasts under the inhibition of Dexamethasone with/without 3-MA, as measured by CCK8 assays; <bold>(B)</bold> Western blot results showed the alteration of mitophagy-related protein expression levels of the above indicators; <bold>(C, D)</bold> Representative images and quantified analysis of immunofluorescence staining of LC3 in osteoblasts; <bold>(E, F)</bold> MDC staining was used to confirm the alteration of the abundance of autophagic vacuoles in aforementioned group; <bold>(G)</bold> Representative images of immunofluorescence double staining of LC3 and Tom20 in osteoblasts; <bold>(H)</bold> Mitophagosomes were detected by transmission electron microscopy (×10,000) in osteoblasts. (Black arrow: mitophagosome). Data was expressed as mean ± SEM, n = 5; *p < 0.01 vs. control group, <sup>#</sup>p < 0.01 vs. DEX group, <sup>&</sup>p < 0.01 vs. DEX+VK2 group.</p></caption><graphic xlink:href=\"fphar-11-01209-g003\"/></fig></sec><sec id=\"s3_4\"><title>Autophagy and Mitophagy Are Both Necessary for Osteoblast Differentiation and Mineralization</title><p>Next the effects Dex and VK2 against the osteogenic differentiation of osteoblasts were evaluated in our study. As shown in <xref ref-type=\"fig\" rid=\"f4\">\n<bold>Figures 4A, B</bold>\n</xref>, compared with untreated controls, Dex treatment significantly reduced osteoblast differentiation (<xref ref-type=\"fig\" rid=\"f4\">\n<bold>Figure 4A</bold>\n</xref>) and mineralization function (<xref ref-type=\"fig\" rid=\"f4\">\n<bold>Figure 4B</bold>\n</xref>) respectively (p<0.01). On the other hand, treatment with VK2 countered the inhibition of Dex on osteoblast differentiation and mineralization (p<0.01). The positive effect of VK2 against Dex was further corroborated by western blot and immunofluorescence analysis of osteoblast marker protein expression. Consistent with cellular effects, the downregulation of Runx2, Ocn, and COL1A1 by Dex can be prevented with treatment with VK2 (<xref ref-type=\"fig\" rid=\"f4\">\n<bold>Figures 4C–E</bold>\n</xref>, p<0.01). However, the pro-osteoblastic effects of VK2 against Dex can be further abolished with treatment with 3-MA. Thus, the results presented here, shows that VK2 can counter the anti-osteoblastic effects of Dex and that functional autophagic/mitophagic activity is necessary for efficient osteoblast differentiation and mineralization function.</p><fig id=\"f4\" position=\"float\"><label>Figure 4</label><caption><p>Autophagy and mitophagy are both necessary for osteoblast differentiation and mineralization. Primary osteoblasts were initially treated with 10 μM Dex for 24 h, then 10<sup>−6</sup> M VK2 without/with 5 mM 3-MA was added into the culture medium for another 48 h. After treatment, the osteoblasts were cultured in osteogenic medium for indicated days. <bold>(A)</bold> Osteoblastic functions were detected by ALP staining on the 7th day; <bold>(B)</bold> and mineralization degree by the ARS staining on the 21st day; <bold>(C)</bold> The osteogenic protein expression levels of the above indicators were detected by western-blotting and <bold>(E)</bold> the analysis of the results; <bold>(D)</bold> Representative immunofluorescence images of the above indicators, counterstained with Dapi. Data was expressed as mean ± SEM, n = 5; *p < 0.01 vs. control group, <sup>#</sup>p < 0.01 vs. DEX group, <sup>&</sup>p< 0.01 vs. DEX+VK2 group.</p></caption><graphic xlink:href=\"fphar-11-01209-g004\"/></fig></sec><sec id=\"s3_5\"><title>Protective Effects of VK2 Against Dex-Induced Bone Loss (GIOP) in Rats</title><p>We next assessed whether the protective effects of VK2 against the deleterious effect of Dex <italic>in vitro</italic> can be recapitulated <italic>in vivo</italic> using the rat GIOP model. SD rats were treated with Dex for 4 weeks to induce bone loss and then administered with VK2 or VK2 + 3-MA for further 8 weeks (<xref ref-type=\"fig\" rid=\"f5\">\n<bold>Figure 5A</bold>\n</xref>). Three-dimensional reconstructions of micro-CT scans showed extensive trabecular bone loss in the distal femur following Dex administration as compared to Sham controls (<xref ref-type=\"fig\" rid=\"f5\">\n<bold>Figure 5B</bold>\n</xref>). This was confirmed by morphometric analyses which showed marked reduction in bone volume (BV/TV) and trabecular number, increased trabecular spacing, but no change in trabecular thickness (<xref ref-type=\"fig\" rid=\"f5\">\n<bold>Figure 5C</bold>\n</xref>, p<0.01). On the other hand, rats that were administered VK2 for 8 weeks after Dex treatment showed significant restoration in trabecular bone architecture (<xref ref-type=\"fig\" rid=\"f5\">\n<bold>Figure 5B</bold>\n</xref>), as well as improvements in BV/TV, Tb.N, and Tb.Sp. The protective effect of VK2 against Dex-induced trabecular bone loss was further confirmed from histological H&E and Masson trichrome staining of femoral bone tissues (<xref ref-type=\"fig\" rid=\"f5\">\n<bold>Figure 5D</bold>\n</xref>). Co-treatment with 3-MA, blocked VK2 protective effect against Dex-induced bone loss suggesting that autophagy/mitophagy is in part accountable for mediating a guardian role of VK2.</p><fig id=\"f5\" position=\"float\"><label>Figure 5</label><caption><p>VK2 against Dex-induced bone loss (GIOP) in rats. <bold>(A)</bold> Schematic of experimental animal arrangement in this study; <bold>(B)</bold> Micro-CT images of the longitudinal sections of the distal femurs were taken; <bold>(C)</bold> Quantitative analysis results of BV/TV, Tb.N, Tb.Sp, and Tb.Th were divided into four groups; <bold>(D)</bold> H&E staining and Masson’s staining of metaphyseal tissue sections of femurs. Data was expressed as mean ± SEM, *p < 0.01 vs SHAM group, <sup>#</sup>p < 0.01 vs. DEX group, <sup>&</sup>p < 0.01 vs. DEX+VK2 group.</p></caption><graphic xlink:href=\"fphar-11-01209-g005\"/></fig><p>To show that restoration of autophagy/mitophagy was indeed involved in mediating the protective effects of VK2 <italic>in vivo</italic>, we carried out IHC and immunofluorescence staining of femoral bone tissues from each treatment group. Compared with Dex treatment only, administration of VK2 following 8-weeks of Dex treatment significantly elevated the expression LC3 and Parkin in bone tissues indicating the restoration of active autophagic/mitophagic activities in osteoblastic cells (<xref ref-type=\"fig\" rid=\"f6\">\n<bold>Figures 6A, B</bold>\n</xref>, p<0.01). In contrast, co-treatment with 3-MA, abolished the autophagy/mitophagy inducing effects of VK2 thereby preventing the restoration of bone architecture following Dex treatment.</p><fig id=\"f6\" position=\"float\"><label>Figure 6</label><caption><p>VK2 restored mitophagy and revived function of osteoblasts <italic>in vivo</italic>. The expression of the proteins in the femurs of the rats were further detected by <bold>(A)</bold> Immunohistochemical staining and quantitative analysis (black arrows: positive area); <bold>(B)</bold> Immunofluorescence and quantitative analysis. Data was expressed as mean ± SEM, *p < 0.01 vs DEX group, <sup>#</sup>p < 0.01 vs. DEX+VK2 group.</p></caption><graphic xlink:href=\"fphar-11-01209-g006\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>In the present study, we showed high dose of Dex treatment markedly attenuated osteoblast autophagy and mitophagy (the selective degradation of damaged mitochondria by autophagy) <italic>via</italic> the downregulation of LC3-II, PINK1, and Parkin protein expression, and the upregulation of p62. Consequently, this led to decreased osteoblast survival, inhibition of osteoblast differentiation and mineralization activity <italic>in vitro</italic>, and induced bone loss in rat model of GIOP. Interestingly, treatment with vitamin K2 (VK2) restored autophagic/mitophagic activities in Dex-treated osteoblasts, and reversed the deleterious effects of Dex on osteoblast survival, differentiation, and mineralization activity <italic>in vitro</italic>. Furthermore, administration of VK2 for 8 weeks following GIOP, protected rats from further bone deterioration and restored bone volume and bone microarchitecture integrity. Preliminary insights into the underlying mechanism suggests that VK2 treatment rescues osteoblastic function in response to Dex <italic>via</italic> restoring autophagic/mitophagic responses. Schematics of our study have been shown in <xref ref-type=\"fig\" rid=\"f7\">\n<bold>Figure 7</bold>\n</xref>.</p><fig id=\"f7\" position=\"float\"><label>Figure 7</label><caption><p>Schematic diagram of proposed mechanism underlying the role of autophagy/mitophagy in mediating the beneficial effect of VK2 against Dexamethasone-induced osteoporosis.</p></caption><graphic xlink:href=\"fphar-11-01209-g007\"/></fig><p>Although the underlying regulatory mechanism(s) of GIOP are unclear, it has been suggested that excess GCs may impeded osteoblast survival and bone formation activity through multiple pathways (<xref rid=\"B5\" ref-type=\"bibr\">Dalle Carbonare et al., 2001</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Hartmann et al., 2016</xref>). In particular, the process of autophagy has gained much attention over the years as a potential driving mechanism in the development of GIOP (<xref rid=\"B24\" ref-type=\"bibr\">Lian et al., 2018</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Shen et al., 2018a</xref>). Autophagy is a fundamental cellular degradative process that functions to remove or recycles dysfunctional protein aggregates and damaged organelles to maintain regular physiological activity (<xref rid=\"B20\" ref-type=\"bibr\">Klionsky and Emr, 2000</xref>; <xref rid=\"B45\" ref-type=\"bibr\">White and Lowe, 2009</xref>). However, when the autophagic process is out of control, that is, either too much or too little, apoptosis can be triggered leading to cell death (<xref rid=\"B26\" ref-type=\"bibr\">Minina et al., 2014</xref>). Meanwhile, mitophagy is an organelle-specific autophagic process whereby damaged or effete mitochondria are delivered to autophagosomes for degradation. It is one of the major mitochondrial quality control process that maintains optimal mitochondrial integrity. It is well known that GC excess interrupts mitochondrial function and prolonged exposure deteriorates mitochondrial integrity leading to release of pro-apoptotic proteins, reactive oxygen species (ROS) accumulation, and ineffective generation of ATP that markedly impedes osteoblast survival. Recent studies have suggested that mitophagy is important in regulating osteoblast survival and function (<xref rid=\"B30\" ref-type=\"bibr\">Pei et al., 2018a</xref>; <xref rid=\"B49\" ref-type=\"bibr\">Zhao et al., 2020</xref>). Nevertheless, the involvement of osteoblast mitophagy in the pathogenesis of GIOP is not clear. The present study showed that high-dose Dex treatment inhibited osteoblast autophagy and mitophagy by markedly downregulating the expression of LC3-II, PINK1 and Parkin. The reduction in the expression of key autophagic and mitophagic proteins impairs the induction of autophagy and mitophagy which is associated with hampered osteoblast differentiation and bone formation function <italic>in vitro</italic> and <italic>in vivo</italic>.</p><p>VK2 has attracted much attention over the years as an auxiliary drug for preventing and treating osteoporotic bone loss (<xref rid=\"B28\" ref-type=\"bibr\">Palermo et al., 2017</xref>). In fact, epidemiological studies have found that a lack of VK2 supplement is associated with a higher risk of osteoporosis and osteoarthritis in older individuals (<xref rid=\"B15\" ref-type=\"bibr\">Hauschka et al., 1975</xref>). Clinical administration of VK2 has been found to potently increase serum osteocalcin and lower the incidence of fractures in osteoporotic patients (<xref rid=\"B39\" ref-type=\"bibr\">Shiraki et al., 2000</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Knapen et al., 2007</xref>). Additionally, VK2 administration was also found to protect against GC-induced bone loss in patients undergoing GC therapy (<xref rid=\"B17\" ref-type=\"bibr\">Inoue et al., 2001</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Sasaki et al., 2005</xref>) and in animal models (<xref rid=\"B13\" ref-type=\"bibr\">Hara et al., 1993</xref>). Accordingly, <xref rid=\"B47\" ref-type=\"bibr\">Zhang et al. (2016)</xref> has proved that supplement with VK2 could prevent bone marrow stem cells from methylprednisolone-induced osteogenic malfunction and apoptosis. VK2 likely exerts its protective effects by enhancing osteoblast differentiation and mineralization function (<xref rid=\"B1\" ref-type=\"bibr\">Atkins et al., 2009</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Rubinacci, 2009</xref>). In our current study, we found out this rescue event is associated with the activation of osteoblastic autophagy and mitophagy. We observed that co-treatment with VK2 potently enhanced osteoblast autophagy and mitophagy. Correspondingly, the deleterious effects of Dex on osteoblast differentiation and function can be ameliorated by treatment with VK2. VK2 was found to reinstate the expression of LC3-II, PINK1 and Parkin, thereby restoring osteoblast autophagy and mitophagy. The positive effects of VK2 on autophagy is consistent with previous report (<xref rid=\"B23\" ref-type=\"bibr\">Li et al., 2019</xref>). To further explore the beneficial roles of VK2, 3-MA, a potent autophagy inhibitor, was used in our study. Consistent with our findings, the administration of 3-MA partly abolished the effects of VK2 both in osteoblasts <italic>in vitro</italic> and <italic>in vivo</italic>.</p><p>Although there are several novel findings in this study, one question that the underlying mechanism of VK2 influence on osteoblast autophagy/mitophagy is still unclear. AMP-activated protein kinase (AMPK), as a critical sensor of cellular energy balance, is reported be crucial in cellular homeostasis (<xref rid=\"B31\" ref-type=\"bibr\">Pei et al., 2018b</xref>). Recent studies have revealed that AMPK phosphorylation promotes autophagy, as well as mitophagy, and plays important parts in mitochondrial quality control (<xref rid=\"B16\" ref-type=\"bibr\">Herzig and Shaw, 2018</xref>). In addition, several studies have reported that AMPK pathway activation is associated with the positive effects of VK2 (<xref rid=\"B3\" ref-type=\"bibr\">Bordoloi et al., 2019</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Duan et al., 2020</xref>). Furthermore, <xref rid=\"B40\" ref-type=\"bibr\">Su et al. (2020)</xref> has demonstrated that VK2 treatment improves mitochondrial function in skeletal muscle <italic>via</italic> activating AMPK-SIRT1 pathway. Therefore, our preliminary insights suggest that AMPK pathway is responsible for the beneficial effects of VK2 in autophagy/mitophagy activation. Hence, signal pathways including AMPK should be investigated to elucidate the function of VK2 in the future studies.</p></sec><sec id=\"s5\"><title>Conclusion</title><p>In summary, we have provided evidence that VK2 can protect osteoblasts against the deleterious effects of Dex <italic>in vitro</italic> and <italic>in vivo</italic> through the restoration of osteoblastic autophagic and mitophagic activities. However, a potential limitation of our study is that we only evaluated the effects of VK2 during the early-mid phases of GIOP, whereas clinically in patients, corticosteroid treatment is usually taken for life. Thus future studies will need to examine the long term effects of VK2 against GIOP. Despite this limitation, our study has shown that osteoblast autophagy and mitophagy are crucial cellular process needed for the osteoblast differentiation and mineralization function. These results provide new insights into the role of osteoblast autophagy and mitophagy in GIOP, and the maintenance of osteoblastic autophagy using VK2 supplementation may significantly improve clinical outcomes of GIOP patients.</p></sec><sec id=\"s6\"><title>Data Availability Statement </title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p></sec><sec id=\"s7\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by Animal Care and Use Committee of Wenzhou Medical University, China.</p></sec><sec id=\"s8\"><title>Author Contributions</title><p>All the listed authors made substantial contributions to the study. LY, LC, JX, and D-YY participated in the experimental design and contributed reagents, materials, and analytical tools. XS, S-JW, J-HT, Z-JX, and Z-YW were also involved in the experiment. LY and LC wrote the manuscript. LC, JX, D-YY, S-JW, J-HT, and B-ZW are involved in data analysis. All authors contributed to the article and approved the submitted version. All the data shown in the figure are from the above authors’ experiments and can be used.</p></sec><sec id=\"s9\"><title>Funding</title><p>This work was funded by a research grant to Science and Technology Department of Zhejiang Province (Grant No.:2016C37122) and National Natural Science Foundation of China (Grant No.:81772348).</p></sec><sec id=\"s10\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\">\n<person-group person-group-type=\"author\"><name><surname>Atkins</surname><given-names>G. J.</given-names></name><name><surname>Welldon</surname><given-names>K. J.</given-names></name><name><surname>Wijenayaka</surname><given-names>A. R.</given-names></name><name><surname>Bonewald</surname><given-names>L. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Chem</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Chem</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Chem.</journal-id><journal-title-group><journal-title>Frontiers in Chemistry</journal-title></journal-title-group><issn pub-type=\"epub\">2296-2646</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850673</article-id><article-id pub-id-type=\"pmc\">PMC7431689</article-id><article-id pub-id-type=\"doi\">10.3389/fchem.2020.00664</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Chemistry</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>The Current Role of Graphene-Based Nanomaterials in the Sample Preparation Arena</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Maciel</surname><given-names>Edvaldo Vasconcelos Soares</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/988062/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Mejía-Carmona</surname><given-names>Karen</given-names></name></contrib><contrib contrib-type=\"author\"><name><surname>Jordan-Sinisterra</surname><given-names>Marcela</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/988237/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>da Silva</surname><given-names>Luis Felipe</given-names></name></contrib><contrib contrib-type=\"author\"><name><surname>Vargas Medina</surname><given-names>Deyber Arley</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/978940/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Lanças</surname><given-names>Fernando Mauro</given-names></name><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/53831/overview\"/></contrib></contrib-group><aff><institution>Laboratory of Chromatography (CROMA), São Carlos Institute of Chemistry (IQSC), University of São Paulo</institution>, <addr-line>São Carlos</addr-line>, <country>Brazil</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Cosimino Malitesta, University of Salento, Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Antonella Profumo, University of Pavia, Italy; Javier Hernández-Borges, University of La Laguna, Spain</p></fn><corresp id=\"c001\">*Correspondence: Fernando Mauro Lanças <email>flancas@iqsc.usp.br</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Analytical Chemistry, a section of the journal Frontiers in Chemistry</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>664</elocation-id><history><date date-type=\"received\"><day>17</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>26</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Maciel, Mejía-Carmona, Jordan-Sinisterra, da Silva, Vargas Medina and Lanças.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Maciel, Mejía-Carmona, Jordan-Sinisterra, da Silva, Vargas Medina and Lanças</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Since its discovery in 2004 by Novoselov et al., graphene has attracted increasing attention in the scientific community due to its excellent physical and chemical properties, such as thermal/mechanical resistance, electronic stability, high Young's modulus, and fast mobility of charged atoms. In addition, other remarkable characteristics support its use in analytical chemistry, especially as sorbent. For these reasons, graphene-based materials (GBMs) have been used as a promising material in sample preparation. Graphene and graphene oxide, owing to their excellent physical and chemical properties as a large surface area, good mechanical strength, thermal stability, and delocalized π-electrons, are ideal sorbents, especially for molecules containing aromatic rings. They have been used in several sample preparation techniques such as solid-phase extraction (SPE), stir bar sorptive extraction (SBSE), magnetic solid-phase extraction (MSPE), as well as in miniaturized modes as solid-phase microextraction (SPME) in their different configurations. However, the reduced size and weight of graphene sheets can limit their use since they commonly aggregate to each other, causing clogging in high-pressure extractive devices. One way to overcome it and other drawbacks consists of covalently attaching the graphene sheets to support materials (e.g., silica, polymers, and magnetically modified supports). Also, graphene-based materials can be further chemically modified to favor some interactions with specific analytes, resulting in more efficient hybrid sorbents with higher selectivity for specific chemical classes. As a result of this wide variety of graphene-based sorbents, several studies have shown the current potential of applying GBMs in different fields such as food, biological, pharmaceutical, and environmental applications. Within such a context, this review will focus on the last five years of achievements in graphene-based materials for sample preparation techniques highlighting their synthesis, chemical structure, and potential application for the extraction of target analytes in different complex matrices.</p></abstract><kwd-group><kwd>graphene</kwd><kwd>graphene oxide</kwd><kwd>cyclodextrin</kwd><kwd>molecularly-imprinted polymer</kwd><kwd>magnetic</kwd><kwd>ionic liquid</kwd><kwd>sample preparation</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Fundação de Amparo à Pesquisa do Estado de São Paulo<named-content content-type=\"fundref-id\">10.13039/501100001807</named-content></funding-source></award-group><award-group><funding-source id=\"cn002\">Conselho Nacional de Desenvolvimento Científico e Tecnológico<named-content content-type=\"fundref-id\">10.13039/501100003593</named-content></funding-source></award-group><award-group><funding-source id=\"cn003\">Coordenação de Aperfeiçoamento de Pessoal de Nível Superior<named-content content-type=\"fundref-id\">10.13039/501100002322</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"8\"/><table-count count=\"5\"/><equation-count count=\"0\"/><ref-count count=\"185\"/><page-count count=\"24\"/><word-count count=\"17421\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Over the last decades, nanotechnology has become a promising tool in relevant scientific fields, allowing humanity to reach top levels of quality in several areas such as engineering, chemistry, medicine, and sports, among others (Lin et al., <xref rid=\"B76\" ref-type=\"bibr\">2019</xref>). One of the most significant achievements in this context was the confirmation of the existence of a single-layered graphene sheet obtained through mechanical exfoliation by Novoselov et al. in 2004 at Manchester University (Novoselov, <xref rid=\"B103\" ref-type=\"bibr\">2004</xref>; Novoselov et al., <xref rid=\"B104\" ref-type=\"bibr\">2005</xref>). The history of graphene (G) starts ~70 years ago when Landau and Peierls stated that strictly 2D crystals were thermodynamically unstable with a slight likelihood even to exist (Peierls, <xref rid=\"B109\" ref-type=\"bibr\">1935</xref>; Landau, <xref rid=\"B64\" ref-type=\"bibr\">1937</xref>). At that time, the scientists thought that the melting point of a thin-film of atoms decreased proportionally to its thickness leading to structure decomposition or segregation (Peierls, <xref rid=\"B109\" ref-type=\"bibr\">1935</xref>; Landau, <xref rid=\"B64\" ref-type=\"bibr\">1937</xref>). Therefore, the atomic monolayers' existence would only be possible to exist as an epitaxially-grown part of 3D complex structures. Nonetheless, this theory was confronted by experimental observations reported in 2004, which preceded the discovery of more than one type of 2D atomic monolayers, highlighting graphene as the most important of them (Geim and Novoselov, <xref rid=\"B46\" ref-type=\"bibr\">2007</xref>). For this reason, graphene theoretically represents a new class of materials possessing a one-atom thickness that, due to its intrinsic properties, are creating new possibilities of practical applications and becoming a hot topic in science.</p><p>Graphene can be defined as a carbon allotrope composed by a structure containing sp<sup>2</sup> hybridized atoms obeying a honeycomb pattern, which is the core for other widely-known allotropic forms (Grajek et al., <xref rid=\"B49\" ref-type=\"bibr\">2019</xref>). In other words, graphene can be stacked to form graphite, rolled to form a carbon nanotube, or even wrapped to become a fullerene, as shown in <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>. In general, it is considered a wonder material due to its nanosheet structure, which has strong σ-orbitals in the 2D plane, ensuring its stiffness. At the same time, the un-hybridized π-orbitals are hinged outwards, superimposing one by one to form the long-range delocalized π electron system, responsible for its outstanding optical, and electrical properties (Grajek et al., <xref rid=\"B49\" ref-type=\"bibr\">2019</xref>). In short, unmodified graphene sheets have a large theoretical surface area (ca. 2630 m<sup>2</sup> g<sup>−1</sup>) distributed along with the thinnest structure of the negligible mass, but high Young's modulus, as already discovered (Fumes et al., <xref rid=\"B43\" ref-type=\"bibr\">2015</xref>). Likewise, its charge carriers have high mobility, possibly traveling micrometers without scattering, becoming an ideal material for producing electronic devices (Hou et al., <xref rid=\"B56\" ref-type=\"bibr\">2019</xref>). Moreover, the excellent thermal and electrical conductivity (~3,000 W mK<sup>−1</sup> and 104 Ω<sup>−1</sup> cm<sup>−1</sup>, respectively), transparency, and impermeability to gases must be underscored (Geim, <xref rid=\"B45\" ref-type=\"bibr\">2009</xref>).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Illustrative drawing of the primary carbon allotropes: <bold>(A)</bold> graphene; <bold>(B)</bold> graphite; <bold>(C)</bold> fullerene; <bold>(D)</bold> carbon nanotubes. Adapted from Maciel et al. (<xref rid=\"B92\" ref-type=\"bibr\">2019</xref>), copyright 2019, with permission from Elsevier.</p></caption><graphic xlink:href=\"fchem-08-00664-g0001\"/></fig><p>Although these top qualities suggest that graphene would be an ideal material with several different potential applications, the manufacturing (especially in industrial-scale) still represents a hurdle to its broad implementation. This occurs because the most utilized manufacturing approach is based on the graphite top-down mechanical exfoliation process by adhesive tape (Allen et al., <xref rid=\"B6\" ref-type=\"bibr\">2010</xref>). Generally, this production method is laborious, non-reproducible, and highly dependent on human handling. It has attracted the chemists' interest in developing scalable alternative routes to produce significant amounts of high-quality graphene nanosheets. Including on this are the chemical exfoliation through liquid solutions, the bottom-up method to produce from organic precursors, among others (Allen et al., <xref rid=\"B6\" ref-type=\"bibr\">2010</xref>; Vadivel et al., <xref rid=\"B147\" ref-type=\"bibr\">2017</xref>). It must be noted that each of these alternative production methods has its proper characteristics. The chemical exfoliation provides interesting results regarding the quality of graphene nanosheets or even due to the existence of intermediary graphene-based compounds similarly attractive for such purposes, such as the graphite oxide or graphene oxide, for instance.</p><p>Even with the graphene existence being confirmed since 2004, its first application in sample preparation was only published in 2011 (Luo et al., <xref rid=\"B89\" ref-type=\"bibr\">2011a</xref>; Zhang and Lee, <xref rid=\"B173\" ref-type=\"bibr\">2011</xref>). The interest of analytical chemists on it and its derivatives is mostly due to the increasing demand for high-performance and selective materials to extract contaminants present in complex matrices containing a large number of interferents. The present outlook of our environment urges researchers to seek technological advances on sample preparation and analytical techniques to tackle even better the increasing use of chemicals by humans in many areas of life: agriculture, health-treatments, and abusive-drugs, among others. Within such a context, the necessity in performing a sample preparation step before the analytical techniques is mandatory due to the complexity related to the mostly analyzed matrices (biological fluids, food, plants, wastewaters, soil, and others). This step is crucial in the analytical workflow responsible for eliminating matrix interferents, isolating, and pre-concentrating target analytes. For these reasons, several different sorption-based sample preparation techniques (e.g., microextraction by packed sorbent [MEPS], stir bar sorptive extraction [SBSE], magnetic solid-phase extraction [MSPE], among others) have been proposed. They are mostly derived from conventional SPE and its main miniaturized mode SPME (Fumes et al., <xref rid=\"B43\" ref-type=\"bibr\">2015</xref>). Generally, they are performed by the employment of an extractive phase, usually named as sorbent. Ideally, this sorbent must present some essential characteristics such as good selectivity to the target compounds, high extraction capability, and even is desirable as a chemical inertia for those interferents present in the analyzed matrix (Toffoli et al., <xref rid=\"B144\" ref-type=\"bibr\">2018</xref>).</p><p>Then, GBMs emerged as promising sorbents to be used in the extraction techniques, due to its chemical structure and properties, which favors the extraction performance by effectively removing the target analytes from complex matrices. Considering all the advantages herein presented, some of them are more interesting from the analytical chemistry standpoint. For example, the flat graphene structure allows potential target analytes to interact on both sides of it, which is advantageous for sorption-based sample preparation techniques. In the case of other carbon allotropes (e.g., carbon nanotubes, graphite, and fullerene), only the external surface is available for such interaction, which potentially diminishes extraction performance due to this steric hindrance associated. Additionally, the delocalized π-electron system favors electrostatic interaction between the graphene and molecules that possess aromatic rings in its structure.</p><p>For this reason, prevalent contaminants such as pesticides, preservatives, pharmaceuticals, and veterinary drugs can be remediated from the environment by using GBMs (Toffoli et al., <xref rid=\"B144\" ref-type=\"bibr\">2018</xref>). Contrariwise, when the potential contaminant does not have aromatic rings, a functional intermediary produced from the chemical exfoliation, namely graphene oxide (GO), can be used instead of graphene. This is owing to its chemical structure that differs from G by the presence of oxygenated groups (e.g., hydroxyl, carbonyl, alkoxy) outside of its 2D-plane, possibly favoring interactions with polar active-sites in other molecules (Smith et al., <xref rid=\"B129\" ref-type=\"bibr\">2019</xref>). Nonetheless, from an operational point of view, the fact that graphene is an ultra-light material makes difficult its deposition by, for example, centrifugation. In this way, some chemical modification or functionalization can be performed to overcome such drawbacks. Nowadays, other carbon-based compounds that possess one-atom planar structures are beginning to spur around, mainly due to the attention given to the scientific community's graphene in the last years. Including in this group are graphyne, graphdiyne, graphone, and graphane, all considered as graphene-derivative compounds (Peng et al., <xref rid=\"B110\" ref-type=\"bibr\">2014</xref>). As an example, graphyne and graphdiyne are 2D-flat allotropic forms of graphene possessing the same honeycomb pattern, which suggests them as suitable for similar applications as its precursor (graphene).</p><p>Conversely, graphone and graphene emerged as hydrogenated graphene-derivative compounds susceptible to chemical modifications onto its surfaces. Despite these compounds already discussed in the literature, their synthesis remains a complicated process; for this reason, they have not yet been applied in sample preparation. However, considering the significant advances in graphene-based technologies since its discovery, these other allotropic forms might gain more attention from scientists throughout the years.</p><p>Following the background regarding the emerging of graphene, this review aims to present the state-of-art about this “wonder material” from a sample preparation viewpoint. In this way, several aspects such as synthesis and functionalization process, the main derivative classes, and its most suitable applications are divided among the next sections. In short, our primary goal was to present a review mostly covering the last 5 years' achievements of the still-evolving field of graphene-based materials in sample preparation and discuss the future trends and potential challenges that chemists should face in the years to come.</p></sec><sec id=\"s2\"><title>Graphene and Graphene Oxide</title><p>As mentioned, graphene (G) is a 2D monolayer of carbon atoms covalently bonded in a honeycomb pattern, displaying a flat sheet conformation (Solís-Fernández et al., <xref rid=\"B130\" ref-type=\"bibr\">2017</xref>). Graphene and related materials are part of the graphene-based materials (GBMs), which comprises graphene (G) nanosheets (in mono, few, and multi-layers), graphene oxide (GO), and reduced graphene oxide (rGO) (De Marchi et al., <xref rid=\"B22\" ref-type=\"bibr\">2018</xref>). A considerable variety of research articles about different synthesis methods, properties, and applications of GBMs are available (Papageorgiou et al., <xref rid=\"B108\" ref-type=\"bibr\">2017</xref>; Lim et al., <xref rid=\"B75\" ref-type=\"bibr\">2018</xref>; Liu and Zhou, <xref rid=\"B77\" ref-type=\"bibr\">2019</xref>). Two different approaches can be used to obtain graphene: (i) the top-down, in which nanostructures are produced from larger dimensions, and (ii) the bottom-up, starting from atoms or small molecules to produce materials of larger dimensions.</p><p>In the top-down approach, graphene is prepared from graphite, by mechanical or chemical exfoliation, or chemical synthesis, separating the graphene thin layers parallelly stacked in the graphite and held together by weak van der Waals forces. Mechanical exfoliation is one of the simplest methods in which a simple direct contact with an adhesive tape (polymer) can take off the graphene layers from the surface of a graphite piece. One of the advantages of mechanical exfoliation is the possibility of different pile-up layers of other 2D materials with several graphene heterostructures (Solís-Fernández et al., <xref rid=\"B130\" ref-type=\"bibr\">2017</xref>). However, it has only been implemented on a small scale and is highly susceptible to contamination. In the same way, organic solvents can be used to separate the graphite layers. Other exfoliation methods include the use of electric fields, sonication, and transfer printing technique (Lim et al., <xref rid=\"B75\" ref-type=\"bibr\">2018</xref>). Several exfoliation methods, including different substrates, thermal released tape, and thermal approaches, have been proposed to improve the quality, size, and homogeneity of graphene (Solís-Fernández et al., <xref rid=\"B130\" ref-type=\"bibr\">2017</xref>).</p><p>Chemical reduction of graphene oxide (GO) is the most popular method to obtain graphene. As shown in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>, GO can be obtained by the oxidation of graphite powder and then exfoliated further to obtain single GO layers, which is subsequently chemically reduced to obtain rGO. Although the final product obtained from this pathway is rGO, its properties are very similar to graphene but are structurally different (Dreyer et al., <xref rid=\"B28\" ref-type=\"bibr\">2010</xref>; Singh et al., <xref rid=\"B127\" ref-type=\"bibr\">2016</xref>). Chemical reduction of GO firstly involves exfoliation in water assisted by ultrasonication, followed by reduction of the oxygenated groups, hence precipitating the rGO from the solution due to its hydrophobicity (Singh et al., <xref rid=\"B127\" ref-type=\"bibr\">2016</xref>). Among the wide variety of chemical reduction agents that can be employed, hydrazine is the most often used because of its high reductive efficiency, even though it is highly environmental toxic. As an alternative, the use of greener reduction agents (De Silva et al., <xref rid=\"B23\" ref-type=\"bibr\">2017</xref>) and thermally-mediated or electrochemical reduction are also employed. Although chemical reduction of GO is a popular upscaling method, it yields a final product containing several structural defects on the sheets, which lead to low-quality materials with variable sizes and edges (Dreyer et al., <xref rid=\"B28\" ref-type=\"bibr\">2010</xref>).</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Graphene chemical reduction pathway.</p></caption><graphic xlink:href=\"fchem-08-00664-g0002\"/></fig><p>Conversely, the bottom-up approaches are another alternative to synthesize high-quality graphene layers. The leading methods used include pyrolysis, chemical vapor deposition (CVD), plasma synthesis, and epitaxial growth (Lim et al., <xref rid=\"B75\" ref-type=\"bibr\">2018</xref>). Graphene sheets are prepared from small building blocks and assembled with dedicated precision, usually employing molecular modeling to build the layers. Among the bottom-up methods, CVD uses high temperatures for the decomposition of hydrocarbons, which are deposited on metal substrates, thus forming thin sheets of graphene (Papageorgiou et al., <xref rid=\"B108\" ref-type=\"bibr\">2017</xref>). The main advantage of bottom-up methods is the production of high-quality graphene sheets. Nevertheless, these methods are not used for large-scale production.</p><p>Graphene oxide (GO) is like a graphene sheet functionalized on both sides with several oxygenated functions such as hydroxyl, carboxyl, and epoxy. These functions impose a hydrophilic character to GO; as a consequence, the interaction between layers is weaker compared to graphene, making GO an easily exfoliated material. GO structure depends principally on the purification methods (Singh et al., <xref rid=\"B127\" ref-type=\"bibr\">2016</xref>). Compared to graphene, the GO structure is still ambiguous; thus, several structural models have been proposed to date (Dreyer et al., <xref rid=\"B28\" ref-type=\"bibr\">2010</xref>; Sun, <xref rid=\"B133\" ref-type=\"bibr\">2019</xref>). The graphene oxide can be obtained by the popular Hummer's method (Hummers and Offeman, <xref rid=\"B58\" ref-type=\"bibr\">1958</xref>), which, until the date, has been subjected to multiple modifications and improvements (Shamaila et al., <xref rid=\"B125\" ref-type=\"bibr\">2016</xref>). The original method proposes the oxidation of graphite powder by KMnO<sub>4</sub> and NaNO<sub>3</sub> in H<sub>2</sub>SO<sub>4</sub> (Hummers and Offeman, <xref rid=\"B58\" ref-type=\"bibr\">1958</xref>). Differences with other modified methods are principally on the type and toxicity of the oxidant reagents, and the quality of the obtained product (Lim et al., <xref rid=\"B75\" ref-type=\"bibr\">2018</xref>).</p><p>A fascinating characteristic raised from the physical and chemical properties of graphene materials is the possibility to perform chemical functionalizations mainly to modify its reactivity yielding a large variety of graphene-based materials (GBMs). Thus, they can be currently used in several applications (Bottari et al., <xref rid=\"B12\" ref-type=\"bibr\">2017</xref>; Mohan et al., <xref rid=\"B98\" ref-type=\"bibr\">2018</xref>). Covalent or non-covalent pathways can functionalize GBMs. Non-covalent functionalization involves first the rupture of the van der Waals forces that stake together with the graphene layers with subsequent formation of non-covalent binding with the substrate by π-π, π-cation, and van der Waals interactions. For that, mechanical liquid exfoliation assisted by ultrasonication is used to overcome these forces; water, organic solvents, ionic liquids, surfactants, mixtures are employed to disperse, and stabilize the graphene sheets in the exfoliation process. For example, non-covalent functionalization can be obtained by forming a stable dispersion of graphene sheets in polymers (ionic, non-ionic, and polysaccharides), water solutions, and organic solvents as polyvinyl alcohol, chitosan, and alginate. They can be employed to obtain graphene aerogels and hydrogels (Dreyer et al., <xref rid=\"B28\" ref-type=\"bibr\">2010</xref>; Bottari et al., <xref rid=\"B12\" ref-type=\"bibr\">2017</xref>).</p><p>On the other hand, the covalent functionalization of graphene (G) yields a low substitution degree due to their stable carbon conjugation. However, covalent functionalization can be done by taking advantage of the oxygenated reactive groups of the graphene oxide (GO) sheets. Therefore, in the same way as the chemical reduction of graphene oxide, the hydroxyl, carboxyl, and epoxy groups can be covalently replaced by other functional groups. The GO surface modification with aliphatic amines to form an amide bond is one of the most common strategies (Dreyer et al., <xref rid=\"B28\" ref-type=\"bibr\">2010</xref>). Undoubtedly, the derivatization of graphene materials improves their electrical, thermal, and mechanical properties as well as their dispersibility (Mohan et al., <xref rid=\"B98\" ref-type=\"bibr\">2018</xref>). Some reasons for the use of functionalized graphene materials as sorbents in sample preparation include: (i) they present improved sorption capacity and recoveries; (ii) easy attachment of graphene onto surfaces to be reusable, and preventing sorbent losses; (iii) avoid the agglomeration of the graphene sheets; and (iv) favored sorbent isolation from the sample (Wang et al., <xref rid=\"B154\" ref-type=\"bibr\">2014</xref>; Ye and Shi, <xref rid=\"B167\" ref-type=\"bibr\">2015</xref>; Chen X. et al., <xref rid=\"B17\" ref-type=\"bibr\">2016</xref>; González-Sálamo et al., <xref rid=\"B48\" ref-type=\"bibr\">2016</xref>).</p><p>Considering the increasing use of GBMs in sample preparation techniques, the most representative and used materials in this arena are discussed in the following sections.</p></sec><sec id=\"s3\"><title>Anchored Graphene-Based Materials</title><sec><title>Alkyl and Aril Groups</title><p>Octadecylsilica particles (for short C18 or ODS) are by far the most commonly used sorbent in solid-phase extraction (SPE) and chromatographic separations. Apart from the conventional C8/C18 reversed phases, today mixed-mode polymeric sorbents are widely used in SPE because they present interactions with several compounds and better performance compared to the conventional ones, and they are also commercially available (Fontanals et al., <xref rid=\"B41\" ref-type=\"bibr\">2020</xref>). Alkyl groups, in general, are commonly used to derivatize sorbents, including GO sheets, to modify their fundamental properties. As a consequence, C18 has also been employed to functionalize GO-based sorbents owing to the high surface area of the GO sheets. Their functionalization with octadecylsilane increases the surface load with C18 groups compared to silica particles (Liang et al., <xref rid=\"B74\" ref-type=\"bibr\">2012</xref>; Xu et al., <xref rid=\"B163\" ref-type=\"bibr\">2012</xref>). Subsequently, the extraction capacity is improved, and hydrophobic interactions increased. Therefore, being applied as a sorbet in reverse-phase, they show an improved extraction efficiency for the extraction of alkanes and PAHs, for instance (Xu et al., <xref rid=\"B163\" ref-type=\"bibr\">2012</xref>). Recently, Qui et al. prepared a solid-phase microextraction (SPME) fiber with C18 particles (3.5 μm) coated with GO and poly(diallyl dimethylammonium chloride) (PDDA)–C18@GO@PDDA as shown in <xref ref-type=\"fig\" rid=\"F3\">Figures 3A,B</xref>. Then, the surface of the fiber was modified by oxidative polymerization by polynorepinephrine (pNE) (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>), which plays the role of a bio-interface, compatible with <italic>in-vivo</italic> sampling (<xref ref-type=\"fig\" rid=\"F3\">Figure 3D</xref>). The prepared SPME fibers showed higher efficiency than commercially available ones such as polydimethylsiloxane (PDMS) and polyacrylate (PA) for the monitoring of acidic drugs in fish samples. Additionally, the fiber exhibited excellent stability, sensitivity, and resistance for <italic>in-vivo</italic> matrices, showing potential for pharmacokinetics applications (Qiu et al., <xref rid=\"B113\" ref-type=\"bibr\">2016</xref>). In another study, the same fiber type was successfully employed to analyze salicylic acid traces in plants <italic>in-vivo</italic> (Fang et al., <xref rid=\"B33\" ref-type=\"bibr\">2018</xref>).</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Preparation and application scheme of C18@GO@PDDA SPME fiber. <bold>(A)</bold> Preparation of the particles (a. C18, b. C18@GO, and c. C18@GO@PDDA). <bold>(B)</bold> A home-made coated PANI fiber by the dip-coating method. <bold>(C)</bold> Bioinspired modification by the NE oxidation polymerization. <bold>(D)</bold>\n<italic>In vivo</italic> sampling in fish dorsal-epaxial muscle with the fiber. Reprinted with permission from Qiu et al. (<xref rid=\"B113\" ref-type=\"bibr\">2016</xref>), copyright 2016, American Chemical Society.</p></caption><graphic xlink:href=\"fchem-08-00664-g0003\"/></fig><p>Although functionalization of GO occupies or replaces part of their original active sites, the sorption capacity of modified-GO materials can be lower or higher compared to GO, which mainly depends on the composite type formed and their specific interaction with the analytes. Even so, GBMs show superior sorptive properties compared to conventional sorbents (e.g., C18), which allows the use of graphene sorbents in small quantities (<100 mg) (Sitko et al., <xref rid=\"B128\" ref-type=\"bibr\">2013</xref>). In this way, Ma et al. functionalized graphene oxide (GO) sheets with different amine-alkyl chains to obtain amine-rGO sorbents <italic>via</italic> solvothermal synthesis. The sorption capacity of the different alkyl-amine-rGO materials was evaluated for the extraction of catechins and caffeine. Results showed that tributylamine-rGO has the highest sorption capacity (203.7 mg g<sup>−1</sup>) for catechins being 11 times higher compared to GO sheets (18.7 mg g<sup>−1</sup>) and other rGO-amino groups (ammonia, ethylenediamine, n-butylamine, tert-butylamine, dodecyl amine, and octadecyl amine). Hence, tributylamine-rGO was employed as a sorbent in a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method achieving a higher clean-up performance compared to traditional sorbents as PSA, C18, and graphitized carbon black (GCB), regularly used in QuERChERS (Ma et al., <xref rid=\"B91\" ref-type=\"bibr\">2018</xref>). A similar comparison was performed by Fumes et al., which employed aminopropyl silica and PSA particles coated by graphene sheets. The extraction performance of the GO-coated particles, used as a sorbent in microextraction by packed sorbent (MEPS) method, were compared with conventional sorbents (C18, strata-X, PSA, amino silica) for the extraction of parabens in wastewater. Aminopropyl silica coated with GO (SiGO) and G (SiG) showed an improved extraction performance compared to conventional sorbents (Fumes and Lanças, <xref rid=\"B42\" ref-type=\"bibr\">2017</xref>). Likewise, a recent work performed by the same research group showed improvements in the extraction capability of aminopropyl silica-GO particles when they are functionalized with C18 and further end-capped. The authors achieved low LODs and LOQs in a complex matrix (coffee samples) by using these particles in a packed in-tube SPME device (Mejía-Carmona and Lanças, <xref rid=\"B97\" ref-type=\"bibr\">2020</xref>). Other interesting graphene-based applications carried out by the Lança's research group also includes tetracyclines' analysis in milk samples (Vasconcelos Soares Maciel et al., <xref rid=\"B148\" ref-type=\"bibr\">2018</xref>) and the determination of triazines in environmental water samples (De Toffoli et al., <xref rid=\"B24\" ref-type=\"bibr\">2018</xref>).</p><p>Several additional complex alkyl and aryl compounds have been used to functionalize GO. For example, Nurerk et al. synthesized a hybrid sorbent based on calix[4]arene-functionalized graphene oxide/polydopamine-coated cellulose acetate fiber (calix[4]arene-GO/PDA-CFs) for the extraction of aflatoxins in corn samples. Calix[4]arene is a macrocyclic molecule of four phenol units bonded by methylene bridges, which can favor the extraction of aflatoxins by H-bonding, hydrophobic, and π-π interactions. The recoveries of aflatoxins (AFs) obtained employing cellulose acetate CFs (35–41%), polydopamine coated CFs (PDA-CFs) (45–55%), calix[4]arene-GO-CFs (60–72%), GO/PDA-CFs (63–82%), and calix[4]arene-GO/PDA-CFs (86–94%) as SPE sorbents showed that together calix[4]arene and GO increased the efficiency of the sorbent phase (Nurerk et al., <xref rid=\"B105\" ref-type=\"bibr\">2018</xref>). Recently, Zhou et al. synthesized a graphene oxide framework (GOF), a 3D nanoporous material, as coating sorbent for stir bar sorptive extraction (SBSE). Graphene oxide was covalently interconnected with a 1,4-phenylene diisocyanate (PPDI) to obtain three-dimensional GOF, which was immobilized onto the surface of stainless-steel wire (SSW) using polydopamine. The stir bar was applied successfully for the extraction of Sudan dyes in lake water and fruit juice (Zhou J. et al., <xref rid=\"B181\" ref-type=\"bibr\">2019</xref>). Other recently published papers on alkyl and aryl modified graphene materials are shown in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Recent applications (2015–2020) of alkyl and aryl functionalized graphene-based materials in sample preparation.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Sorbent</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Analytes</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Matrix</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Sample preparation</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Analysis</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>LOD</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>References</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Diallyl dimethyl ammonium chloride-assembled GO-coated C18 (C18@GO@PDDA)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Salicylic acid and derivates</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Aloe plants (<italic>in-vivo</italic> sampling)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPME fiber</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.8–2.8 μg g<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fang et al., <xref rid=\"B33\" ref-type=\"bibr\">2018</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Aminopropyl silica coated GO- functionalized Octadecylsilane/end-capped (SiGOC18ecap)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Xanthines</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Coffee</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">In-tube SPME</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC- MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.1–0.2 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mejía-Carmona and Lanças, <xref rid=\"B97\" ref-type=\"bibr\">2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Graphene derivatized silica</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fluoroquinolones</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-FLD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Speltini et al., <xref rid=\"B132\" ref-type=\"bibr\">2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Guanidyl-functionalized GO-grafted silica (Guanidyl@GO@sil)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Herbicides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Lycium barbarum</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.5–2.0 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hou et al., <xref rid=\"B55\" ref-type=\"bibr\">2018b</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polypyrrole-coated GO and C18 incorporated in chitosan cryogel (PPY/GOx/C18/CS)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Carbamate pesticides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fruit juices</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.5–2.0 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Klongklaew et al., <xref rid=\"B63\" ref-type=\"bibr\">2018</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Alkyl-NH<sub>2</sub>/rGO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pesticides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tea</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Modified QuEChERS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GC-MS/MS<break/> UHPLC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.33–9.26 μg kg<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ma et al., <xref rid=\"B91\" ref-type=\"bibr\">2018</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Calix[4]arene-functionalized GO/polydopamine-coated cellulose acetate fiber (calix[4]arene-GO/PDA-CFs)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Aflatoxins</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Corn</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-FLD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.01–0.05 μg kg<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nurerk et al., <xref rid=\"B105\" ref-type=\"bibr\">2018</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Graphene oxide supported on aminopropyl silica (Si-GO)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tetracyclins</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bovine milk</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MEPS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.03–0.21 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Vasconcelos Soares Maciel et al., <xref rid=\"B148\" ref-type=\"bibr\">2018</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Graphene oxide supported on aminopropyl silica (Si-GO)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Triazines</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">In-tube SPME</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.1–2.9 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">De Toffoli et al., <xref rid=\"B24\" ref-type=\"bibr\">2018</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Graphene-C18 Reinforced Hollow Fiber (G-C18-HF)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chlorophenols</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Honey</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HF-LPME</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.5–1.5 μg kg<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sun et al., <xref rid=\"B135\" ref-type=\"bibr\">2014</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Poly(diallyldimethylammoniumchloride) assembled GO-coated C18 particles (C18@GO@PDDA)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Acidic pharmaceuticals</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fish (<italic>in-vivo</italic> sampling)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPME fiber</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.13–7.56 μg kg<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Qiu et al., <xref rid=\"B113\" ref-type=\"bibr\">2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Graphene oxide/silica modified with nitro-substituted tris(indolyl)methane</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Organic acids</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Honey and nongfu spring drink</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.5–1.0 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Wang N. et al., <xref rid=\"B151\" ref-type=\"bibr\">2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Graphene supported on aminopropyl silica (Si-G) and primary-secondary amine (PSA) silica (PSA-G)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Parabens</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MEPS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UHPLC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.06–0.09 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fumes and Lanças, <xref rid=\"B42\" ref-type=\"bibr\">2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Acrylamide-functionalized graphene</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Monoamine acidic metabolites</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Urine and plasma</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">μSPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.08–0.25 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yang et al., <xref rid=\"B164\" ref-type=\"bibr\">2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tannic acid functionalized graphene</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Beryllium</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Wastewater and street dust</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">d-SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Atomic absorption</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.84 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yavuz et al., <xref rid=\"B166\" ref-type=\"bibr\">2018</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GO framework interconnected by 1,4-phenylene diisocyanate (PPDI)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sudan dyes (G, I, II, and III)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lake water and fruit juice</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SBSE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.15–0.3 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Zhou J. et al., <xref rid=\"B181\" ref-type=\"bibr\">2019</xref></td></tr></tbody></table></table-wrap></sec><sec><title>Cyclodextrins</title><p>Other emergent graphene-based materials for sample preparation are those combined with cyclodextrins (CD), which are cyclic oligosaccharides formed by starch enzymatic degradation, and are linked by several α-1,4-anhydroglucopyranose. In nature, they are made up of 6, 7, or 8 glucose units and categorized as α, β, and γ-CD, respectively. CDs have cone shapes with a hydrophobic cavity and a hydrophilic external surface. This specific structure allows them to form inclusion complexes with specific molecules such as polyphenolics compounds through hydrogen bonding, hydrophobic, and Van der Waals interactions (Pinho et al., <xref rid=\"B112\" ref-type=\"bibr\">2014</xref>; Zhu et al., <xref rid=\"B185\" ref-type=\"bibr\">2016</xref>). Reactive OH groups can also be replaced to modify their solubility, improve inclusion ability, or induce desired properties as functionalization for immobilization on a solid support. Consequently, more than 100 CDs are commercially available, and more than 1,500 derivatives have been synthesized for different purposes (Szejtli, <xref rid=\"B136\" ref-type=\"bibr\">2004</xref>; Gentili, <xref rid=\"B47\" ref-type=\"bibr\">2020</xref>).</p><p>The reliable recognition capacity of phenolic compounds, due to the excellent size match, becomes common to find works reporting a combination between graphene and cyclodextrins used as electrochemical detectors (Wang C. et al., <xref rid=\"B150\" ref-type=\"bibr\">2016</xref>), electrocatalytic detector (Pham et al., <xref rid=\"B111\" ref-type=\"bibr\">2016</xref>), and electrocatalytic material, for instance (Ran et al., <xref rid=\"B115\" ref-type=\"bibr\">2017</xref>). Thus, there are many different strategies available in the literature to synthesize GBMs functionalized by cyclodextrins. GO functionalization with CD can be very simple, as reported by Cao et al. (<xref rid=\"B14\" ref-type=\"bibr\">2019</xref>). They prepared a suspension containing a graphene-based material and β-cyclodextrin, with it subsequently stirred under a heated water bath at a temperature of 60°C for 4 h. In this case, the resulting material was GO; if it is necessary to reduce graphene oxide to graphene, it can be done using hydrazine (Pham et al., <xref rid=\"B111\" ref-type=\"bibr\">2016</xref>; Tan and Hu, <xref rid=\"B138\" ref-type=\"bibr\">2017</xref>). As an example, modifications in the synthesis route can be made with (3-aminopropyl)triethoxysilane (APTES) to support an amino group to both graphene oxide nanosheets or cyclodextrins walls (Deng et al., <xref rid=\"B25\" ref-type=\"bibr\">2017</xref>). This procedure results in an amido bonding between epoxy and –COOH groups of GO and –NH<sub>2</sub> from the APTES. <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref> shows a scheme to exemplify the most common bond between GO and cyclodextrin, and how it is expected to be the structure of the graphene-based material functionalized by cyclodextrin.</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Scheme illustrating a graphene-based sorbent functionalized with cyclodextrins through peptide bonds.</p></caption><graphic xlink:href=\"fchem-08-00664-g0004\"/></fig><p>The combination of graphene and cyclodextrins properties becomes the resulting material attractive to be employed in sample preparation techniques. An interesting application was carried out by Deng et al. The novel β-CD–GO-coated SPME fiber was prepared using a sol-gel technique and immobilizing onto a pre-functionalized stainless steel wire (Deng et al., <xref rid=\"B25\" ref-type=\"bibr\">2017</xref>). They applied this material as a sorbent in a headspace technique (HS-SPME), aiming to extract organophosphate flame retardants in water samples to be analyzed by gas chromatography with nitrogen phosphorus detector (NPD). The method showed functional recovery (82.1–116.9%), linear range with correlation coefficients (<italic>R</italic>) ranging from 0.9955 to 0.9998. The LODs and LOQs for the nine analytes ranged from 1.1–60.4 to 2.7–170.5 ng L<sup>−1</sup>, respectively; RSD was 2.2–9.6%, and enrichment factors obtained from 22.5 to 1307.5. This high enrichment factor is attributed to the combined advantages of β-CD and GO. When compared with the commercial fibers and some published methods, the GO/β-CD sol-gel coating fiber showed a higher extraction efficiency, except for those organophosphate flame retardants containing a benzene ring. Similarly, Cao et al. combined the advantages of graphene and cyclodextrins with ionic liquids and ILs (Cao et al., <xref rid=\"B14\" ref-type=\"bibr\">2019</xref>). They synthesized a VOIm<sup>+</sup>\n<inline-formula><mml:math id=\"M1\"><mml:msubsup><mml:mrow><mml:mtext>AQSO</mml:mtext></mml:mrow><mml:mrow><mml:mn>3</mml:mn></mml:mrow><mml:mrow><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula> functionalized β-cyclodextrin/magnetic graphene oxide material (Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>/GO/β-CD/IL), which was used as a sorbent to extract plant growth regulators from vegetable samples using magnetic solid-phase extraction (MSPE) followed by UHPLC-MS/MS analysis. This approach showed fast separation, high surface area, high adsorption capability, and environmental friendliness. The comparison between Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>/GO, Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>/GO/β-CD, and Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>/GO/IL showed that Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>/GO/β-CD/IL had higher extraction efficiency and selective adsorption capacity.</p><p>For food analysis, β-CD combined with GO were used in the sample preparation during the analysis of organochlorine pesticide residues in honey (Mahpishanian and Sereshti, <xref rid=\"B95\" ref-type=\"bibr\">2017</xref>). The prepared material was applied as a sorbent in vortex-assisted magnetic solid-phase extraction (MSPE) before gas chromatography-electron capture detection (GC-ECD) analysis. The method was optimized and evaluated, showing linearity ranging from 2 to 10,000 ng kg<sup>−1</sup> and <italic>R</italic><sup>2</sup> > 0.9966, RSDs < 7.8%, LODs from 0.52–3.21 ng kg<sup>−1</sup>, and LOQ from 1.73–10.72 ng kg<sup>−1</sup>. For the real samples, the proposed sorbent showed good recoveries in the range of 78.8–116.2% with RSDs (<italic>n</italic> = 3) below 8.1%.</p><p>These works demonstrated that cyclodextrins' functionalized GBMs possess great supramolecular recognition, high extraction efficiency, good recoveries, and enrichment capability. It is noteworthy that in all reported works, the chosen graphene-based material is actually the graphene oxide (GO). This trend is justified by the presence of epoxy and -COOH groups on the GO surface, favoring the bonding with the CDs. Although some works reported the employment of graphene combined with cyclodextrins as sorbent, these materials were obtained by graphene oxide reduction (Ragavan and Rastogi, <xref rid=\"B114\" ref-type=\"bibr\">2017</xref>; Tan and Hu, <xref rid=\"B138\" ref-type=\"bibr\">2017</xref>). In this strategy, the reduction stage can be performed before or after the support between graphene-based and CDs. In this way, considering that the oxygenated groups present in the GO structure can improve interaction with molecules of β-CD (Tan and Hu, <xref rid=\"B138\" ref-type=\"bibr\">2017</xref>), it is presumable that the reduction of graphene oxide after β-CD coupling is the best synthesis route to maximize the amount of cyclodextrin coupled.</p><p>To complement this topic, <xref rid=\"T2\" ref-type=\"table\">Table 2</xref> presents recently published works using graphene-based materials combined with cyclodextrins for sample preparation. It must be highlighted that all applications employed β-cyclodextrin. Considering the existence of over 100 commercially available and more than 1,500 derivative materials already described, it is clear that sorbents based on graphene functionalized with cyclodextrins are a broad research field to be still explored. Finally, the GBMs/CD's excellent characteristics for supramolecular recognition, high extraction efficiencies, good recoveries, and enrichment capability should contribute to its widespread development in the coming years.</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Recent applications (2017–2020) of graphene-based materials functionalized by cyclodextrins to sample preparation.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Sorbent</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Analytes</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Matrix</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Sample preparation</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Analysis</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>LOD</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>References</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">G-Fe<sub>3</sub>O<sub>4</sub>-β-CD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bisphenol-A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MSPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UV-vis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN1\"><sup>**</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ragavan and Rastogi, <xref rid=\"B114\" ref-type=\"bibr\">2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GNS/β-CD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phenolphthalein</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">d-SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UV-vis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN1\"><sup>**</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tan and Hu, <xref rid=\"B138\" ref-type=\"bibr\">2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe<sub>4</sub>O<sub>3</sub>-GO-β-CD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neonicotinoid pesticide</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MSPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN1\"><sup>**</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Liu G. et al., <xref rid=\"B79\" ref-type=\"bibr\">2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">β-CD/MrGO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Organochlorine pesticides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Honey</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MSPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GC-ECD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.52–3.21 ng kg<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mahpishanian and Sereshti, <xref rid=\"B95\" ref-type=\"bibr\">2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GO/β-CD sol-gel coating fiber</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Organophosphate flame retardants</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Environmental water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HS-SPME</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GC-NPD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.1–60.4 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Deng et al., <xref rid=\"B25\" ref-type=\"bibr\">2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>/GO/β-CD/IL</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Plant growth regulators</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Vegetables</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MSPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UHPLC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.01–0.18 μg kg<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cao et al., <xref rid=\"B14\" ref-type=\"bibr\">2019</xref></td></tr></tbody></table><table-wrap-foot><fn id=\"TN1\"><label>**</label><p><italic>Not specified</italic>.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Magnetic Materials</title><p>Although graphene and its derivatives are considered to be cutting-edge materials in modern sorbent-based sample preparation (Toffoli et al., <xref rid=\"B144\" ref-type=\"bibr\">2018</xref>; Grajek et al., <xref rid=\"B49\" ref-type=\"bibr\">2019</xref>; Hou et al., <xref rid=\"B56\" ref-type=\"bibr\">2019</xref>), their use can be related to some drawbacks in both bed-packed and dispersive microextraction. Their strong van der Waals interactions may cause irreversible aggregation of the material, causing graphene swelling, which often occurs due to the continuous water/solvent deposition between the graphene nanosheets (Zheng et al., <xref rid=\"B180\" ref-type=\"bibr\">2017</xref>; Iakunkov et al., <xref rid=\"B59\" ref-type=\"bibr\">2019</xref>). For these reasons, columns and microextraction devices packed with GBMs are usually susceptible to clogging and high backpressures. Likewise, for dispersive techniques, graphene nanosheets are well-suspended in solution, creating difficulty for the sorbent recovery, even after filtration and centrifugation (Hou et al., <xref rid=\"B56\" ref-type=\"bibr\">2019</xref>; Li F. et al., <xref rid=\"B67\" ref-type=\"bibr\">2020</xref>).</p><p>Within such a context, a modern, and advantageous strategy to overcome those drawbacks is the magnetic solid-phase extraction (MSPE) which is considered an efficient and environment-friendly sample preparation technique (Šafariková and Šafarik, <xref rid=\"B120\" ref-type=\"bibr\">1999</xref>). MSPE extraction mechanism relies on the use of extraction sorbents supported over magnetic materials (Laura et al., <xref rid=\"B66\" ref-type=\"bibr\">2019</xref>). In general, MSPE is a dispersive technique—thin-films or blocks format are also possible—in which the sorbent collection from the sample bulk is easily performed by application of an external magnetic field (Ibarra et al., <xref rid=\"B60\" ref-type=\"bibr\">2015</xref>; Evrim et al., <xref rid=\"B31\" ref-type=\"bibr\">2019</xref>). The use of graphene-based materials for MSPE not only efficiently eliminates the clogging problems from the packed-dispositive but can also enhance the extraction capacity due to GBMs' properties. Also, MSPE possibly eliminates additional centrifugation and filtration steps (Li et al., <xref rid=\"B72\" ref-type=\"bibr\">2018</xref>). For these reasons, the use and development of magnetic sorbents incorporating GBMs have become a key-point in sample preparation in recent years. Nowadays, this combination has been applied in the MSPE of a wide diversity of organic and inorganic analytes from several complex samples, including the treatment of solid matrices (Feriduni, <xref rid=\"B38\" ref-type=\"bibr\">2019</xref>).</p><p>These graphene-based magnetic sorbents are currently obtained by physical or chemical anchoring of the magnetic carries onto the graphene sheets. The most common carries include iron (Fe), cobalt, (Co), and nickel (Ni) oxides, highlighting the magnetite (Fe<sub>3</sub>O<sub>4</sub>) and maghemite (γ-Fe<sub>2</sub>O<sub>3</sub>) as the magnetic nanoparticles (MNPs) more frequently used (Laura et al., <xref rid=\"B66\" ref-type=\"bibr\">2019</xref>). The Fe<sub>3</sub>O<sub>4</sub> and γ-Fe<sub>2</sub>O<sub>3</sub> have superparamagnetic properties, are easy to prepare, and disperse very well in aqueous solutions. Besides, those are MNPs feasible to be modified and functionalized (Yu M. et al., <xref rid=\"B171\" ref-type=\"bibr\">2019</xref>). More popular methods for the preparation of Fe<sub>3</sub>O<sub>4</sub> and γ-Fe2O3 based magnetic sorbents include chemical co-precipitation, hydrothermal synthesis, sol-gel reactions, solvothermal synthesis, thermal decomposition, microemulsion, and sonochemical approaches (Filik and Avan, <xref rid=\"B40\" ref-type=\"bibr\">2019</xref>).</p><p>The most straightforward type of graphene magnetic sorbents is prepared by direct immobilization of the MNPs on the surface of the G. In this context, two synthetic routes can be employed for obtaining them: (i) the chemical co-precipitation and (ii) the hydrothermal synthesis. The chemical co-precipitation is based on the deposition of iron ions over the nanosheets by adding an alkaline solution to an aqueous dispersion of graphene Fe<sup>3+</sup>/Fe<sup>2+</sup> salts, at elevated temperature and vigorous stirring. For example, this method was employed by Yang et al. to prepare superparamagnetic GO/Fe<sub>3</sub>O<sub>4</sub> nanoparticles (Yang et al., <xref rid=\"B165\" ref-type=\"bibr\">2009</xref>). After primary treatment of GO sheets, a dispersion of GO, FeCl<sub>3</sub> was stirred under inert atmosphere by several hours. After that, Fe<sup>3+</sup>/Fe<sup>2+</sup> ions were coordinated by the carboxylate anions on the GO sheets, and then GO/Fe<sub>3</sub>O<sub>4</sub> nanoparticles were precipitated by the addition of an aqueous NaOH solution (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>). As Fe<sup>3+</sup> shows higher affinity than Fe<sup>2+</sup> for carboxylic groups, the ratio of those ions should be controlled—generally, 2:1 is used—and the content of the carboxylic acid groups on the GO sheets needs to be previously determined by acid-base titration (Yang et al., <xref rid=\"B165\" ref-type=\"bibr\">2009</xref>). The chemical co-precipitation method can also be employed to prepare magnetic reduced graphene oxide sorbents (rGO/Fe<sub>3</sub>O<sub>4</sub>). As an example, Chandra et al. prepared an rGO/Fe<sub>3</sub>O<sub>4</sub> for arsenic removal from surface water samples (Chandra et al., <xref rid=\"B15\" ref-type=\"bibr\">2010</xref>). In this case, after dispersion and precipitation with ammonia, GO/Fe<sub>3</sub>O<sub>4</sub> particles were reduced to rGO/Fe<sub>3</sub>O<sub>4</sub> by slowly adding hydrazine hydrate under stirring conditions 90°C.</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>Schematic representation of GO loaded with Fe3O4 nanoparticles. Reprinted with permission from Yang et al. (<xref rid=\"B165\" ref-type=\"bibr\">2009</xref>), copyright 2009, Royal Society of Chemistry.</p></caption><graphic xlink:href=\"fchem-08-00664-g0005\"/></fig><p>An important issue is that the morphology of graphene magnetic sorbents prepared via chemical co-precipitation can be challenging to control. Thus, the resulting magnetic material sometimes presents low absorptivity due to the uneven distribution and agglomeration of the Fe<sub>3</sub>O<sub>4</sub> particles on the nanosheets. Within such a context, hydrothermal synthesis has been proposed as an alternative to yield sorbents with better Fe<sub>3</sub>O<sub>4</sub> particles distribution with more G exposed adsorption sites and then, improved adsorption capacity. This method is based on the reduction of Fe<sup>3+</sup> and GO in sodium acetate and polyethyleneglycol in an autoclave at elevated temperature (Li et al., <xref rid=\"B72\" ref-type=\"bibr\">2018</xref>). In this way, Wu and cookers prepared rGO/Fe<sub>3</sub>O<sub>4</sub> particles (Wu et al., <xref rid=\"B158\" ref-type=\"bibr\">2013</xref>), sonicating GO first in ethylene glycol, and then in the presence of FeCl<sub>3.</sub> After complete dispersion, the obtained clear solution was spiked with sodium acetate, and the mixture was sealed in a Teflon-lined stainless-steel autoclave and maintained at 200°C for 8 h. The authors reported regular morphology particles.</p><p>Also, more reproducible, stable, and versatile graphene magnetic sorbents can be prepared from silica-coated magnetite particles (Fe<sub>3</sub>O<sub>4</sub>@SiO2). In this case, before graphene anchoring, Fe<sub>3</sub>O<sub>4</sub> particles are modified with a silane coupling agent, consisting of tetraethyl orthosilicate (TEOS) and (3-aminopropyl) triethoxysilane (APTES) (Li et al., <xref rid=\"B72\" ref-type=\"bibr\">2018</xref>). Sequentially, the graphene can be coupled to the particles by physical adsorption or by covalent bonding. Luo et al. prepared Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>/G particles for extraction of sulfonamides from water samples, by physical immobilization of graphene nanosheets on silica-coated magnetite (Luo et al., <xref rid=\"B90\" ref-type=\"bibr\">2011b</xref>). The procedure obtained by pure dispersion under sonication for several hours renders particles not stable enough to continue reusing. Therefore, the chemical bonding of the graphene to the silanol groups is the preferred synthetic method. Amino groups are introduced on the surface of the Fe3O4@SiO2 particles and GO, anchored via an amidation reaction with the aid of cross-linking agents such as 1-(3-dimethyl aminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) and -hydroxysuccinimide (NHS). This process is schematically represented in <xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref> (Li et al., <xref rid=\"B72\" ref-type=\"bibr\">2018</xref>). For example, Zhang et al. prepared Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>/GO particles by mixing Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub> and 3-aminopropyltriethoxysilane in isopropanol, under N<sub>2</sub> atmosphere at 70°C, followed by addition of a GO solution containing NHS and EDC, stirring overnight (Zhang et al., <xref rid=\"B176\" ref-type=\"bibr\">2014</xref>). Like Fe<sub>3</sub>O<sub>4</sub>/rGO composites, Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>/rGO particles can be obtained by the posterior reduction of GO with hydrazine.</p><fig id=\"F6\" position=\"float\"><label>Figure 6</label><caption><p>Schematic procedures for the preparation of Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>/G. Reprinted from Li et al. (<xref rid=\"B72\" ref-type=\"bibr\">2018</xref>) by permission from Elsevier, 2018.</p></caption><graphic xlink:href=\"fchem-08-00664-g0006\"/></fig><p>The adsorption capacity of these magnetic particles is mostly based on the hydrophobic interactions of the honeycomb-like lattice into the carbon atoms. Consequently, bare graphene magnetic particles are not suitable MSPE sorbents for polar or ionic compounds. The main advantage of the Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>/G particles is the possibility to covalently bond additional functional moieties, which improve the adsorption capacity, selectivity, and applicability of them. In this manner, Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>/G have been covalently functionalized with ionic surfactants, ionic liquids (ILs), deep eutectic solvents, boronate affinity materials (BAM), supramolecules (crown ethers, cyclodextrins, calixarenes, cucurbiturils, and pillararenes), aptamers, polymers, and metal-organic frameworks (MOFs). For those magnetic GBMs, their preparation and applications were recently comprehensively reviewed by Li et al. They provided a summary of the state of the art of them, highlighting application as MSPE sorbents of organic compounds, biomolecules, and metal ions (Li et al., <xref rid=\"B72\" ref-type=\"bibr\">2018</xref>). For this reason, herein, we provide an updated overview of the reported magnetic graphene sorbents between 2019 and 2020, and their applications in the determination of small organic molecules by chromatographic analysis.</p><p>In this context, magnetic graphene sorbents have found spread applications in the extraction of biomolecules organic compounds and metal ions. An assessment in the Scopus database, using the keywords “graphene” and “magnetic solid-phase extraction,” yield 248 results from 2010. Among them, 26 correspond to review papers and the rest to research papers mainly dedicated to describing the application of magnetic graphene sorbent in different areas of the analytical chemistry. As mentioned previously, Li et al. recently published a summary of the applications of graphene-based MSPE. In <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>, we provide a summary of the magnetic graphene sorbent applications published from 2019 to date. It is noteworthy that a vast number of researchers using graphene-derived magnetic materials focus their efforts in areas as environmental surveillance and food security applications. This includes a wide diversity of analytes such as drug residues, pesticides, hormones, food additives, and active plant ingredients.</p><table-wrap id=\"T3\" position=\"float\"><label>Table 3</label><caption><p>Recent applications (2019–2020) of graphene-based MSPE to the determination of organic compound by chromatography and mass spectrometry.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Sorbent</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Analytes</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Matrix</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Analysis</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>LOD</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>References</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Magnetic N-doped 3D graphene-like framework carbon (Fe3O4@N-3DFC)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cephalosporin antibiotics</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">River water and zebrafish samples</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.20–0.45 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Niu et al., <xref rid=\"B102\" ref-type=\"bibr\">2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fluorine and nitrogen functionalized magnetic graphene (G-NH-FBC/Fe2O3)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Perfluoroalkyl and polyfluoroalkyl substances</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water and functional beverage</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-Orbitrap HRMS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Xian et al., <xref rid=\"B159\" ref-type=\"bibr\">2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Graphene-like MoS2-modified magnetic carbon-dot nanoflowers (MoS2@Fe3O4@C-dot NFs)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ibuprofen</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pharmaceutical, environmental water and synthetic urine samples</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">11 × 10<sup>−6</sup> μg mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yilmaz and Sarp, <xref rid=\"B168\" ref-type=\"bibr\">2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Oxide/lanthanum phosphate nanocomposite (MGO@LaP)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chlorpyrifos pesticides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water and fruit samples</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GC–ECD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.67 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Asadi et al., <xref rid=\"B9\" ref-type=\"bibr\">2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe3O4/rGO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Oral anticoagulants</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human plasma</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.003 μg mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ferrone et al., <xref rid=\"B39\" ref-type=\"bibr\">2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Iron crosslinked alginate encapsulated magnetic graphene oxide (Fe/alginate/Fe3O4/rGO)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Endocrine-disrupting compounds</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Superficial water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8–14 ng L-1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Shah and Jan, <xref rid=\"B124\" ref-type=\"bibr\">2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">chitosan functionalized magnetic graphene oxide nanocomposite (Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>@CS/GO)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Alkaloids</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chinese herb (<italic>Pericarpium papaveris</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UHPLC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.016–0.092 μg kg<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tang et al., <xref rid=\"B142\" ref-type=\"bibr\">2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe<sub>3</sub>O<sub>4</sub>/rGO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Oral anticoagulants</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human plasma</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UHPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.003 μg mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tang et al., <xref rid=\"B142\" ref-type=\"bibr\">2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Magnetic carbon nanodot/graphene oxide hybrid material (Fe<sub>3</sub>O<sub>4</sub>@C-nanodot@GO)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ibuprofen</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human blood</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8.0 ng mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yuvali et al., <xref rid=\"B172\" ref-type=\"bibr\">2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe<sub>3</sub>O<sub>4</sub>/GO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Psychoactive drugs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Urine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UHPLC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.02–0.2 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lu et al., <xref rid=\"B84\" ref-type=\"bibr\">2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe<sub>3</sub>O<sub>4</sub>/GO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Melamine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water and dairy products</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.03 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Abdolmohammad-zadeh, <xref rid=\"B3\" ref-type=\"bibr\">2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Three-dimensional graphene aerogel combined with Fe<sub>3</sub>O<sub>4</sub> nanoparticles (3DG-Fe<sub>3</sub>O<sub>4</sub>@Sp)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cholecalciferol (vitamin D3)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bovine milk</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3.01 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sereshti et al., <xref rid=\"B122\" ref-type=\"bibr\">2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe3O4/GO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">45 multi-class pesticides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Vegetables (cabbage, leek, and radicchio)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GC-MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.4–4.0 μg kg−1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chatzimitakos et al., <xref rid=\"B16\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Aptamer-functionalized Fe<sub>3</sub>O<sub>4</sub>/graphene oxide (Fe<sub>3</sub>O<sub>4</sub>/GO/Apt)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chloramphenicol</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Honey and Milk</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.24 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tu et al., <xref rid=\"B145\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Flower-like hybrid material composed of Fe<sub>3</sub>O<sub>4</sub>, graphene oxide and CdSe nanodots (Fe<sub>3</sub>O<sub>4</sub>/GO/CdSe)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ibuprofen</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pharmaceuticals, water, and urine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.36 ng mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sarp and Yilmaz, <xref rid=\"B121\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe<sub>3</sub>O<sub>4</sub>/rGO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Aflatoxin B1 and B2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Vegetable Oils</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-PCD-FLD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.01–0.02 μg kg<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yu L. et al., <xref rid=\"B170\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe<sub>3</sub>O<sub>4</sub>/rGO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N-nitrosamines</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mainstream cigarette smoke</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.018–0.057 ng cigarette<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pang et al., <xref rid=\"B107\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GO@NH<sub>2</sub>@Fe<sub>3</sub>O<sub>4</sub></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Quinolones</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MALDI-TOF MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.010 mg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tang H. et al., <xref rid=\"B139\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GO@NH<sub>2</sub>@Fe<sub>3</sub>O<sub>4</sub></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Triazines</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DART-MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.6–152.1 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Jing et al., <xref rid=\"B62\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Magnetic nitrogen-doped reduced graphene oxide (Fe<sub>3</sub>O<sub>4</sub>@N-rGO)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Endocrine disruptors</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Carbonated beverages</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.1–0.2 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Li et al., <xref rid=\"B70\" ref-type=\"bibr\">2019b</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Molecular imprinted polymer (MIP) material combined with magnetic graphene oxide (Fe<sub>3</sub>O<sub>4</sub>/GO-MIP)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Quercetin and luteolin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Green tea and serum samples</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.09–4.5 ng mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dramou et al., <xref rid=\"B27\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Magnetic graphene-like molybdenum disulfide nanocomposite</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Triazines and sulfonylurea herbicides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UHPLC-MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">20 and 170 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Zhou Y. et al., <xref rid=\"B184\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ionic liquid magnetic graphene (IL@MG)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Microcystins</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UHPLC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.414 ng L<sup>−1</sup> and 0.216 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Liu X. et al., <xref rid=\"B83\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Covalent organic framework-derived hydrophilic magnetic graphene Composite (magG@PDA@TbBd)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phthalate esters</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Milk</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CapillaryLC-MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.004–0.02 ng mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lu et al., <xref rid=\"B85\" ref-type=\"bibr\">2019a</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Curcumin loaded magnetic graphene oxide</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Parabens</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Toothpaste and mouthwash</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.4–1.0 ng mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Razavi and Es, <xref rid=\"B116\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe<sub>3</sub>O<sub>4</sub>/rGO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Non-steroidal Anti-inflammatory Drugs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Animal food</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.1–0.5 μg kg<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Wang et al., <xref rid=\"B155\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe<sub>3</sub>O<sub>4</sub>/rGO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phenolic compounds</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Oil seeds</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">LC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.02–90.00 μg kg<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lang et al., <xref rid=\"B65\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe<sub>3</sub>O<sub>4</sub>/rGO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pesticides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.2–1.6 ng mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Madej et al., <xref rid=\"B93\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Three-dimensional hierarchical frameworks based on molybdenum disulfide-graphene oxide-supported magnetic nanoparticles (Fe<sub>3</sub>O<sub>4</sub>/GO/MoS<sub>2</sub>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fluoroquinolone antibiotics</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.25–0.50 ng mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Xiao et al., <xref rid=\"B160\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Magnetic nanoparticles/graphene oxide (TPN/Fe<sub>3</sub>O<sub>4</sub>NPs/GO) nanocomposite</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pesticides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.17–1.7 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Moradi et al., <xref rid=\"B99\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Silver-modified Fe<sub>3</sub>O<sub>4</sub>/graphene nanocomposite (Ag@Fe<sub>3</sub>O<sub>4</sub>@G)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Aromatic amines</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.10–0.20 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Alasl et al., <xref rid=\"B4\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe<sub>3</sub>O<sub>4</sub>/GO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chlorophenols</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sewage water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GC-ECD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3.0–28.4 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Esfandiarnejad and Sereshti, <xref rid=\"B29\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ternary nano-composite, magnetite/reduced graphene oxide/silver (Fe<sub>3</sub>O<sub>4</sub>/rGO/Ag)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Morphine and codeine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Blood and urine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV-VIs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.8–2.1 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Abdolmohammad-zadeh et al., <xref rid=\"B2\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Magnetic amino-functionalized zinc metal-organic framework based on a magnetic graphene oxide composite (M-IRMOF/Fe<sub>3</sub>O<sub>4</sub>/GO)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Heterocyclic fungicides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lettuce</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.21–1.0 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Liu G. et al., <xref rid=\"B78\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe<sub>3</sub>O<sub>4</sub>/rGO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chiral pesticides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cucumber, tomato, cabbage, grape, mulberry, apple, and pear</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chiral HPLC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.02–10.0 μg g<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Zhao et al., <xref rid=\"B179\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reduced graphene oxide-carbon nanotubes composite (Fe<sub>3</sub>O<sub>4</sub>/rGO-CNTs)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sulfonamides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Milk</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.35–1.32 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Feng et al., <xref rid=\"B37\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Magnetic graphene oxide/multiwalled carbon nanotube core-shell (GO/MWCNT/Fe<sub>3</sub>O<sub>4</sub>/SiO<sub>2</sub>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paracetamol and caffeine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Synthetic urine and wastewater</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.48–3.32 ng mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ibrahim et al., <xref rid=\"B61\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Magnetized graphene oxide functionalized with hydrophilic phytic acid and titanium(IV) (magGO@PEI@PA@Ti<sup>4+</sup>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nucleobases, nucleosides, and nucleotides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Medicinal mushroom <italic>C. Sinensis</italic>, and natural foods</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.8–2.8 ng mL <sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Zhang Q. et al., <xref rid=\"B175\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MagG@SiO<sub>2</sub>@ZIF-8 composites</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phthalate Easers</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human Plasma</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GC-MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.003–0.01 ng mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lu et al., <xref rid=\"B86\" ref-type=\"bibr\">2019b</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Multi-templates molecularly imprinted polymer (MIP) on the surface of mesoporous silica-coated magnetic graphene oxide (MGO@mSiO<sub>2</sub>),</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Alkylphenols</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.010–0.013 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Xie et al., <xref rid=\"B161\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phthalate esters</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bottled, injectable and tap waters</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.004–0.013 mg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Abdelghani et al., <xref rid=\"B1\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe<sub>3</sub>O<sub>4</sub>/rGO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Non-steroidal anti-inflammatory drugs and bisphenol-A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tap water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.031 mg L<sup>−1</sup> and 0.023 mg L<sup>−1</sup><break/> 0.1785 mg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ungku Abdullah et al., <xref rid=\"B146\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">rGO/ZnFe<sub>2</sub>O<sub>4</sub> nanocomposite</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Estrogens</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water, soil, and fish</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UHPLC-QTOF-MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.01–0.02 ng mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Li W. et al., <xref rid=\"B73\" ref-type=\"bibr\">2020</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polydopamine functionalized magnetic graphene (PDA@MG)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Triazole fungicides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4.8–8.4 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Xiong et al., <xref rid=\"B162\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">zeolitic imidazolate framework-7@graphene oxide (mag-ZIF-7@GO)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fungicides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water and soil samples</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-Orbitrap HRMS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.58–2.38 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Zhang S. et al., <xref rid=\"B177\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Magnetic polyethyleneimine modified reduced graphene oxide (Fe<sub>3</sub>O<sub>4</sub>@PEI-rGO)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polar non-steroidal anti-inflammatory drugs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.2 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Li et al., <xref rid=\"B71\" ref-type=\"bibr\">2019a</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Guanidinium ionic liquid modified magnetic chitosan/graphene oxide nanocomposites (GIL-MCGO)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DNA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human whole blood and <italic>E. coli</italic> cell lysate</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN2\"><sup>**</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN2\"><sup>**</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Liu M. et al., <xref rid=\"B81\" ref-type=\"bibr\">2019</xref></td></tr></tbody></table><table-wrap-foot><fn id=\"TN2\"><label>**</label><p><italic>Not specified</italic>.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Molecularly-Imprinted Polymers</title><p>Another group of materials explored for sample preparation involves the molecularly-imprinted polymers (MIPs) combined with GBMs. In this context, MIPs are prepared from a template (mold) which behaves structurally similarly as the target analytes, to achieve a selective interaction through a template-complementary binding site (Pan et al., <xref rid=\"B106\" ref-type=\"bibr\">2018</xref>). These materials are traditionally prepared by copolymerization of the complex formed between the template and a functional monomer. This can occur through either covalent (hydrogen bonds) or non-covalent bonds (ionic and hydrophobic interactions) with a cross-linking agent in the presence of a suitable porogenic solvent (Zhou T. et al., <xref rid=\"B182\" ref-type=\"bibr\">2019</xref>). After that, the molecular template is eliminated, resulting in a rigid three-dimensional cavity selective to the target analytes. As an example, <xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref> depicts the synthesis steps employed by Xie et al. (<xref rid=\"B161\" ref-type=\"bibr\">2019</xref>). The authors selected 4-vinylbenzoic acid as a functional monomer to prepare the template taking advantage of the presence of the carboxyl ring and benzene, which can bind to BPA, 4- tert-OP, and 4-NP through hydrogen bonds and π-π interactions in the polymerization process.</p><fig id=\"F7\" position=\"float\"><label>Figure 7</label><caption><p>Schematic diagram representing the synthesis of VTTS-MGO@mSiO2@MIP. Reprinted with permission from Xie et al. (<xref rid=\"B161\" ref-type=\"bibr\">2019</xref>), copyright 2019, Elsevier.</p></caption><graphic xlink:href=\"fchem-08-00664-g0007\"/></fig><p>Due to this high selectivity and relatively easy preparation, these materials have been widely used for molecular recognition and separation in different fields (sensors, drug delivery, protein recognition, and chromatography). In this section, their application in the field of sample preparation combined with GBMs is covered, highlighting preparation strategies of aqueous-recognition MIPs cleverly to achieve such functionalization (Zhou T. et al., <xref rid=\"B182\" ref-type=\"bibr\">2019</xref>). For example, Luo et al. (<xref rid=\"B88\" ref-type=\"bibr\">2017</xref>) synthesized boronic acid-functionalized with graphene oxide, with a subsequent immobilization of ovalbumin as MIP-template, to obtain a high-selective sorbent, namely GO-APBA/MIPs. This strategy was chosen to overcome such difficulties related to specific recognition and separation of glycoproteins in complex biological samples. A comparison between the resulting material (GO-APBA/MIPs) and a bare GO-MIPs, without insertion of boronic acid, showed more extraction performance for the hybrid obtained sorbent. In this way, the outstanding recognition capacity by linking boronic acid and MIP cavities together with a high surface area of GO can represent a promising strategy to produce high performative sorbent material for biological glycoproteins.</p><p>Another interesting example was reported by Cheng et al. (<xref rid=\"B20\" ref-type=\"bibr\">2017</xref>), consisting of a more straightforward strategy using GO combined with MIP to extract and efficiently pre-concentrate bis(2-ethylhexyl) phthalate (DEHP) in environmental water samples. Contrary to the prior study (Luo et al., <xref rid=\"B88\" ref-type=\"bibr\">2017</xref>), considering the smaller complexity of the target compound and water samples, the employment of only GO-MIP was already enough to achieve excellent extraction performance. In this case, dispersive solid-phase microextraction combined with HPLC-UV reported enrichment factors of more than 100-fold compared to the directly injected extract, highlighting this simple GO-MIP as a suitable sorbent in such cases.</p><p>An attractive approach to obtain high performative material involves the use of the graphene-based MIP functionalized with a magnetic particle to favor the extraction and sorbent isolation. Within such a context, Ning et al. (<xref rid=\"B101\" ref-type=\"bibr\">2014</xref>) proposed a novel nanosized substrate imprinted polymer (GO-MIP-Fe<sub>3</sub>O<sub>4</sub>) on magnetic graphene oxide (GO-Fe<sub>3</sub>O<sub>4</sub>) surface to remove 17β-estradiol (17β-E<sub>2</sub>) from food samples. The resulting sorbent has shown a functional extraction recovery of 84.20% at a low concentration level of 0.5 μmol L<sup>−1</sup>. Furthermore, due to the magnetic properties of the GO-MIP-Fe<sub>3</sub>O<sub>4</sub>, a simple, fast, and efficient separation of 17β-E<sub>2</sub> were achieved, suggesting the combination between these materials as an excellent way to obtain hybrid sorbents. Following the same trend, Barati et al. (<xref rid=\"B10\" ref-type=\"bibr\">2017</xref>) synthesized a MIP based on magnetic-chitosan functionalize with GO to extract fluoxetine from environmental and biological samples. From our viewpoint, the outstanding characteristic of this work is the excellent pre-concentration factor of 500 related to such sorbent, which reinforces the combination of MIP, GBMs, and magnetic materials as an excellent way to improve sample preparation performance. Finally, another study based on a similar approach was presented by Fan et al., who prepared, through a chemical co-precipitation method, a novel hybrid sorbent based on MIP, GO, and superparamagnetic Fe<sub>3</sub>O<sub>4</sub> particles (GO-MIP-Fe<sub>3</sub>O<sub>4</sub>). In this case, the author worked with natural samples, specifically alkaloids (evodiamine and rutaecarpine) extract from <italic>Evodiae Fructus</italic>, suggesting a great versatility of such magnetic GO-MIP sorbent. Also, its analytes' recovery achieves values over 82% considering as good values from our viewpoint.</p><p>Moreover, <xref rid=\"T4\" ref-type=\"table\">Table 4</xref> presents other published studies to complement the discussion regarding hybrid sorbents combining MIP with GBMs.</p><table-wrap id=\"T4\" position=\"float\"><label>Table 4</label><caption><p>Applications of MIPs-GO composite in sample pretreatment.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Sorbent</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Analytes</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Matrix</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Sample preparation</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Analysis</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>LOD</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>References</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GO-APBA/MIP</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ovalbumin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Egg white</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SDS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gel-Electrophoresis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN3\"><sup>**</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Luo et al., <xref rid=\"B88\" ref-type=\"bibr\">2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">bis(2-ethylhexyl) phthalate (DEHP)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Trace DEHP phthalate</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPME</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.92 ng mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cheng et al., <xref rid=\"B20\" ref-type=\"bibr\">2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MIPs-GO-Fe<sub>3</sub>O<sub>4</sub></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">17β-estradiol</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Milk powder</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">External magnet</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN3\"><sup>**</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.035 and 0.10 μmol L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ning et al., <xref rid=\"B101\" ref-type=\"bibr\">2014</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GO-QDs-MIPs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p-t-octylphenol</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UHPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.15 μmol L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Han et al., <xref rid=\"B51\" ref-type=\"bibr\">2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">(TFMAA)-GO (EGDMA)-GO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cefadroxil</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">d-SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.01 μg mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chen and Ye, <xref rid=\"B18\" ref-type=\"bibr\">2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MGR@MIPs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4-nitrophenol</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lake water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MIP</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN3\"><sup>**</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Luo et al., <xref rid=\"B87\" ref-type=\"bibr\">2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MIP-GO/Chm</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fluoxetine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tap, well and spring water, and urine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MSPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UV–Vis spectrophotometry</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.03 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Barati et al., <xref rid=\"B10\" ref-type=\"bibr\">2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MIP@Fe<sub>3</sub>O<sub>4</sub>@GO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Evodiamine and rutaecarpine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Evodiae fructus</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">External magnet</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN3\"><sup>**</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fan et al., <xref rid=\"B32\" ref-type=\"bibr\">2017</xref></td></tr></tbody></table><table-wrap-foot><fn id=\"TN3\"><label>**</label><p><italic>Not specified</italic>.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Ionic Liquids</title><p>In 1914, a work with ionic liquids (ILs) was reported for the first time, although at that time, the author had no idea of the importance that these would take in the scientific area almost a century later (Walden, <xref rid=\"B149\" ref-type=\"bibr\">2003</xref>). From the 1980's to the present, the interest in studying ionic liquids grew exponentially (Evans et al., <xref rid=\"B30\" ref-type=\"bibr\">1981</xref>; Shi et al., <xref rid=\"B126\" ref-type=\"bibr\">2015</xref>; Welton, <xref rid=\"B156\" ref-type=\"bibr\">2018</xref>). Although they have been applied in several scientific areas, in the analytical chemistry the studies have focused mainly on the use of them for extraction, and separation purposes (Rodríguez-Sánchez et al., <xref rid=\"B117\" ref-type=\"bibr\">2014</xref>; Tang et al., <xref rid=\"B140\" ref-type=\"bibr\">2014</xref>; García-Alvarez-Coque et al., <xref rid=\"B44\" ref-type=\"bibr\">2015</xref>; Marcinkowski et al., <xref rid=\"B96\" ref-type=\"bibr\">2015</xref>; Hu et al., <xref rid=\"B57\" ref-type=\"bibr\">2016</xref>; Yu et al., <xref rid=\"B169\" ref-type=\"bibr\">2016</xref>; Nawała et al., <xref rid=\"B100\" ref-type=\"bibr\">2018</xref>; Rykowska et al., <xref rid=\"B119\" ref-type=\"bibr\">2018</xref>).</p><p>The ILs are compounds with a dual nature acting as nonpolar for nonpolar analytes and, inversely, for those with a strong proton donor group, depending on the separation mechanism that it presents (Berthod et al., <xref rid=\"B11\" ref-type=\"bibr\">2018</xref>). Therefore, their excellent properties (high thermal stability, good solubility, and easily functionalization) have been modified in several different ways. One approach consists of replacing the anionic or cationic part with another material, of automatically regulating the IL property or nature (hydrophobicity, hydrophilicity, viscosity, and among others) (Ho et al., <xref rid=\"B52\" ref-type=\"bibr\">2014</xref>; An et al., <xref rid=\"B8\" ref-type=\"bibr\">2017</xref>). In this way, improvements in their sensitivity and selectivity to the extraction of the different analytes can be achieved. One of these desired enhancements is obtained by combining the ILs with GBMs, owing to their different types of chemical interactions with the analytes (e.g., n-π, π-π, hydrogen bonding, dipolar, ionic charge/charge) (Chen Y. et al., <xref rid=\"B19\" ref-type=\"bibr\">2016</xref>; Feng et al., <xref rid=\"B36\" ref-type=\"bibr\">2020</xref>). For these reasons, IL-GBMs present excellent extraction efficiency for a wide variety of analytes in several complex matrices (e.g., environmental, food, drinks, biological, and among others), as presented. Additionally, <xref ref-type=\"fig\" rid=\"F8\">Figure 8</xref> illustrates a typical synthesis process performed to achieve such hybrid sorbents.</p><fig id=\"F8\" position=\"float\"><label>Figure 8</label><caption><p>Preparation procedure of Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>@G@PIL. Reprinted with permission from Chen Y. et al. (<xref rid=\"B19\" ref-type=\"bibr\">2016</xref>), copyright 2016, Elsevier.</p></caption><graphic xlink:href=\"fchem-08-00664-g0008\"/></fig><p>The employment of ILs combined with GMBs includes the work reported by Liu X. et al. (<xref rid=\"B83\" ref-type=\"bibr\">2019</xref>) to determine the environmentally-dangerous monocyclic heptapeptides (microcystins, MC) in natural water samples. They synthesized the IL-G by the co-precipitation route. For the analytes' extraction, the MSPE technique was used while for its separation, determination, and quantification, the authors employed UHPLC-MS/MS. For the optimization of the experimental parameters, univariate analysis, and orthogonal screening were used. The analysis time was 18 min, with excellent linearity. The LODs were 0.414 and 0.216 ng L<sup>−1</sup> for MC-LR and MC-RR, respectively, reporting traces of these two compounds in natural water samples, and it can be concluded that the method used is promising for the study of other types of microcystins in water samples.</p><p>Other interesting applications based on IL-GBMs include work published by Chen Y. et al. (<xref rid=\"B19\" ref-type=\"bibr\">2016</xref>). They synthesized magnetite nanoparticles (Fe<sub>3</sub>O<sub>4</sub>) of controlled size by a co-precipitation method to obtain the Fe<sub>3</sub>O<sub>4</sub>-SiO<sub>2</sub>-G-PIL hybrid sorbent; the obtained material was used to determine preservatives in vegetables by QuEChERS following GC-MS analysis. In this study, it was possible to take advantage of the functional properties related to magnetic nanoparticles and also by its coupling to the GO with the polymeric ILs (e.g., high surface area, and solvent effects). Moreover, the method's detection limits varied between 0.82 and 6.64 μg kg<sup>−1</sup> for the 20 preservatives studied. Similarly, Tashakkori et al. (<xref rid=\"B143\" ref-type=\"bibr\">2019</xref>) synthesized a series of ionic liquids grafted onto stainless steel wires, which were previously coated with GO, using a sol-gel technique. The authors used this hybrid material as a sorbent in an on-line DI-SPME-GC-MS approach to determine phthalate esters (PAE) in several samples such as tap water and seawater and coffee. They reported low detection limits (5–30 ng L<sup>−1</sup>) and the lab-made SPME fibers being used more than 120 times, which is a useful feature when compared with the commercially available ones such as PA and CAR/PDMS. These results reinforce the great interest in developing hybrid GBMs combined with ILS.</p><p>Additionally, biological samples were analyzed by Ding et al. (<xref rid=\"B26\" ref-type=\"bibr\">2015</xref>). They functionalized magnetic chitosan with a series of ionic liquids of guanidinium and graphene oxide, aiming to extract trypsin, lysozyme, ovalbumin, and albumins from bovine serum. In this case, the analytical results obtained by this hybrid guanidinium-IL functionalized with MCGO were compared with those achieved by employing just GO, magnetic chitosan, or MCGO. In this case, the hybrid guanidinium-IL-MCGO exhibits higher extraction performance compared to the other sorbents, which suggests the combination of GBMs and ILs as a suitable strategy to achieve more performative extractive phases. Also, Hou et al. (<xref rid=\"B53\" ref-type=\"bibr\">2016</xref>) proposed a poly(1-vinyl-3-hexylimidazolium bromide)-GO-grafted silica [poly(VHIm<sup>+</sup>Br<sup>−</sup>)-GO-Sil] as a hybrid IL sorbent to extract flavonoids in urine samples. They synthesized the material by an interesting process consisting of an <italic>in situ</italic> radical chain-transfer polymerization and then <italic>in situ</italic> anion exchange. In this case, the procedure started with the silica coating by GO, using a manufacturing layer by layer fabrication method. Then, the poly (VHIm<sup>+</sup>Br<sup>−</sup>)-GO-Sil anion was transformed into hexafluorophosphate (PF<sup>6−</sup>) by <italic>in situ</italic> anion exchange. The method based on SPE-HPLC-UV showed acceptable extraction recoveries for four flavonoids, with limits of detection in the range of 0.1–0.5 μg L<sup>−1</sup>. The proposed material showed ecological and cost-effective advantages. It can be applied successfully to the extraction and enrichment of flavonoids in human samples, even allowing the study of metabolic kinetics.</p><p>Finally, considering other types of samples, an interesting study published by Zhou et al. (<xref rid=\"B183\" ref-type=\"bibr\">2016</xref>) was carried out to analyze phthalates (PAE) in eraser samples. The Office of Quality and Technology Supervision of the province of Jiangsu in China required PAE analyses in several samples to maintain concentrations inside the accepted limits registered by law. For this reason, it is necessary to monitor residues of this compound not only in food but also in everyday objects used by people (Zhou et al., <xref rid=\"B183\" ref-type=\"bibr\">2016</xref>). Within such a context, a new graphene oxide compound modified with ionic liquids (GO-[AEMIM][Br]) was synthesized through an amidation reaction between the amino groups of the ILs and the carboxyl groups of GO. The high extraction capacity reported by this hybrid sorbent suggests the combination of the properties of ILs and GBMs (tunability and high surface area) as a positive relationship that assisted the PAE extraction in such a non-common sample.</p><p>Apart from the interesting application herein discussed, other relevant studies based on hybrid sorbents combining IL and GBMs are shown in <xref rid=\"T5\" ref-type=\"table\">Table 5</xref>.</p><table-wrap id=\"T5\" position=\"float\"><label>Table 5</label><caption><p>Applications of Ils-GO composite in sample pretreatment.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Sorbent</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Analyte</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Matrix</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Sample preparation</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Analysis</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>LOD</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>References</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-GO@Silica</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chlorophenols</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN4\"><sup>**</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Wang et al., <xref rid=\"B152\" ref-type=\"bibr\">2017a</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL@MG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Microcystins</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MSPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UHPLC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.414 ng L<sup>−1</sup> and 0.216 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Liu X. et al., <xref rid=\"B83\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[C<sub>4</sub>C<sub>12</sub>im]@GO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hg</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AAS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sotolongo et al., <xref rid=\"B131\" ref-type=\"bibr\">2018</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">[BMim]@MGO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Heavy metal ions</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ICP-OES</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ICP-OES</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.1–1.0 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rofouei et al., <xref rid=\"B118\" ref-type=\"bibr\">2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MGONPs-C<sub>16</sub>mimBr</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chlorophenols</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MSPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.10–0.13 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Liu W. et al., <xref rid=\"B82\" ref-type=\"bibr\">2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PGO-MILN</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chlorophenols</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MSPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">LC-MS/MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.2–2.6 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cai et al., <xref rid=\"B13\" ref-type=\"bibr\">2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GO-1-butyl-3-aminopropyl imidazolium chloride.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Anabolic steroids<break/> β-blockers</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7–23 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Serrano et al., <xref rid=\"B123\" ref-type=\"bibr\">2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GO-PILs monolith</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phenolic</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPME</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.2–0.5 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sun et al., <xref rid=\"B134\" ref-type=\"bibr\">2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>@G@PIL</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Preservatives</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Vegetables</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">QuEChERS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GC-MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.82–6.64 μg kg<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chen Y. et al., <xref rid=\"B19\" ref-type=\"bibr\">2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">rGO/ILN-ETD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Estrogens</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Milk</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ETD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.09–0.30 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chu et al., <xref rid=\"B21\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1-(3-aminopropyl)-3-vinyl imidazolium bromide/tetrafluoroborate) grafted</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phthalate Esters</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Coffee<break/> Tap Water<break/> Seawater</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPME</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GC-MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5–30 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tashakkori et al., <xref rid=\"B143\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PANI-MWCNTs-rGO-IL</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Alcohols</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tea</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPME</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GC-FID</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2.2–28.3 ng L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Li et al., <xref rid=\"B69\" ref-type=\"bibr\">2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-TGO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Auxins</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Soybean sprouts</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PT-SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-DAD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.004–0.026 μg g<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Zhang H. et al., <xref rid=\"B174\" ref-type=\"bibr\">2018</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MGO-C<sub>16</sub>MIM-DMG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Trace nickel</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Spinach<break/> Cacao powder<break/> Tea<break/> Cigarette</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MSPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FAAS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.16 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Aliyari et al., <xref rid=\"B5\" ref-type=\"bibr\">2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MCGO@guanidinium IL</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protein</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bovine serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MSPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UV-vis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN4\"><sup>**</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ding et al., <xref rid=\"B26\" ref-type=\"bibr\">2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">poly(VHIm<sup>+</sup>Br<sup>−</sup>)@GO@Sil</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Flavonoids</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Urine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.1–0.5 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hou et al., <xref rid=\"B53\" ref-type=\"bibr\">2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-coated Fe<sub>3</sub>O<sub>4</sub>/GO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hemin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Serum</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FAAS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3.0 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Farzin et al., <xref rid=\"B35\" ref-type=\"bibr\">2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1-(3-aminopropyl)imidazole chloride modified MGO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polysaccharides</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Brown alga</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MSPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN4\"><sup>**</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Wang et al., <xref rid=\"B153\" ref-type=\"bibr\">2017b</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PILs@GO@Sil</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phenolic acids</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Black wolfberry yogurt and urine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.20–0.50 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hou et al., <xref rid=\"B54\" ref-type=\"bibr\">2018a</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Magnetic GO/PPy</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Methotrexate</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Urine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">d-SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7 ng mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hamidi et al., <xref rid=\"B50\" ref-type=\"bibr\">2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3D-IL-Fe<sub>3</sub>O<sub>4</sub>-GO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PAHs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human blood</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PT-SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GC-MS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.002–0.004 μg L<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Zhang Y. et al., <xref rid=\"B178\" ref-type=\"bibr\">2018</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PIL(Br)-G/SiO<sub>2</sub></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human serum albumin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human whole blood</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref ref-type=\"table-fn\" rid=\"TN4\"><sup>**</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Liu et al., <xref rid=\"B80\" ref-type=\"bibr\">2018</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fe<sub>3</sub>O<sub>4</sub>/GO NPs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cephalosporins</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Urine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MSPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.6 and 1.9 ng mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Wu et al., <xref rid=\"B157\" ref-type=\"bibr\">2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GO-[AEMIM][Br]</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phthalates</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Eraser</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HPLC-UV</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.02–0.88 ng mL<sup>−1</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Zhou et al., <xref rid=\"B183\" ref-type=\"bibr\">2016</xref></td></tr></tbody></table><table-wrap-foot><fn id=\"TN4\"><label>**</label><p><italic>Not specified</italic>.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Miscellaneous</title><p>This section aims to present some alternative graphene-based materials that could be an alternative for the sample preparation in the future. For this reason, there are not many publications apart from the materials already discussed in the previous sections. An original example recently published by Tahmasebi (<xref rid=\"B137\" ref-type=\"bibr\">2018</xref>) consisted of intercalating aluminum polyoxocations (Al<sub>30</sub>) between the graphene oxide (GO) nanosheets and further support this onto polycaprolactone nanofibers, aiming to enhance temperature, and pH resistance to the sorbent phase. This novel material was applied in SPE extraction of four statin drugs showing acceptable analytical performance. The author emphasized the excellent ability of this sorbent to interact with acidic polar species, possibly due to the resistance to pH variations. Another interesting application was performed by Li et al. (<xref rid=\"B68\" ref-type=\"bibr\">2014</xref>) whose synthesized a magnetic ionic liquid/chitosan/GO (MCGO-IL) as a biodegradable/bio-sorbent for the mitigation of Cr (IV) in the treatment of residual in environmental water samples. A solid-liquid separation was performed in the presence of an external magnetic field, involving a pseudo-second-order kinetic sorption step. The maximum adsorption capacity was 145.35 mg g<sup>−1</sup>, obtained thanks to the intermolecular hydrogen bond between MCGO-IL and Cr(IV), due to the hydroxy and amine groups to which the ions bind metallic. This result demonstrates the potential of this hybrid sorbent material in the cleaning processes of contaminating metal ions in complex matrices such as water. Additionally, the authors emphasize its low producing cost as a useful characteristic since commercially available sorbents often are expensive and non-reusable.</p><p>Additionally, the more significant interlayer distance between GO sheets, due to Al<sub>30</sub> insertions, can enhance the interaction with the π-electron system onto the GO surface, resulting in enhanced extraction performance. Similarly, Amiri et al. (<xref rid=\"B7\" ref-type=\"bibr\">2019</xref>) synthesized another hybrid sorbent but this time supporting GO nanosheets with polyoxotungstate (POT), instead of Al, to enhance chemical stability and pH resistance once POT act as charge-compensating and space-filling compound. This strategy is proposed to enhance the POT water dispersibility and surface area of the sorbent, possibly favoring the extraction performance while ensuring its high thermal and pH stability. Another strategy, underscored by Farajvand et al. (<xref rid=\"B34\" ref-type=\"bibr\">2018</xref>), was to covalently-bond an electrically conducting polymers onto the GO surface to diminish its self-aggregation in aqueous as well as enhance adsorption capacity. In this case, polyaniline was used considering its various oxidation states possessing distinct charge carriers which allow chemical interactions with several heavy metals. In this case, this sorbent was tested for Cd isolation from environmental water samples by SPE and subsequent dispersive liquid-liquid extraction.</p><p>In general, all these modifications performed in the G or GO chemical structures aim to improve its performance. However, a work published by Ashori et al. seems to follow the reverse trend focusing on using the graphene oxide as a reinforcement for glass fiber and epoxy composites. For this reason, the primary goals were to improve chemical reactivity, toughness, and adhesion to polymeric matrices, including the GO's widely-known properties. Although this material had not been applied for sample preparation yet, its high mechanical strength might be a promising tool particularly for miniaturized sample preparation techniques once they often demand high-pressure procedures causing clogging problems when GO or G are applied as sorbents. Recently, another interesting compound, namely graphene-aerogel, has gained attention mainly due to its superior and tunable volume as well as surface area as compared to graphene. The aerogel only itself exhibits poor extraction performance for water-soluble analytes, thus demanding some modification on it, as the functionalization of graphene-based compounds. In this way, graphene-based aerogels are suitable for sample preparation since they can relate the great qualities of graphene with the impressive compressibility of aerogels. Therefore, high-performance sorbents packed into sample preparation hardware can be reusable many times, considering the compressibility factor, which can help unpack and recover these extractive phases. As examples, Maggira et al. (<xref rid=\"B94\" ref-type=\"bibr\">2019</xref>) and Tang S. et al. (<xref rid=\"B141\" ref-type=\"bibr\">2019</xref>) reported two self-recoverable graphene-aerogels which were successfully applied to the analysis of sulfonamides and phenolic compounds in complex matrices, respectively.</p><p>Considering the impressive arising of graphene-based sorbents throughout the last years, herein, we aimed to pinpoint some of the unusual approached to perform such modifications and production of extractive phase. However, novel sorbent phases can be expected to be developed daily, considering the great qualities of nanomaterials for analytical chemistry purposes, highlighting the graphene.</p></sec></sec><sec id=\"s4\"><title>Concluding Remarks</title><p>Bearing in mind the great importance of sample preparation to clean-up samples, extract, and pre-concentrate target analytes become easier to understand the increasing interest of the analytical chemistry in developing modern strategies to optimize such a critical stage. In this context, one of the most relevant and promising fields is the development of more performative and environmentally-friend sorption-based techniques and, by consequence, the sorbents commonly used on them. As it is known, a good sorbent material must have some essential characteristics, including (i) selectivity for specific analytes and thus present chemical inertia for matrix interferents; (ii) good recovery and enrichment factors; and (iii) simple and non-expensive production. Once fulfilling these requirements, graphene-based materials have increasingly seemed to be the right candidate since their first application in sample preparation around 2011. Its large surface area, together with the π-π delocalized electron system, aid in improving so much the extraction performance of target compounds possessing aromatic rings as several chemical classes (e.g., pesticides, pharmaceuticals, and others) as herein discussed on section Graphene and Graphene Oxide.</p><p>Although the successful application in its “bare” form (G and GO), sometimes the functionalization of them seems to enhance even more the performance of such sorbents. For this reason, in this work, we have shown several different applications based on the use of hybrid materials consisting of GBMs anchored to other extractive sorbents such as alkyl and aryl groups, cyclodextrins, magnetic particles, molecularly imprinted polymers, ionic liquids, and among others. The functionalization is often achieved by forming covalent or hydrogen bondings, using sol-gel or polymerization router, or even electrochemical deposition. This interesting approach is encouraged by the possibility to ally the advantages of each class in only one. Apart from this goal, the clogging and backpressure problems often related to G and GO when packed in sorbent-based sample preparation techniques are overcome by the addition of other compounds. For example, several works underscored in sections Alkyl and Aril Groups, Cyclodextrins, and Magnetic Materials based on magnetic particles, or anchoring <italic>in silica</italic> reported this result.</p><p>Similarly, increasing on extraction selectivity and by consequence, performance is observed when GBMs were functionalized with both MIPS or the tunable-ILs, as can be seen in sections Molecularly-Imprinted Polymers and Ionic Liquids. This background explains the significant tendency to work with hybrid graphene-based materials instead of its bare form. GBMs are an excellent sorbent, often surpassing the commercially available phases such as C8 and C18.</p><p>After all, by assessing the recent literature and considering the vast number of applications involving graphene in the sample preparation arena, as herein discussed, an increasing tendency to expand the footprint of GBMs functionalized with several classes must continue in the years to come. This conclusion is mainly supported by the unique favorable GBMs physical-chemical properties, which—together with the advancements on the materials synthesis routes, extraction techniques, and related subjects—evidenced this field as one of the most critical developments in the sample preparation area nowadays. Also, an increasing number of papers reporting the employment of hybrid GBMs and miniaturized sample preparation techniques must be expected. This trend can be projected considering the high potential obtained by combining the well-established benefits of automation/miniaturization with the use of more selective and performative materials, possibly leading to greener sample preparation techniques by following the Green Chemistry concept.</p></sec><sec id=\"s5\"><title>Author Contributions</title><p>EM wrote sections Introduction, Miscellaneous, and Concluding Remarks, as well as edited the whole manuscript. KM-C wrote sections Graphene and Graphene Oxide and Alkyl and Aril Groups, built <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, and revised the whole manuscript. MJ-S wrote sections Molecularly-Imprinted polymers and Ionic Liquids, as well as built <xref rid=\"T4\" ref-type=\"table\">Tables 4</xref>, <xref rid=\"T5\" ref-type=\"table\">5</xref>. LS wrote section Cyclodextrins and built <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>. DV wrote section Magnetic Materials and built <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>. FL conceptualized, supervised, and edited all versions of the manuscript, as well as provide all required facilities. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s6\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> The authors are grateful to FAPESP (Grants 2017/02147-0, 2015/15462-5, and 2014/07347-9) and CNPq (307293/2014-9) for the financial support provided. This research project was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001.</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abdelghani</surname><given-names>J. I.</given-names></name><name><surname>Freihat</surname><given-names>R. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Cell Dev. Biol.</journal-id><journal-title-group><journal-title>Frontiers in Cell and Developmental Biology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-634X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850861</article-id><article-id pub-id-type=\"pmc\">PMC7431690</article-id><article-id pub-id-type=\"doi\">10.3389/fcell.2020.00758</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cell and Developmental Biology</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Tumor Microenvironment in Ovarian Cancer: Function and Therapeutic Strategy</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Yang</surname><given-names>Yanfei</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1050175/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Yang</surname><given-names>Yang</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1049754/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Yang</surname><given-names>Jing</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/748764/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zhao</surname><given-names>Xia</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wei</surname><given-names>Xiawei</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/874406/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Gynecology and Obstetrics, Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, West China Second Hospital, Sichuan University</institution>, <addr-line>Chengdu</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Laboratory of Aging Research and Cancer Drug Target, State Key Laboratory of Biotherapy and Cancer Center, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University</institution>, <addr-line>Chengdu</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Cecilia Ana Suarez, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Esther Hoste, Ghent University, Belgium; Priyanka Gupta, University of Alabama at Birmingham, United States</p></fn><corresp id=\"c001\">*Correspondence: Xia Zhao, <email>xia-zhao@126.com</email></corresp><corresp id=\"c002\">Xiawei Wei, <email>xiaweiwei@scu.edu.cn</email>; <email>weixiaweiscu@126.com</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>These authors have contributed equally to this work</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Cell and Developmental Biology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>758</elocation-id><history><date date-type=\"received\"><day>02</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>20</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Yang, Yang, Yang, Zhao and Wei.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Yang, Yang, Yang, Zhao and Wei</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Ovarian cancer is one of the leading causes of death in patients with gynecological malignancy. Despite optimal cytoreductive surgery and platinum-based chemotherapy, ovarian cancer disseminates and relapses frequently, with poor prognosis. Hence, it is urgent to find new targeted therapies for ovarian cancer. Recently, the tumor microenvironment has been reported to play a vital role in the tumorigenesis of ovarian cancer, especially with discoveries from genome-, transcriptome- and proteome-wide studies; thus tumor microenvironment may present potential therapeutic target for ovarian cancer. Here, we review the interactions between the tumor microenvironment and ovarian cancer and various therapies targeting the tumor environment.</p></abstract><kwd-group><kwd>ovarian cancer</kwd><kwd>tumor microenvironment</kwd><kwd>anti-angiogenesis therapy</kwd><kwd>tumor-associated macrophage-targeted strategies</kwd><kwd>immune checkpoint inhibitors</kwd></kwd-group><counts><fig-count count=\"3\"/><table-count count=\"3\"/><equation-count count=\"0\"/><ref-count count=\"287\"/><page-count count=\"31\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Highlights</title><list list-type=\"simple\"><list-item><label>–</label><p>The tumor microenvironment plays important roles in the progression of ovarian cancer.</p></list-item><list-item><label>–</label><p>Current “-omic” technology is revealing the molecular landscape of ovarian cancer tumor microenvironment, facilitating future therapeutic strategy.</p></list-item><list-item><label>–</label><p>Ovarian cancer therapies targeting tumor microenvironment is rapidly developing, targets mainly focusing on cancer-associated fibroblasts, tumor-associated macrophages, angiogenesis and immune checkpoint blockade.</p></list-item></list></sec><sec id=\"S2\"><title>Introduction</title><p>Ovarian cancer is one of the leading causes of common, lethal gynecologic malignancy (<xref rid=\"B55\" ref-type=\"bibr\">Cortez et al., 2018</xref>). In 2018 worldwide, there were an estimated 295,414 cases and 184,799 deaths from ovarian cancer (<xref rid=\"B31\" ref-type=\"bibr\">Bray et al., 2018</xref>). Because of the lack of an early diagnosis method and the absence of specific early warning symptoms, patients with ovarian cancer are usually diagnosed at an advanced stage and have a poor prognosis (<xref rid=\"B221\" ref-type=\"bibr\">Scarlett and Conejo-Garcia, 2012</xref>).</p><p>Based on histological origin, ovarian tumors can be categorized into epithelial, germ cell, sex cord, or stromal tumors (<xref rid=\"B123\" ref-type=\"bibr\">Jayson et al., 2014</xref>). Around 90% of primary ovarian tumors are of epithelial origin (<xref rid=\"B53\" ref-type=\"bibr\">Colombo et al., 2010</xref>; <xref rid=\"B145\" ref-type=\"bibr\">Ledermann et al., 2013</xref>), so we mainly focus on evidence of epithelial ovarian cancer in this review. The World Health Organization (WHO) classified epithelial ovarian cancer (EOC) into the following types: serous, mucinous, endometrioid, clear cell, transitional cell, mixed epithelial, undifferentiated, and unclassified (<xref rid=\"B145\" ref-type=\"bibr\">Ledermann et al., 2013</xref>). According to architectural features, EOC is also classified into 3 grades by the International Federation of Gynecology and Obstetrics (FIGO) system (<xref rid=\"B53\" ref-type=\"bibr\">Colombo et al., 2010</xref>); in serous EOC, FIGO grade 1 is defined as low-grade while FIGO grade 2 and 3 are combined as high-grade (<xref rid=\"B25\" ref-type=\"bibr\">Bodurka et al., 2012</xref>). The classification with histosubtypes and grades are with prognostic significance (<xref rid=\"B145\" ref-type=\"bibr\">Ledermann et al., 2013</xref>).</p><p>The current standardized treatment for ovarian cancer is optimal cytoreductive surgery plus platinum-based chemotherapy with the carboplatin-paclitaxel regimen (<xref rid=\"B27\" ref-type=\"bibr\">Bolton et al., 2012</xref>). However, with the development of chemotherapy-resistant and refractory diseases, the sensitivity of chemotherapy has decreased (<xref rid=\"B148\" ref-type=\"bibr\">Lim and Ledger, 2016</xref>). Therefore, the long-term survival rate for ovarian cancer has decreased, and the recurrence rate has increased (<xref rid=\"B148\" ref-type=\"bibr\">Lim and Ledger, 2016</xref>). <xref rid=\"B112\" ref-type=\"bibr\">Hennessy et al. (2009)</xref> reported that despite benefiting from first-line therapy, 75% of patients with advanced ovarian cancer (stage III or IV) have tumor relapse at a median of 15 months from diagnosis. Moreover, for patients with early-stage disease (stage I or II), the long-term survival rates (>10 years) are 80–95%. In contrast, patients with advanced disease (stage III or IV) had a 10–30% long-term survival rate. Therefore, there is an urgent need to find new targeted therapies to improve the treatment efficacy of ovarian cancer. In recent years, the tumor microenvironment (TME) has been reported to play a vital role in the tumorigenesis of ovarian cancer and is considered a possible therapeutic target for ovarian cancer. Here, we review the interactions between the TME and ovarian cancer and various therapies targeting the tumor environment.</p></sec><sec id=\"S3\"><title>TME in Ovarian Cancer</title><p>The TME comprises (1) the extracellular matrix (ECM), which consists of chemokines, inflammatory cytokines, integrins, matrix metalloproteinases (MMPs) and other secreted molecules, and (2) stromal cells, including cancer cells, cancer stem cells, pericytes, cancer-associated fibroblasts (CAFs), endothelial cells (ECs) and immune cells (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>) (<xref rid=\"B108\" ref-type=\"bibr\">Hanahan and Weinberg, 2011</xref>). In this part, we reviewed current findings on the impact of some key components above on ovarian cancer progression.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Cell components and functions in the tumor microenvironment (TME). Cell components in the TME can be classified into cancer cells, immune cells and stromal cells; these cells actively interact with each other by molecules they secrete [including cytokines, chemokines, damage-associated molecular patterns (DAMPs) etc.] and receptors they express, such as histocompatibility complex class (MHC) molecules, programmed cell death protein 1 (PD-1), etc., forming an evolving microenvironment. On the continuous spectrum from anti-tumor to pro-tumor effect, different cell components can locate at distinct positions, and the same group of cells may also be re-polarized depending on signals in the TME. The progression or regression of a single tumor site depends on the overall effect of the complex cellular and molecular regulating network in the TME.</p></caption><graphic xlink:href=\"fcell-08-00758-g001\"/></fig><sec id=\"S3.SS1\"><title>Cancer-Associated Fibroblasts</title><p>Fibroblasts, which differentiate from mesenchymal-derived cells, are part of the TME (<xref rid=\"B122\" ref-type=\"bibr\">Ishii et al., 2016</xref>). They produce various MMPs, tissue inhibitor of metalloproteinases (TIMPs) and most of the proteins comprising the ECM, such as collagens, fibronectin and laminin (<xref rid=\"B130\" ref-type=\"bibr\">Kalluri and Zeisberg, 2006</xref>; <xref rid=\"B75\" ref-type=\"bibr\">Erdogan and Webb, 2017</xref>). These fibroblasts in the tumor milieu are also called “CAFs.” Additionally, CAFs can transdifferentiate from other cells, such as pericytes, epithelial cells and ECs, via exposure to platelet-derived growth factor (PDGF), tumor-derived transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), MMPs and reactive oxygen species (ROS) (<xref rid=\"B38\" ref-type=\"bibr\">Cai et al., 2012</xref>; <xref rid=\"B273\" ref-type=\"bibr\">Yu Y. et al., 2014</xref>; <xref rid=\"B62\" ref-type=\"bibr\">Denton et al., 2018</xref>).</p><p>CAFs are known to promote tumor progression via various mechanisms. CAFs can enhance tumor cell proliferation, invasion and migration. <xref rid=\"B233\" ref-type=\"bibr\">Sjoberg et al. (2016)</xref> showed that CAFs highly expressed CXCL14, which was an important factor in promoting cancer growth. CAFs also express the fibroblast activation protein α (FAP). Yang’s study indicated that FAPα enhanced the migration and invasion ability of HO-8910PM cells (a highly metastatic ovarian cancer cell line) Additionally, FAPα increased HO-8910PM cell proliferation.</p><p>CAFs promote immune inhibition and angiogenesis. <xref rid=\"B96\" ref-type=\"bibr\">Givel et al. (2018)</xref> found that CAFs increase the infiltration of FOXP3+ regulatory T lymphocytes (Tregs) at the tumor site, which exerts immune suppression effect in the tumor milieu. Additionally, in Orimo’s research, CAFs have high expression of stromal cell-derived factor-1 (SDF-1). Released SDF-1 promotes angiogenesis and tumor proliferation in a paracrine fashion (<xref rid=\"B185\" ref-type=\"bibr\">Orimo et al., 2005</xref>).</p><p>CAFs also increase platinum resistance and accelerate recurrence. Fauceglia’s study showed that CAFs expressed the FAP α. By analyzing 338 EOC tissues, they found that the overexpression of FAP acted as a hallmark for platinum resistance. Additionally, patients with FAP+ stroma had a shortened recurrence compared to that of patients with FAP- stroma (<xref rid=\"B171\" ref-type=\"bibr\">Mhawech-Fauceglia et al., 2015</xref>).</p><p>Several studies have indicated that CAFs was a biomarker of poor prognosis in ovarian cancer (<xref rid=\"B268\" ref-type=\"bibr\">Yang et al., 2013</xref>; <xref rid=\"B171\" ref-type=\"bibr\">Mhawech-Fauceglia et al., 2015</xref>; <xref rid=\"B283\" ref-type=\"bibr\">Zhao et al., 2017</xref>; <xref rid=\"B96\" ref-type=\"bibr\">Givel et al., 2018</xref>). In Givel’s study of CAFs in high-grade serous ovarian cancers (HGSOC), the results showed that the expression of CXCL12β and the infiltration of CAF-S1 (a subtype of CAFs) implied a dismal prognosis (<xref rid=\"B96\" ref-type=\"bibr\">Givel et al., 2018</xref>). Despite that accumulating studies have demonstrated the pro-tumor progression impact of CAFs, it is worthy of notifying that there are different subtypes of CAFs with heterogenous function status. Recently, <xref rid=\"B120\" ref-type=\"bibr\">Hussain et al. (2020)</xref> found 2 CAF subsets distinguished by the FAP expression level. The FAP-high CAF subtype, instead of the FAP-low subtype, was found to aggressively enhance tumor progression and negatively influence patient outcomes, which shed light on therapeutic strategies involving CAF modulation to consider CAF status in patient selection.</p><p>CAFs are a crucial cell population in the tumor microenvironment. CAFs promote the proliferation, invasion and migration of cancer cells and stimulate angiogenesis by coordinating with other cells. A deeper understanding of CAFs is needed to better understand how CAFs affect the tumor microenvironment.</p></sec><sec id=\"S3.SS2\"><title>Endothelial Cells</title><p>Endothelial cells (ECs) are components of the TME. Lining the vessels, ECs are crucial for transporting oxygen and nutrients and are closely associated with angiogenesis (<xref rid=\"B41\" ref-type=\"bibr\">Carmeliet and Jain, 2000</xref>; <xref rid=\"B108\" ref-type=\"bibr\">Hanahan and Weinberg, 2011</xref>). As we all know, angiogenesis is a complicated process accommodated by angiogenesis activators and inhibitors. Angiogenesis activators include VEGF, FGF-2, PDGF, TGFα and TGFβ, TNF-α, prostaglandin E2 and Interleukin 8 (IL-8). The angiogenesis inhibitors contain angiopoietin (Angs), Thrombospondin 1 (TSP-1) and endostatin. Moreover, the signal-transducing network of endothelial cells is associated with VEGF, FGF and Angs signals (<xref rid=\"B56\" ref-type=\"bibr\">Cross and Claesson-Welsh, 2001</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Ahmed and Bicknell, 2009</xref>).</p><p>VEGF is a protein family consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and PLGF (placental growth factor). It is regulated by the ischaemia/hypoxia-induced genes (HIFs), epidermal growth factor (EGF) and PDGF (<xref rid=\"B224\" ref-type=\"bibr\">Semenza, 2000</xref>). There are three receptors for VEGF: VEGF receptors 1 (VEGFR1), VEGFR2 and VEGFR3. VEGFR1 and VEGFR2 are mainly expressed on ECs and are receptors for VEGF-A (<xref rid=\"B103\" ref-type=\"bibr\">Hagberg et al., 2010</xref>). Additionally, VEGFR1 is also a receptor for VEGF-B and PLGF. VEGFR3 is a receptor for VEGF-C and VEGF-D. Neuropilins (NRP1 and NRP2) are coreceptors for the VEGF family. With the help of NRP1, the binding affinity between VEGF-A and PLGF and VEGFR2 increases (<xref rid=\"B81\" ref-type=\"bibr\">Ferrara and Adamis, 2016</xref>). Similarly, with the effect of NRP2, VEGF-C, and VEGF-D have increased binding affinity with VEGFR-3. It is well known that the VEGF family is implicated in the adjustment of angiogenesis and lymphangiogenesis (<xref rid=\"B238\" ref-type=\"bibr\">Tammela and Alitalo, 2010</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Apte et al., 2019</xref>). Among them, VEGF-A is crucial for angiogenesis, while VEGF-C and VEGF-D regulate lymphangiogenesis (<xref rid=\"B238\" ref-type=\"bibr\">Tammela and Alitalo, 2010</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Apte et al., 2019</xref>).</p><p>Angs also a protein family consisting of Ang-1, Ang-2, Ang-3, and Ang-4. Through combined with the receptors of Angs “TIEs,” they perform different functions in angiogenesis. Ang-1 and -4 can bind to TIE2 and stimulate the tyrosine phosphorylation of TIE2. On the contrary, Ang-2 and -3 can competitive combined TIE2 without stimulating tyrosine phosphorylation, which stopping the signal transduction of angiogenesis (<xref rid=\"B217\" ref-type=\"bibr\">Sallinen et al., 2010</xref>; <xref rid=\"B151\" ref-type=\"bibr\">Lin et al., 2011</xref>; <xref rid=\"B216\" ref-type=\"bibr\">Sallinen et al., 2014</xref>). However, other study indicated that with Ang-1, Ang-2 block the TIE2 signaling, while Ang-2 induce the TIE2 signaling without Ang-1 (<xref rid=\"B275\" ref-type=\"bibr\">Yuan et al., 2009</xref>). Additionally, Ang-1 promotes the maturation and stabilization of vessel, while Ang-2 destabilize the stabilized vessel (<xref rid=\"B250\" ref-type=\"bibr\">Tse et al., 2003</xref>).</p><p>VEGF indicate poor clinical outcomes (<xref rid=\"B261\" ref-type=\"bibr\">Wimberger et al., 2014</xref>; <xref rid=\"B227\" ref-type=\"bibr\">Shen et al., 2017</xref>; <xref rid=\"B235\" ref-type=\"bibr\">Sopo et al., 2019</xref>). Wimberger et al. found that by Kaplan-Meier analyses, VEGFR1 expression was closely related to decreased overall survival (OS) and progression-free survival (PFS) (<xref rid=\"B261\" ref-type=\"bibr\">Wimberger et al., 2014</xref>). Sopo’ study discovered that VEGFR1, VEGF-A and VEGF-D were highly expressed in omental metastases compared to expression in primary ovarian epithelial tumors. Interestingly, patients with low VEGF-A expression were more likely to have a poor prognosis. Patients with high VEGF-C expression were related to a short PFS (<xref rid=\"B235\" ref-type=\"bibr\">Sopo et al., 2019</xref>).</p><p>The CD146 expression in the membrane of ECs promotes the migration of ECs and angiogenesis. CD146 is an endothelial biomarker and the extracellular domain of CD146 directly interacts with VEGFR2. Yan’s study demonstrated that CD146 can promote angiogenesis (<xref rid=\"B265\" ref-type=\"bibr\">Yan et al., 2003</xref>). Subsequently, Jiang’s report indicates CD146 promotes the migration of ECs and the formation of microvasculature by enhancing VEGFR2 phosphorylation and downstream signaling (AKT/p38 MAPKs/NF-κB) activation (<xref rid=\"B124\" ref-type=\"bibr\">Jiang et al., 2012</xref>). Interestingly, Zhou’s research indicated that the gene and protein levels of CD146 and VEGFRA were increased in patients with EOC compared to those of non-cancer patients (<xref rid=\"B287\" ref-type=\"bibr\">Zhou et al., 2019</xref>).</p><p>Enhanced Angs expression increase relapse and decrease survival time (<xref rid=\"B217\" ref-type=\"bibr\">Sallinen et al., 2010</xref>, <xref rid=\"B216\" ref-type=\"bibr\">2014</xref>; <xref rid=\"B151\" ref-type=\"bibr\">Lin et al., 2011</xref>). Sallinen et al. observed that patients with ovarian carcinoma had higher Ang2 levels compared to those of patients with benign ovarian tumors. Furthermore, by analyzing the Kaplan–Meier curves, they found that increased Ang-2 levels (>2.7 ng/ml) were a biomarker for poor recurrence-free survival (<xref rid=\"B217\" ref-type=\"bibr\">Sallinen et al., 2010</xref>). Subsequently, they discovered that the expression levels of Ang-1 and Ang-2 were 26 and 44%, respectively, higher in women with ovarian cancer than in normal women. Increased Ang-2 expression was significantly related to advanced stage and grade of cancer and relapse of ovarian cancer. Additionally, elevated Ang-2 expression is a predictor of poor OS and short PFS (<xref rid=\"B216\" ref-type=\"bibr\">Sallinen et al., 2014</xref>).</p><p>As an important part of the tumor microenvironment, ECs are closely related to angiogenesis. VEGF and Angs are crucial regulators in angiogenesis. Both VEGF and Angs are associated with poor clinical outcomes, which provide possible targets for treatment.</p></sec><sec id=\"S3.SS3\"><title>Immune Cells</title><p>Immune cells include macrophages, dendritic cells (DCs), neutrophils, mast cells, myeloid-derived suppressor cells (MDSCs) and lymphocytes (<xref rid=\"B108\" ref-type=\"bibr\">Hanahan and Weinberg, 2011</xref>). They play significant roles both in tumor progression and tumor suppression, participating in evolving processes of tumorigenesis, metastasis, and angiogenesis by producing various signaling molecules, such as EGF, VEGF, MMP-9, IFNs, ILs, etc.</p><sec id=\"S3.SS3.SSS1\"><title>Macrophages</title><p>Macrophages are an essential population of immune cells that participate in inflammation and tumourigenesis (<xref rid=\"B101\" ref-type=\"bibr\">Grivennikov et al., 2010</xref>). Among them, macrophages residing in tumors are termed as tumor-associated macrophages (TAMs). TAMs can derive from resident macrophages or infiltrating macrophages from bone marrow monocytes circulating in the blood (<xref rid=\"B95\" ref-type=\"bibr\">Ghosn et al., 2010</xref>).</p><p>Depending on stimuli in the TME, TAMs can present two main phenotypes: the anti-tumor M1 macrophages and pro-tumor M2 macrophages (<xref rid=\"B229\" ref-type=\"bibr\">Sica et al., 2008</xref>; <xref rid=\"B101\" ref-type=\"bibr\">Grivennikov et al., 2010</xref>; <xref rid=\"B198\" ref-type=\"bibr\">Qian and Pollard, 2010</xref>; <xref rid=\"B228\" ref-type=\"bibr\">Sica, 2010</xref>; <xref rid=\"B102\" ref-type=\"bibr\">Gupta et al., 2018</xref>). When stimulated with interferon-gamma (IFN-γ), bacterial lipopolysaccharide (LPS) and granulocyte-macrophage-colony-stimulating factor (GM-CSF), monocytes differentiated into M1 macrophages, which can secrete IL-1, IL-12, TNFα and CXCL12 (<xref rid=\"B229\" ref-type=\"bibr\">Sica et al., 2008</xref>; <xref rid=\"B201\" ref-type=\"bibr\">Ramanathan and Jagannathan, 2014</xref>). M1 macrophages possess cytotoxicity, tumor suppression and immune-stimulation functions (<xref rid=\"B90\" ref-type=\"bibr\">Galdiero et al., 2013</xref>).</p><p>When stimulated with cytokines, including IL-4, IL-10, and IL-13, monocytes differentiated into M2 macrophages (<xref rid=\"B146\" ref-type=\"bibr\">Leyva-Illades et al., 2012</xref>; <xref rid=\"B252\" ref-type=\"bibr\">van Dalen et al., 2018</xref>). In the immune escape stage, the tumor macroenvironment maintains immunosuppression due to the secretion of many growth factors and cytokines, such as IL-4 and IL-13, by cancer cells. The immunosuppressive state accelerates monocytes to M2 macrophages; M2 macrophages, in turn, can promote tumor growth (<xref rid=\"B99\" ref-type=\"bibr\">Gordon, 2003</xref>; <xref rid=\"B210\" ref-type=\"bibr\">Roy and Li, 2016</xref>).</p><p>In ovarian cancer, TAMs are predominantly M2 macrophages, associating with tumor invasion, angiogenesis, metastatic disease and early recurrence (<xref rid=\"B194\" ref-type=\"bibr\">Pollard, 2004</xref>; <xref rid=\"B203\" ref-type=\"bibr\">Reinartz et al., 2014</xref>; <xref rid=\"B270\" ref-type=\"bibr\">Yin et al., 2016</xref>). They produce and secrete cytokines, which have immunosuppressive effects, such as IL-1R decoy, IL-10, CCL17 and CCL22 (<xref rid=\"B99\" ref-type=\"bibr\">Gordon, 2003</xref>). Via several mechanisms, they suppress adaptive immunity (<xref rid=\"B147\" ref-type=\"bibr\">Li et al., 2007</xref>; <xref rid=\"B183\" ref-type=\"bibr\">Noy and Pollard, 2014</xref>). Firstly, M2 macrophages can inhibit the proliferation of T cells and accelerate the immunosuppression of Treg cell transport to tumors by producing the chemokine CCL22 (<xref rid=\"B147\" ref-type=\"bibr\">Li et al., 2007</xref>). Secondly, M2 macrophages express the ligand receptors for CTLA-4 and PD-1. The activation of PD-1 and CTLA-4 inhibits cytotoxic function and regulates the cell cycle of T cells (<xref rid=\"B183\" ref-type=\"bibr\">Noy and Pollard, 2014</xref>). Then, M2 macrophages can also inhibit the activation of T cells through the depletion of <sc>L</sc>-arginine, which plays an essential role in T cell function (<xref rid=\"B90\" ref-type=\"bibr\">Galdiero et al., 2013</xref>). Arginase I (ARG1), a hallmark of M2 macrophages, is an <sc>L</sc>-arginine processing enzyme. In the TME, ARG1 decomposes <sc>L</sc>-arginine into <sc>L</sc>-ornithine and urea. The depletion of <sc>L</sc>-arginine suppresses the re-expression of the CD3 ζ chain, which is internalized by antigen stimulation and T cell receptor (TCR) signaling (<xref rid=\"B208\" ref-type=\"bibr\">Rodriguez et al., 2004</xref>).</p><p>Aside from immune suppression, M2 macrophages also take part in tissue repair, ECM remodeling and angiogenesis, which are processes involved in tumor progression as well (<xref rid=\"B165\" ref-type=\"bibr\">Mantovani et al., 2002</xref>; <xref rid=\"B51\" ref-type=\"bibr\">Coffelt et al., 2010</xref>; <xref rid=\"B211\" ref-type=\"bibr\">Ruffell et al., 2012</xref>; <xref rid=\"B82\" ref-type=\"bibr\">Finkernagel et al., 2016</xref>; <xref rid=\"B210\" ref-type=\"bibr\">Roy and Li, 2016</xref>). They can restructure ECM and regulate ECM components by degrading ECM via producing MMPs, serine proteases and cathepsins (<xref rid=\"B211\" ref-type=\"bibr\">Ruffell et al., 2012</xref>), which may facilitate tumor cell migration, invasion and metastasis. Additionally, they can secrete VEGF-A, which is an angiogenic factor, and produce proangiogenic cytokines, such as IL-1β, TNFα and uPA (urokinase-type plasminogen activator) (<xref rid=\"B210\" ref-type=\"bibr\">Roy and Li, 2016</xref>). In M2 macrophages, there is a subtype expressing TIE2, a tyrosine kinase receptor. The TIE2 macrophages are involved in angiogenesis (<xref rid=\"B79\" ref-type=\"bibr\">Fagiani and Christofori, 2013</xref>). These TIE2 macrophages recruited by CCL3, CCL5, CCL8, and TIE2-ligand Ang 2 are considered the most important reason for tumor vascularization because the deficiency of this cell type restricts the angiogenic switch (<xref rid=\"B182\" ref-type=\"bibr\">Ngambenjawong et al., 2017</xref>).</p><p>TAMs are plastic. The simple dichotomy of M1/M2 macrophages cannot account for the complexity of TAM heterogeneity (<xref rid=\"B187\" ref-type=\"bibr\">Ostuni et al., 2015</xref>). Transcriptome analysis uncovered a spectrum model of TAMs (<xref rid=\"B263\" ref-type=\"bibr\">Xue et al., 2014</xref>). M1 and M2 macrophages can be regarded as two ends of a continuum with wide ranges of functional states (<xref rid=\"B165\" ref-type=\"bibr\">Mantovani et al., 2002</xref>; <xref rid=\"B187\" ref-type=\"bibr\">Ostuni et al., 2015</xref>); the sub-populations of TAMs in between the two ends can share features of both M1 and M2 types (<xref rid=\"B198\" ref-type=\"bibr\">Qian and Pollard, 2010</xref>). For example, recently <xref rid=\"B232\" ref-type=\"bibr\">Singhal et al. (2019)</xref> found that TAMs could co-express M1/M2 markers, together with T cell co-inhibitory and co-stimulatory receptors.</p><p>The dynamic nature of the TME cellular environment gives a basis for the plasticity of TAMs. Macrophages present reversible changes in their functional phenotypes and distribution in response to different microenvironmental stimuli, including various cytokines and locally derived molecules, which are tissue- and tumor-specific (<xref rid=\"B237\" ref-type=\"bibr\">Stout et al., 2005</xref>; <xref rid=\"B184\" ref-type=\"bibr\">Okabe and Medzhitov, 2014</xref>; <xref rid=\"B187\" ref-type=\"bibr\">Ostuni et al., 2015</xref>; <xref rid=\"B134\" ref-type=\"bibr\">Kim and Bae, 2016</xref>). Therefore, in different histotypes of tumors (<xref rid=\"B279\" ref-type=\"bibr\">Zhang et al., 2014</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Cassetta et al., 2019</xref>) and different microregions of the same tumor (<xref rid=\"B165\" ref-type=\"bibr\">Mantovani et al., 2002</xref>; <xref rid=\"B134\" ref-type=\"bibr\">Kim and Bae, 2016</xref>; <xref rid=\"B266\" ref-type=\"bibr\">Yang M. et al., 2018</xref>), there can be TAMs with different extent of infiltration and functional status.</p><p>In ovarian cancer, <xref rid=\"B279\" ref-type=\"bibr\">Zhang et al. (2014)</xref> found the density and the cancer islet/stroma ratio of TAMs vary among serous, mucinous, endometrioid, clear cell and undifferentiated histotypes. In the stroma and lumina of a small number of patient ovarian tumor samples, limited frequencies of iNOS expressing TAMs were found, which were thought to be cytotoxic (<xref rid=\"B137\" ref-type=\"bibr\">Klimp et al., 2001</xref>); in contrast, in the malignant ascites of ovarian cancer, abundant TAMs can be found, which are primarily M2-like with pro-tumor capacity (<xref rid=\"B102\" ref-type=\"bibr\">Gupta et al., 2018</xref>). As the tumor grows, stimuli in the TME alter, resulting in changes in TAM infiltration and polarization in a tumor progression level-dependent manner. In ovarian cancer studies, TAM and M2 macrophage density were found to increase as cancer stage and ascites volume increased or as lymphatic invasion appeared (<xref rid=\"B279\" ref-type=\"bibr\">Zhang et al., 2014</xref>; <xref rid=\"B133\" ref-type=\"bibr\">Ke et al., 2016</xref>; <xref rid=\"B276\" ref-type=\"bibr\">Yuan et al., 2017</xref>; <xref rid=\"B102\" ref-type=\"bibr\">Gupta et al., 2018</xref>); contrarily, M1/M2 ratio decreased as cancer stage increased (<xref rid=\"B279\" ref-type=\"bibr\">Zhang et al., 2014</xref>).</p><p>Despite expressing similar markers, TAMs may not always have similar functional implications. In colon cancer study, TAMs expressing PD-1 presented weakened phagocytic potency, associating with reduced survival (<xref rid=\"B100\" ref-type=\"bibr\">Gordon et al., 2017</xref>), while in early lung cancer study the PD-1+ TAMs did not affect tumor-specific T cell attack against tumor (<xref rid=\"B232\" ref-type=\"bibr\">Singhal et al., 2019</xref>). This indicates the necessity of future studies focusing on TAM functional status in the context of tumor tissue types and stages of the disease; this is especially true with ovarian cancer as it has many histotypes and high heterogeneity.</p><p>Several studies revealed the prognostic value of TAMs in ovarian cancer. The M1/M2 and M2/TAM ratio have been reported to be positively associated with PFS and OS, while the overall TAM density in ovarian tumors indicated no prognostic significance (<xref rid=\"B142\" ref-type=\"bibr\">Lan et al., 2013</xref>; <xref rid=\"B279\" ref-type=\"bibr\">Zhang et al., 2014</xref>; <xref rid=\"B276\" ref-type=\"bibr\">Yuan et al., 2017</xref>). M2 density in the ascites or tumor samples is associated with reduced relapse-free survival (<xref rid=\"B203\" ref-type=\"bibr\">Reinartz et al., 2014</xref>) and PFS (<xref rid=\"B142\" ref-type=\"bibr\">Lan et al., 2013</xref>; <xref rid=\"B276\" ref-type=\"bibr\">Yuan et al., 2017</xref>). However, there is a controversy in the relationship between M2 density alone and OS: <xref rid=\"B142\" ref-type=\"bibr\">Lan et al. (2013)</xref> reported a negative association between the two factors, while <xref rid=\"B279\" ref-type=\"bibr\">Zhang et al. (2014)</xref> found no significant relevance. This may be due to the difference of included tumor histotypes.</p></sec><sec id=\"S3.SS3.SSS2\"><title>Dendritic Cells</title><p>Dendritic cells (DCs) capture endogenous or exogenous antigens, process them, and present the antigenic peptides to other immune cells (<xref rid=\"B12\" ref-type=\"bibr\">Banchereau et al., 2000</xref>), acting as a bridge connecting the innate and the adaptive immune system (<xref rid=\"B247\" ref-type=\"bibr\">Timmerman and Levy, 1999</xref>; <xref rid=\"B206\" ref-type=\"bibr\">Riboldi et al., 2005</xref>). There are two main subtypes of DCs: the conventional DC (cDC) that is specialized in antigen presentation, and the plasmacytoid DC (pDC) that produces IFN upon antigen stimulation aside from activating lymphocytes and other myeloid cells (<xref rid=\"B141\" ref-type=\"bibr\">Labidi-Galy et al., 2011</xref>; <xref rid=\"B255\" ref-type=\"bibr\">Vu Manh et al., 2015</xref>). cDCs comprise 5–10% of myeloid cells in most tumors; pDCs are rare in mouse tumors but found in most human tumors (<xref rid=\"B240\" ref-type=\"bibr\">Tang et al., 2017</xref>).</p><p>DCs play key roles in anti-tumor immunity because it is indispensable for T cell immune responses against tumors (<xref rid=\"B42\" ref-type=\"bibr\">Casey et al., 2015</xref>). DCs are responsible for tumor antigen recognition, which is the initiating event of the tumor-specific adaptive immune response. In both the animal ovarian cancer model and human HGSOC patients, DCs can sense damage-associated molecular patterns (DAMPs) released from dead cancer cells, such as double-stranded DNA (dsDNA) fragments and calreticulin, an endoplasmic reticulum (ER) chaperone, eliciting Th1 polarized immunity (<xref rid=\"B67\" ref-type=\"bibr\">Ding et al., 2018</xref>; <xref rid=\"B132\" ref-type=\"bibr\">Kasikova et al., 2019</xref>).</p><p>After capturing antigens, DCs present peptides processed from those antigens to CD4+ and CD8+ T cells via major histocompatibility complex class II (MHC II) and MHC I molecules respectively, which subsequently initiate a series of T cell activity (<xref rid=\"B72\" ref-type=\"bibr\">Dudek et al., 2013</xref>; <xref rid=\"B212\" ref-type=\"bibr\">Sabado et al., 2017</xref>). This process has been reported to be significant for tumor development prevention (<xref rid=\"B161\" ref-type=\"bibr\">MacKie et al., 2003</xref>; <xref rid=\"B91\" ref-type=\"bibr\">Galon et al., 2006</xref>).</p><p>Besides T cell activation, DCs are also crucial for the augmentation of cytotoxic T lymphocytes (CTLs) population in the TME. It is reported that intratumoral cDCs are responsible for intratumoral CTL proliferation both <italic>in vivo</italic> and <italic>in vitro</italic> (<xref rid=\"B65\" ref-type=\"bibr\">Diao et al., 2011</xref>), and they are the only group of phagocytosing tumor myeloid cells that can stimulate CD8+ T cell proliferation (<xref rid=\"B32\" ref-type=\"bibr\">Broz et al., 2014</xref>). As the major determinant of success in tumor deterrent, from the immune aspect (<xref rid=\"B35\" ref-type=\"bibr\">Budhu et al., 2010</xref>), is to increase the functional tumor-infiltrated CTL population, the significance of cDCs in the TME for anti-tumor responses is self-evident.</p><p>Effective T cell activation by DCs require DC maturation, a process happens after DC exposing to antigen, characterized by increased membrane expression of MHC and co-stimulatory molecules (CD80, CD86, CD40) (<xref rid=\"B26\" ref-type=\"bibr\">Bol et al., 2016</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Bhatia et al., 2019</xref>), alteration of chemokine receptors to favor DC lymph node (LN) migration (<xref rid=\"B69\" ref-type=\"bibr\">Drakes and Stiff, 2018</xref>); mature DCs produce cytokines that favor Th1 (anti-tumor) immunity. <xref rid=\"B249\" ref-type=\"bibr\">Truxova et al. (2018)</xref> found in cohorts of HGSOC patients that tumor-infiltrated mature LAMP+ DCs is robustly associated with Th1 immune responses, clinically favorable cytotoxic activities in the TME and favorable OS.</p><p>The process of DC maturation can be hampered by multiple factors, leaving DC immatured, potentially developing into a tolerogenic status and promote immune tolerance (<xref rid=\"B64\" ref-type=\"bibr\">Dhodapkar et al., 2001</xref>). Immature DCs express low levels of co-stimulatory molecules and cytokines and mount limited immune activities (<xref rid=\"B69\" ref-type=\"bibr\">Drakes and Stiff, 2018</xref>). Factors that lead to DC dysfunction, including the inhibition of DC maturation, involve the immune-modulating molecules in the TME, such as IL-6, IL-10 and VEGF, tumor-derived soluble mediators and exosomes, the activation of oncogene STAT3 in DCs, the ER stress response, and the abnormal intracellular lipid accumulation (<xref rid=\"B57\" ref-type=\"bibr\">Cubillos-Ruiz et al., 2015</xref>; <xref rid=\"B240\" ref-type=\"bibr\">Tang et al., 2017</xref>; <xref rid=\"B63\" ref-type=\"bibr\">DeVito et al., 2019</xref>). These factors suppress DC functions by reducing the expression of co-stimulatory molecules and the secretion of pro-inflammatory cytokines, inhibiting DC lymph node chemotaxis, dampening DC differentiation, inducing tolerogenic phenotypes on DCs and shortening the lifespan of DCs (<xref rid=\"B240\" ref-type=\"bibr\">Tang et al., 2017</xref>).</p><p>Tolerogenic DCs suppresses anti-tumor immunity via several mechanisms. First, they produce less pro-inflammatory cytokines and induce immune suppressive cytokines. <xref rid=\"B141\" ref-type=\"bibr\">Labidi-Galy et al. (2011)</xref> found in a cohort of 44 ovarian cancer patients that intra-tumoural tolerogenic pDCs secreted fewer IFN-α, TNF-α, IL-6, macrophage inflammatory protein-1β and CCL5, while induced IL-10 from CD4+ T cells, promoting immune tolerance in these patients. Second, they harbor enzymes negatively regulating T effector cell functions, such as nitric oxide synthase (NOS) and Indoleamine 2,3-Dioxygenase (IDO) (<xref rid=\"B42\" ref-type=\"bibr\">Casey et al., 2015</xref>). IDO is an enzyme catalyzing tryptophan degradation, capable of suppressing tumor-infiltrated lymphocyte proliferation, promoting Treg differentiation, inducing T cell anergy, and promoting tumor angiogenesis as well as metastasis (<xref rid=\"B181\" ref-type=\"bibr\">Munn et al., 2005</xref>; <xref rid=\"B243\" ref-type=\"bibr\">Tanizaki et al., 2014</xref>; <xref rid=\"B180\" ref-type=\"bibr\">Munn and Mellor, 2016</xref>). In EOC patients, there was significantly increased frequency of IDO+ DCs in tumor draining LN compared to the normal donor LN; besides, <italic>in vitro</italic> study revealed IDO significantly inhibited proliferation of tumor-associated lymphocytes derived from EOC patients (<xref rid=\"B199\" ref-type=\"bibr\">Qian et al., 2009</xref>).</p><p>Many factors are affecting the actual DC functions and behaviors, which are with high plasticity, contributing to either pro-tumor or anti-tumor effect. Tumor expressing molecules are associated with mature DC infiltration. Recently, <xref rid=\"B159\" ref-type=\"bibr\">MacGregor et al. (2019a)</xref> found higher surface expression of B7-H4, a B7 family molecule, was correlated with higher mature DC (CD11c+HLA-DRhigh) infiltration in EOC patient samples, which may be associated with increased expression of CXCL17, a monocyte and DC chemoattractant in those tumors. This group have also found that tumour-to-stroma ratio (TSR), which represents the percentage of malignant cell component relative to the stroma in the tumor tissue, have an impact on infiltrated DC phenotype: high TSR was associated with elevated PD-L1 expression on mature DCs (CD11c+HLA-DRhigh) infiltrating in ovarian tumor tissue (<xref rid=\"B160\" ref-type=\"bibr\">MacGregor et al., 2019b</xref>).</p><p>DC functions can be regulated by their interactions with the proximal milieu, so different locations of DCs may result in different function. <xref rid=\"B141\" ref-type=\"bibr\">Labidi-Galy et al. (2011)</xref> discovered that in ovarian cancer patients, tumor pDCs produced less pro-inflammatory cytokines than pDCs from ascites or peripheral blood.</p><p>Also, DC performance can vary by different tumor development stage. In an ovarian cancer mouse model, at the early stage, tumor growth was prevented by infiltrating DCs and DC depletion at this stage accelerated tumor expansion; at the advanced stage, however, DCs become immunosuppressive in the TME, abrogating enduring activity of anti-tumor T cells, and DC depletion at this stage significantly delayed disease progression (<xref rid=\"B222\" ref-type=\"bibr\">Scarlett et al., 2012</xref>). Similarly, in a mouse model of ovarian cancer, <xref rid=\"B140\" ref-type=\"bibr\">Krempski et al. (2011)</xref> also found progressively gained immunosuppressive phenotype of infiltrating DCs as the tumor progressed over time, represented by gradually increased PD-1 expression.</p><p>More studies are favored in the future to reveal facts on how DCs functions are regulated, thereby providing clues for therapeutic strategies in maintaining their anti-tumor potential.</p></sec><sec id=\"S3.SS3.SSS3\"><title>Myeloid-Derived Suppressor Cells</title><p>Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells that co-express the myeloid surface markers GR-1 and CD11b (<xref rid=\"B10\" ref-type=\"bibr\">Atretkhany and Drutskaya, 2016</xref>). MDSCs consist of three phenotypes: PMN-MDSC, M-MDSC and a small group of cells that have myeloid colony-forming activity, including myeloid progenitors and precursors (<xref rid=\"B88\" ref-type=\"bibr\">Gabrilovich, 2017</xref>). PMN-MDSCs are similar to neutrophils in phenotype and morphology and represent over 80% of MDSCs, while M-MDSCs are similar to monocyte (<xref rid=\"B88\" ref-type=\"bibr\">Gabrilovich, 2017</xref>). Studies have confirmed that MDSCs promote tumor progression by various mechanisms. First, MDSCs are implicated in immune suppression (<xref rid=\"B186\" ref-type=\"bibr\">Ostrand-Rosenberg and Fenselau, 2018</xref>). Despite their involvement in the inhibition of many cells in the immune system, MDSCs mainly target T cells. We summarized the mechanisms involved in immune suppression. (1) MDSCs accelerate lymphocyte nutrient depletion (<xref rid=\"B208\" ref-type=\"bibr\">Rodriguez et al., 2004</xref>; <xref rid=\"B236\" ref-type=\"bibr\">Srivastava et al., 2010</xref>). Both <sc>L</sc>-arginine and <sc>L</sc>-cysteine are essential amino acids that are important for T cell activation and function. MDSCs produce ARG1 and depletion of <sc>L</sc>-arginine through an ARG1-dependent manner (<xref rid=\"B208\" ref-type=\"bibr\">Rodriguez et al., 2004</xref>). MDSCs also sequester <sc>L</sc>-cysteine (<xref rid=\"B236\" ref-type=\"bibr\">Srivastava et al., 2010</xref>). Therefore, the amount of ζ-chain in the TCR complex is downregulated, and the proliferation of antigen-activated T cells is suppressed. (2) MDSCs disturb lymphocyte trafficking and viability (<xref rid=\"B110\" ref-type=\"bibr\">Hanson et al., 2009</xref>; <xref rid=\"B215\" ref-type=\"bibr\">Sakuishi et al., 2011</xref>). Galectin 9, which is expressed in MDSCs, binds to TIM3 on lymphocytes, which induces the apoptosis of T cells (<xref rid=\"B215\" ref-type=\"bibr\">Sakuishi et al., 2011</xref>). Similarly, MDSCs express ADAM17, which can decrease the <sc>L</sc>-selectin level on T cells and limit T cell recruitment in lymph nodes (<xref rid=\"B110\" ref-type=\"bibr\">Hanson et al., 2009</xref>). (3) MDSCs promote Treg cell activation and expansion (<xref rid=\"B89\" ref-type=\"bibr\">Gabrilovich et al., 2012</xref>). MDSCs stimulate CD4+ T cells to translate into induced Treg (iTreg) cells and expand natural Treg (nTreg) cells. These processes are associated with CD40-CD40L interactions, IFN-γ, IL-10, and TGFβ. (4) MDSCs stimulate the generation of oxidative stress. Oxidative stress is linked to ROS and RNS (reactive nitrogen species) (<xref rid=\"B88\" ref-type=\"bibr\">Gabrilovich, 2017</xref>). Superoxide reacts with NO and generates PNT (peroxynitrite), which nitrates T-cell receptors and limits the response of antigen-MHC complexes, thus suppressing T cells directly. PNT also nitrates T-cell-specific chemokines, which decreases the combination of antigenic peptides to MHC and limits the migration of T cells (<xref rid=\"B175\" ref-type=\"bibr\">Molon et al., 2011</xref>).</p><p>Moreover, MDSCs facilitate neovascularization through different mechanisms. Hypoxia in tumors induces MDSCs to produce VEGF, FGF2 and MMP9. Interestingly, the activation of STAT3 in MDSCs also stimulates neovascularization through IL-1β, CCL2 and CXCL2 release (<xref rid=\"B34\" ref-type=\"bibr\">Bruno et al., 2019</xref>). Additionally, these factors stimulate invasion and metastasis by producing MMPs (<xref rid=\"B186\" ref-type=\"bibr\">Ostrand-Rosenberg and Fenselau, 2018</xref>).</p><p>MDSC is an important part of the tumor microenvironment. MDSC promote tumor progression by regulating immune suppression and facilitating neovascularization. Moreover, the different tumor microenvironment is related to different functions and differentiation of MDSC. Nevertheless, the mechanism is still not clear.</p></sec><sec id=\"S3.SS3.SSS4\"><title>Lymphocytes</title><p>Lymphocytes, a major component of the TME, include B lymphocytes and T lymphocytes and mediate innate and adaptive immunity, respectively (<xref rid=\"B213\" ref-type=\"bibr\">Sadelain et al., 2017</xref>). B lymphocytes accelerate tumor progression by producing protumorigenic cytokines and regulating the Th1: Th2 ratio (<xref rid=\"B200\" ref-type=\"bibr\">Quail and Joyce, 2013</xref>). T lymphocytes, a major component of the TME, are crucial for adaptive immunity (<xref rid=\"B213\" ref-type=\"bibr\">Sadelain et al., 2017</xref>). T cells develop in the thymus. Before encountering the initial antigen, T cells are regarded as naïve (TN) cells. After antigen encounter, naïve (TN) cells are activated and start differentiation (<xref rid=\"B234\" ref-type=\"bibr\">Smith-Garvin et al., 2009</xref>). They proliferate rapidly and release inflammatory cytotoxic granules and cytokines, which activate the immune response. According to the cytokine environment, T cells differentiate into various subsets (<xref rid=\"B257\" ref-type=\"bibr\">Wang M. et al., 2017</xref>).</p><p>Due to the exclusive expression of CD4 or CD8 markers, mature T cells are categorized into CD3+CD4+, CD3+CD8+ T cells and CD4+ Treg cells (<xref rid=\"B136\" ref-type=\"bibr\">Kishton et al., 2017</xref>). CD3+CD4+ T cells are also called helper T cells (Th cells) and regulate immune responses by releasing cytokines that promote or inhibit inflammation (<xref rid=\"B129\" ref-type=\"bibr\">Joyce and Pollard, 2009</xref>). CD3+CD4+ T cells can be divided into Th1 and Th2 cells. Among them, Th1 cells produce and release pro-inflammatory cytokines and assist CD3+CD8+ T cells in tumor rejection. Therefore, Th1 cells are antitumorigenic. However, Th2 cells release anti-inflammatory cytokines and promote tumor progression (<xref rid=\"B129\" ref-type=\"bibr\">Joyce and Pollard, 2009</xref>; <xref rid=\"B200\" ref-type=\"bibr\">Quail and Joyce, 2013</xref>). CD3+CD8+ T cells, called cytotoxic T lymphocytes (CTLs), produce inflammatory cytokines and cell lytic molecules such as perforin and granzyme, which specifically recognize and destroy pathogen-infected or malignant cells (<xref rid=\"B129\" ref-type=\"bibr\">Joyce and Pollard, 2009</xref>; <xref rid=\"B280\" ref-type=\"bibr\">Zhang and Bevan, 2011</xref>).</p><p>Treg cells (CD4+CD25+Foxp3+) also play a crucial role in the immune response (<xref rid=\"B190\" ref-type=\"bibr\">Patsoukis et al., 2016</xref>). During development in the thymus, Treg cells universally express Foxp3, representing 5–10% of CD4+ T cells. When responding to TCR and TGF-β, Treg cells show suppression. Treg cells protect hosts against autoimmune diseases through inhibiting self and autoreactive cells (<xref rid=\"B60\" ref-type=\"bibr\">de Aquino et al., 2015</xref>). Additionally, Treg cells play a tumourigenic role mainly through immunosuppression monitoring (<xref rid=\"B152\" ref-type=\"bibr\">Lindau et al., 2013</xref>). Treg cells regulate the immune response through four mechanisms (<xref rid=\"B254\" ref-type=\"bibr\">Vignali et al., 2008</xref>; <xref rid=\"B78\" ref-type=\"bibr\">Facciabene et al., 2012</xref>): (1) Secreting immunosuppressive molecules. Treg cells suppress effector T cell functions by secreting cytokines such as IL-10, IL-35, and TGFβ. Additionally, IL-10 and TGFβ are reported as key mediators that limit antitumor immunity and promote tumor progression (<xref rid=\"B78\" ref-type=\"bibr\">Facciabene et al., 2012</xref>). Interestingly, these cytokines not only inhibit the function of effector cells but also promote DC polarization to tolerogenic phenotypes. Additionally, Treg cells secrete VEGF, which is also an immunosuppressive molecule (<xref rid=\"B254\" ref-type=\"bibr\">Vignali et al., 2008</xref>). Through VEGF, Treg cells exert inhibition and regulate the differentiation of DCs. (2) Cytolysis. Treg cells induce the apoptosis of effector cells by secreting granzyme B and perforin (<xref rid=\"B254\" ref-type=\"bibr\">Vignali et al., 2008</xref>). (3) Metabolic disruption. Several mechanisms have been reported for the metabolic disruption regulated by Treg cells. However, it is still controversial. Treg cells deplete the local level of IL-2, which causes effector cells to starve and results in the apoptosis of effector cells. Moreover, with the expression of CD73 and CD39, Treg cells catalyze ATP to adenosine, which inhibits the function of effector T cells (<xref rid=\"B61\" ref-type=\"bibr\">Deaglio et al., 2007</xref>; <xref rid=\"B254\" ref-type=\"bibr\">Vignali et al., 2008</xref>). (4) Modulation of DC maturation and function. CTLA-4 (cytotoxic T-lymphocyte antigen 4) is expressed on Treg cells, and CD80 and CD86 are expressed on DCs. Treg cells induce DCs through CTLA4–CD80/CD86 interactions, which induces the release of IDO (indoleamine 2,3-dioxygenase). IDO expression depletes essential tryptophan and inhibits the function of effector T cells (<xref rid=\"B80\" ref-type=\"bibr\">Fallarino et al., 2003</xref>). Furthermore, Treg cells suppress the function of DCs by depleting costimulatory molecules and inhibiting LAG3 (lymphocyte-activation gene 3) binds to MHC class II molecules (<xref rid=\"B254\" ref-type=\"bibr\">Vignali et al., 2008</xref>). It has been reported that TLR (Toll-like receptor), GITR (glucocorticoid-induced TNF receptor), CTLA-4, and FR (folate receptor) directly or indirectly regulate the function of Treg cells (<xref rid=\"B189\" ref-type=\"bibr\">Pasare and Medzhitov, 2003</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Callahan et al., 2010</xref>; <xref rid=\"B60\" ref-type=\"bibr\">de Aquino et al., 2015</xref>). TLR activation decreases the suppressive effect of Treg cells partially through IL-6. GITR, a costimulatory molecule, has a high expression level on Treg cells (<xref rid=\"B189\" ref-type=\"bibr\">Pasare and Medzhitov, 2003</xref>). Treatment with anti-GITR mAb downregulates the inhibition of Treg cells. Similarly, CTLA-4 and FR4 are expressed on Treg cells. When blocking CTLA-4 or deleting FR4, the inhibition of Treg cells decreased and active Treg cells were depleted (<xref rid=\"B39\" ref-type=\"bibr\">Callahan et al., 2010</xref>).</p><p>Several researches implied that lymphocytes were correlated with clinical outcomes in ovarian cancer. Plasma cell and B cell infiltration impacted the prognosis of ovarian cancer. CD138 and CD20 are markers for plasma cells and mature B cells, respectively. <xref rid=\"B156\" ref-type=\"bibr\">Lundgren et al. (2016)</xref> found that patients with high expression of CD138 and CD20 were related to advanced tumor grade. Additionally, the Kaplan–Meier analysis suggested that high expression of CD138 was linked to worse OS and OCSS (ovarian cancer-specific survival).</p><p>Tumor-infiltrating T cells are associated with clinical outcomes in ovarian cancer (<xref rid=\"B278\" ref-type=\"bibr\">Zhang et al., 2003</xref>). Through evaluating 186 frozen tissue samples from patients with advanced ovarian cancer, Zhang‘study demonstrated that the 5-year OS rate was higher in patients whose tumors had T cell infiltration compared to survival of patients whose tumors did not have T cell infiltration. They also confirmed that intratumor T cells were significantly associated with delayed relapse (<xref rid=\"B278\" ref-type=\"bibr\">Zhang et al., 2003</xref>).</p><p>CD8+ T lymphocyte infiltration extended survival time. In Hamanishi’s research, they demonstrated that patients with CD8+ T lymphocyte infiltration had prolonged PFS and OS (<xref rid=\"B106\" ref-type=\"bibr\">Hamanishi et al., 2007</xref>). Similarly, <xref rid=\"B220\" ref-type=\"bibr\">Sato et al. (2005)</xref> noticed that patients with high percentages of CD8+ T cells had a greater survival rate than that of patients with low percentages (55 months vs. 26 months). <xref rid=\"B50\" ref-type=\"bibr\">Clarke et al. (2009)</xref> also reported that in patients with advanced stage, CD8+ T lymphocyte infiltration was linked to increased PFS, OS and disease-specific survival. Interestingly, <xref rid=\"B269\" ref-type=\"bibr\">Ye et al. (2014)</xref> observed that CD137, a TNFR-family member, is expressed on both CD4+ and CD8+ T lymphocytes. Patients with CD137 expression had improved survival in ovarian cancer.</p><p>In contrast, Treg cell infiltration indicated poor clinical outcomes. In Curiel’s study, they evaluated 104 women with ovarian carcinoma and found that patients with advanced disease stage had a higher percentage of CD4+CD25+FOXP3+ Treg cells. Furthermore, Treg cells in the tumor sites were linked with decreased survival and a high death hazard (<xref rid=\"B58\" ref-type=\"bibr\">Curiel et al., 2004</xref>).</p><p>Lymphocytes play important roles in both innate immune responses and adaptive immune responses. Different lymphocytes have different functions. In ovarian cancer, specific lymphocytes infiltration is directly related to patient prognosis. At present, the mechanism of lymphocyte regulation is not completely understood, thereby deserving further investigation.</p></sec></sec></sec><sec id=\"S4\"><title>Novel Molecular Discoveries in Ovarian TME</title><p>Despite the advances of immunostaining technology that has provided important biological features in the ovarian cancer TME, it is still underpowered to adequately detail the multi-variant cellular and molecular interactions in it.</p><p>In the past decade, enormous technical development has been realized in sensitive detection and accurate quantification of genomic, transcriptomic, and proteomic features. The “-omic” techniques give scientists a scope from a higher dimension to reveal the TME molecular landscape as a whole. With information from this landscape, multilevel analysis has shed light on the heterogeneous nature of ovarian cancer, the complex and dynamic molecular events in the evolving TME, facilitating the advancement and validation of biomarkers for disease diagnosis, prognosis, treatment targets and treatment response prediction (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Recent molecular findings by “-omic technology” in the ovarian cancer tumor microenvironment (TME). With the tools of genomic, transcriptomic, proteomic technology, a comprehensive multi-level landscape of ovarian cancer TME is revealed. With data are drawn from the “-omic scope,” the molecular profile of ovarian cancer is further identified in terms of tumor heterogeneity and treatment impact; new discoveries on factors that are either positively or negatively associated with tumor progression are also identified, providing clues for treatment target exploration and novel biomarker designing for disease screening, staging, diagnosis, and prognosis; also, correlations between certain therapeutic regimens and ovarian cancer TME profiles are investigated, providing implication for precision medicine by precise patient selection.</p></caption><graphic xlink:href=\"fcell-08-00758-g002\"/></fig><p>Here we review the latest evidence provided by “-omic” technology, revealing the molecular characteristics of ovarian cancer TME in disease development and treatment intervention.</p><sec id=\"S4.SS1\"><title>TME Heterogeneity in Ovarian Cancer</title><p>Ovarian cancer is a kind of highly heterogenous tumor; the diversified TME of ovarian cancer is one manifestation for this fact. With immunogenomic approaches, <xref rid=\"B127\" ref-type=\"bibr\">Jiménez-Sánchez et al. (2017)</xref> observed changes of different metastatic tumor sites of an HGSOC patient who received multiple times of chemotherapy. They found both progressing and regressing metastases during treatment in the same patient, characterized by immune cell exclusion and T cell infiltration with the oligoclonal expansion of specific subsets respectively. The progressing tumor presented with molecular patterns of immune suppression associated with Wnt signaling and higher HLA mutation and neoepitope loads while regressing sites showed patterns of immune activation with the expression of HLA, IFN-γ, CXCL9 etc. and enriched TCR signaling (<xref rid=\"B127\" ref-type=\"bibr\">Jiménez-Sánchez et al., 2017</xref>). By similar approach, recently the same group further discovered co-existence of both immune-cell-excluded and inflammatory microenvironment in the same tumor sites of the same patients with HGSOC, indicating ubiquitous variation in immune cell infiltration even in the micro-niches of the same tumor entity (<xref rid=\"B126\" ref-type=\"bibr\">Jiménez-Sánchez et al., 2020</xref>).</p></sec><sec id=\"S4.SS2\"><title>TME Molecular Factors Positively Associated With Tumor Progression</title><p>By -omic studies, there is a growing body of evidence unveiling the tumor driving signaling, interactions among cell components and immune profiles in the ovarian cancer TME. Comprehensive analysis of TME cell components, genomic alterations and gene expression revealed that amplification of Myc target genes and Wnt signaling were associated with impaired immune cell infiltration (<xref rid=\"B126\" ref-type=\"bibr\">Jiménez-Sánchez et al., 2020</xref>). Using next-generation sequencing technology, <xref rid=\"B11\" ref-type=\"bibr\">Au Yeung et al. (2016)</xref> identified high levels of microRNA-21 (miR21) from the exosomes secreted by cancer-associated adipocytes (CAAs) and CAFs, which later were transferred to ovarian cancer cells, suppressing cancer cell apoptosis and conferring chemoresistance. In order to investigate Treg antigen specificity, <xref rid=\"B3\" ref-type=\"bibr\">Ahmadzadeh et al. (2019)</xref> adopted TCR β chain deep sequencing in Tregs from multiple tumor samples, including ovarian cancer and discovered that TCR repertoires were distinct from conventional T cells, displaying tumor- and neoantigen-specific reactivity. This finding suggested Tregs clonally expand in an antigen-selective manner in the TME.</p><p>Recently, the interplay between metabolism and tumor progression as well as immune suppression among players in the TME has been reported with -omic tools. By phosphoproteomic techniques, <xref rid=\"B59\" ref-type=\"bibr\">Curtis et al. (2019)</xref> identified increased activation of the p38-MAPK pathway in CAFs that are co-cultured with ovarian cancer cells and verified that this activation lay the premise for glycogen mobilization in cancer cells, which as an energy source fueled metastatic tumor growth. Another proteomic study identified the central metabolic regulator of CAF, the methyltransferase nicotinamide <italic>N</italic>-methyltransferase (NNMT) being a prominent signature in metastatic stroma tissue of HGSOC patients, which is necessary for the differentiation of competent CAF phenotype, supporting cancer cell migration and proliferation (<xref rid=\"B73\" ref-type=\"bibr\">Eckert et al., 2019</xref>). In a mouse model of metastatic ovarian cancer, <xref rid=\"B98\" ref-type=\"bibr\">Goossens et al. (2019)</xref> used Gene Ontology (GO) enrichment analysis and found that at a later time after tumor inoculation (day 21), there was an up-regulation of cholesterol metabolic gene clusters in TAMs, resulting in membrane-cholesterol efflux and depletion of lipid rafts from TAMs, leading to IL-4-mediated immune-suppressive TAM reprogramming. All these discoveries provide novel therapeutic targets that could facilitate the current treatment strategy.</p><p>There are increasing -omic studies showing an association between TME cellular and molecular profile and patient clinical features. <xref rid=\"B14\" ref-type=\"bibr\">Barnett et al. (2010)</xref> earlier have found increased Treg infiltration, represented by several enriched immunologic pathway gene signatures, was associated with higher grade and advanced stage in serous ovarian cancer. From secretome analysis of fibroblasts and cancer cells, <xref rid=\"B113\" ref-type=\"bibr\">Hernandez-Fernaud et al. (2017)</xref> identified abundance chloride intracellular channel protein 3 (CLIC3) in the TME of aggressive ovarian cancers, which correlates with poor clinical outcome. Aside from single biomarkers, -omic research enables scientists to discover a correlation between a set of molecular patterns and patient prognosis. With proteomic technology, <xref rid=\"B83\" ref-type=\"bibr\">Finkernagel et al. (2019)</xref> identified 779 proteins in the ascites of HGSOC patients and identified protein marker sets to predict patient survival, with CAPG, LCK and TNFAIP6 as the core type 2 signature that has 91.2% correctness in identifying short-term survivors. Another multi-level -omic study with HGSOC primary tumor samples, discovered the association between short-term survival and copy number gain of CCNE1, lack of BRCA mutation signature, low homologous recombination deficiency scores, and the presence of ESR1-CCDC170 gene fusion (<xref rid=\"B267\" ref-type=\"bibr\">Yang S. Y. C. et al., 2018</xref>). One study with HGSOC metastatic samples revealed expression pattern of 22 matrisome genes and thereby generated a “matrix index” with their expression; this index was significantly correlated with Treg and Th2 cell signatures and can identify the patient group with shorter OS (<xref rid=\"B191\" ref-type=\"bibr\">Pearce et al., 2018</xref>).</p></sec><sec id=\"S4.SS3\"><title>TME Molecular Factors Negatively Associated With Tumor Progression</title><p>Together with novel findings of tumor promoting factors, there are also tumor-suppressing molecular patterns uncovered by recent -omic studies. In a study concentrating on RNA binding proteins (RBPs), the authors referred to published literature as well as oncogenic databases and conducted functional verification studies; they identified the sorbin and SH3 domain containing 2 (SORBS2) out of a pool of RBPs, as a suppressor of metastatic colonization of ovarian cancer, which exerted tumor suppressive function by dampening cancer invasiveness and repolarizing MDSCs and TAMs (<xref rid=\"B284\" ref-type=\"bibr\">Zhao L. et al., 2018</xref>). Another study investigated the correlation between tumor-infiltrating lymphocytes (TILs) and malignant diversity in HGSOC samples, where the authors found epithelial CD8+ TILs were negatively associated with tumor clonal heterogeneity, suggesting neoantigen-specific depletion of tumor clones and spatial antigen-specific T cell tracking of tumors (<xref rid=\"B277\" ref-type=\"bibr\">Zhang et al., 2018</xref>).</p><p>Molecular signatures correlating with good clinical prognosis have been reported as well. In <xref rid=\"B83\" ref-type=\"bibr\">Finkernagel et al.’s (2019)</xref> ascites proteomic study mentioned above, authors reported markers of BCAM, HSPA1A, and DKK1 as the core type 1 signature with 90.9% of correctness in the identification of long-term survivors. Similarly, in <xref rid=\"B267\" ref-type=\"bibr\">Yang S. Y. C. et al. (2018)</xref> study with primary HGSOC samples, they reported increased somatic mutation burden, BRCA1/2 biallelic inactivation, and enriched infiltration of activated as well as memory T cells in long-term survivors. Lastly, in <xref rid=\"B277\" ref-type=\"bibr\">Zhang et al.’s (2018)</xref> multi-level study with HGSOC tumor samples, they discovered the combinatorial prognostic value of high immune activity and low mutation prevalence of foldback inversions, which lead to best clinical outcomes.</p><p>Detailed mechanistic studies are needed in the future to determine the way of targeting the tumor suppressing molecular patterns that can potentiate therapeutic strategy.</p></sec><sec id=\"S4.SS4\"><title>Treatment Impact and Response Prediction in Ovarian Cancer TME</title><p>The tumouricidal treatment causes cell death, incurring multiple changes in the TME. After neoadjuvant chemotherapy (NACT), researchers adopted immunogenic analysis in HGSOC tumors and found increased NK cell infiltration and oligoclonal expansion of T cells, suggesting chemotherapy can potentiate immunogenicity of the primary tumor (<xref rid=\"B126\" ref-type=\"bibr\">Jiménez-Sánchez et al., 2020</xref>). Another gene expression analysis revealed ovarian cancer patients treated with paclitaxel had an enriched gene signature linked to M1 TAM activation (<xref rid=\"B256\" ref-type=\"bibr\">Wanderley et al., 2018</xref>). However, this immune activation property of chemotherapy may just exist in the early stage right after the treatment; when bulky tumor cells are eradicated, the chemo-resistant cancer stem cells (CSCs) remain and subsequently bring about recurrence. The positively selected CSCs by chemotherapy showed altered lipid metabolism signatures, resulting in accumulation of lactate, which acidifies ascites, leading to T cell dysfunction and Treg polarization (<xref rid=\"B4\" ref-type=\"bibr\">Ahmed et al., 2018</xref>). By comparing the gene expression profile of ascites-derived tumor cells from treatment naive (CN) and recurrent (CR) ovarian cancer patients, Ahmed and colleagues found massively reduced MHC I molecule (HLA-C and -B) expression and IFN response-related gene expression, including IFIT2, TMEM173 and MX2 in CR patients, suggesting an immuno-compromised ascites TME in CR after chemotherapy. Hence, CSCs could become a key target in treatment exploitation for CR patients (<xref rid=\"B4\" ref-type=\"bibr\">Ahmed et al., 2018</xref>). More comparative studies between CN and CR patients are needed in the future for the discovery of clues that can overcome treatment resistance and targets that can further improve the current regimens.</p><p>Due to the heterogeneous nature of ovarian cancer, responses of treatment targeting different tumor driving molecules vary among individuals. With “-omic” tools, the identification of tumor biomarkers that are associated with exceptional clinical response or resistance has become possible. <xref rid=\"B162\" ref-type=\"bibr\">Mak et al. (2016)</xref> conducted a genomic and proteomic analysis of EMT signatures in multiple cancers, including ovarian cancer; they identified a set of 77 EMT-related genes, generating an EMT score according to their expression signature; they found EMT score was positively correlated with expression levels of immune checkpoint genes, implicating predictive value of EMT score in treatment response of immune checkpoint blockade. Another study investigated immunogenomic profile in predicting combination treatment response of a PARP inhibitor (PARPi) and an anti-PD-1 antibody in ovarian cancer patients; the authors found two determinants associated with a positive response: mutational signature of defective homologous recombination DNA repair and exhausted CD8+ T cell primed by IFN in the TME. These findings are important for the patient selection of certain treatments, paving the way for future precision medicine.</p><p>In conclusion, more mechanistic and phenotypic investigation is required to decipher the roles of certain patterns of molecular alteration in disease development and treatment intervention, so as to facilitate the deployment of more individualized and molecularly informed treatments for ovarian cancer patients.</p></sec></sec><sec id=\"S5\"><title>Therapeutic Strategies Targeting the TME</title><sec id=\"S5.SS1\"><title>Therapies Targeting CAFs</title><p>Several therapies are targeting CAFs in ovarian cancer: (1) direct deletion FAP+ fibroblasts; (2) reverting the activated CAFs into a quiescent state; (3) targeting CAF-specific pathways (<xref rid=\"B47\" ref-type=\"bibr\">Chen and Song, 2019</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Barrett and Puré, 2020</xref>; <xref rid=\"B248\" ref-type=\"bibr\">Truffi et al., 2020</xref>).</p><sec id=\"S5.SS1.SSS1\"><title>Direct Deletion FAP+ Fibroblasts</title><p>It is well known that CAF has phenotypic heterogeneity. Activated CAFs can selectively express a variety of different biomarkers in specific TMEs environments, such as alpha-SMA FAP, S100A4 and PDGFR (<xref rid=\"B15\" ref-type=\"bibr\">Barrett and Puré, 2020</xref>). FAP is a serine protease, which regulates the recruitment, proliferation and differentiation of myofibroblasts. FAP is an important surface marker in CAFs, which exists in more than 90% of CAFs (<xref rid=\"B47\" ref-type=\"bibr\">Chen and Song, 2019</xref>). FAP+ cells cannot only promote tumor progression but also block immunotherapy by producing ECM and direct signaling pathways (<xref rid=\"B197\" ref-type=\"bibr\">Puré and Blomberg, 2018</xref>). Studies have shown that inhibition of FAP can reduce the infiltration of CAF (<xref rid=\"B219\" ref-type=\"bibr\">Santos et al., 2009</xref>). Therefore, targeted therapy for FAP on CAF was proposed.</p><p>In Paulette’s research, they examined the gene expression of FAP in high-grade serous EOCs and found that a higher FAP expression in tumor tissue than normal control. They also reported that patients with high FAP expression showed poor OS. Then they blocked the FAP via a FAP-specific siRNA, the results demonstrated that the proliferation of cells was reduced 9–13% (<xref rid=\"B171\" ref-type=\"bibr\">Mhawech-Fauceglia et al., 2015</xref>). Thereby, downregulated FAP+ fibroblasts could reduce the proliferation of tumor cells and might be a new treatment for ovarian cancer.</p></sec><sec id=\"S5.SS1.SSS2\"><title>Reverting the Activated CAFs Into a Quiescent State</title><p>There are two main states of CAFs: quiescent state and active state. In general, most CAFs are in a quiescent state and have low proliferative and metabolic capacity (<xref rid=\"B109\" ref-type=\"bibr\">Hansen et al., 2016</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Chen and Song, 2019</xref>). However, once the homeostasis is broken, CAFs is activated in order to return to the quiescent state (<xref rid=\"B109\" ref-type=\"bibr\">Hansen et al., 2016</xref>). In the tumor microenvironment, not only cancer cells can enhance the activation of CAFs, but some cytokines can also activate CAFs (<xref rid=\"B47\" ref-type=\"bibr\">Chen and Song, 2019</xref>). Recent years, some scientists target therapy of CAF by restoring the activation state of tumorigenic CAFs to a static state (<xref rid=\"B209\" ref-type=\"bibr\">Rossmann et al., 1967</xref>; <xref rid=\"B84\" ref-type=\"bibr\">Froeling et al., 2011</xref>). But it has not been used in ovarian cancer.</p></sec><sec id=\"S5.SS1.SSS3\"><title>Targeting CAFs Associated Signal Molecules</title><p>Cancer-associated fibroblasts regulate immune cells and ECM via a series of signal molecules. CAFs regulate the function of the myeloid cell and T cell (<xref rid=\"B15\" ref-type=\"bibr\">Barrett and Puré, 2020</xref>). CAFs decrease the number of MDSCs through inhibiting CXCL12/CXCR4 signal pathway and promote the differentiation of myeloid cells into DCs via stimulating IL6/STAT3 signal pathway (<xref rid=\"B97\" ref-type=\"bibr\">Gok Yavuz et al., 2019</xref>; <xref rid=\"B248\" ref-type=\"bibr\">Truffi et al., 2020</xref>). Besides, CAFs inhibit T cells through increase the expression of PD-L1/2, while CAFs activate T cells via stimulating the production of IL-6 (<xref rid=\"B13\" ref-type=\"bibr\">Barnas et al., 2010</xref>; <xref rid=\"B49\" ref-type=\"bibr\">Cho et al., 2011</xref>). CAFs can active the ECM through secreting growth factors (such as VEGF) and cytokines (such as TGF-β, IL-6 and IL-10) (<xref rid=\"B139\" ref-type=\"bibr\">Kohlhapp et al., 2015</xref>).</p><p>Recently, several therapies targeting CAFs associated signal molecules have been developed, including TGF-β inhibitors, PDGF inhibitor, Hedgehog inhibitors, FAK inhibitors and IL-6 inhibitors.</p><p>TGF-β1 is a cytokine produced by CAFs that plays a significant role in promoting tumorigenesis (<xref rid=\"B77\" ref-type=\"bibr\">Fabregat et al., 2014</xref>). A-83-01 is a TGF-β inhibitor. In Yamamura’s research, they found that <italic>in vitro</italic> TGF-β1 treatment stimulated HM-1 cell motility, invasion and adhesion. However, A-83-01 could counteract the effect of TGF-β1. Interestingly, <italic>in vivo</italic>, they found that mice treated with A-83-01 had a longer survival time than that of the control group (<xref rid=\"B264\" ref-type=\"bibr\">Yamamura et al., 2012</xref>). Similarly, in Gao’s study, they investigated the tumor-suppressive activity of LY2109761, a TGF-β type I (TβRI) and type II (TβRII) kinase. The results demonstrated that LY2109761 augmented ovarian cancer cell apoptosis. Moreover, combined cisplatin with LY2109761 enhanced the lethal effect of cisplatin in normal and cisplatin-resistant ovarian cancer cells. Furthermore, <italic>in vivo</italic> treatment with cisplatin and LY2109761 reduced the tumor volume in a cisplatin-resistant ovarian cancer model. Therefore, they confirmed that LY2109761 increases the antitumor activity of cisplatin (<xref rid=\"B92\" ref-type=\"bibr\">Gao et al., 2015</xref>).</p><p>PDGF is a factor that can stimulate other cell trans-differentiation for CAFs. In Matei’s report, they found that PDGFR was expressed in 39% of ovarian cancers. When ovarian cancer cells were treated with imatinib (a PDGFR inhibitor), cells were arrested in the G0–G1 phase. Therefore, they confirmed that imatinib could suppress the proliferation of ovarian cancer cells (<xref rid=\"B166\" ref-type=\"bibr\">Matei et al., 2004</xref>). Imatinib also showed its clinical activity on platinum-resistant ovarian carcinoma. In Matei’s study, in patients treated with imatinib mesylate and docetaxel, they found that 1 patient had a complete response and 4 patients had partial responses (ORR: 21.7%) (<xref rid=\"B167\" ref-type=\"bibr\">Matei et al., 2008</xref>). Overall, the inhibition of PDGF could suppress tumor proliferation.</p><p>Targeting Hedgehog, FAK and IL-6 is also an effective treatment for ovarian cancer. IPI-126 is a Hedgehog inhibitor. When treated with IPI-126, Hh signaling was suppressed, thereby inhibiting the proliferation of serous ovarian cancer (<xref rid=\"B169\" ref-type=\"bibr\">McCann et al., 2011</xref>). VS-6063, a FAK inhibitor, suppressed the phosphorylation of FAK. VS-6063 increase the chemosensitivity in taxane-resistant ovarian cancer cells, thus decrease the tumor load (<xref rid=\"B131\" ref-type=\"bibr\">Kang et al., 2013</xref>). Tocilizumab is a monoclonal antibody against IL-6R. In a phase I clinical trial, the results showed that patients treated with tocilizumab increase the serum IL-6 and soluble IL-6R. Moreover, increased sIL-6 indicated a longer survival time. Therefore, tocilizumab prolongs survival time in recurrent ovarian cancer (<xref rid=\"B66\" ref-type=\"bibr\">Dijkgraaf et al., 2015</xref>).</p><p>Cancer-associated fibroblasts is an important component of the tumor microenvironment, which play a pro-tumor function in the process of cancer development, making it a hot spot of targeted therapy. Nevertheless, targeted CAFs therapy still has challenges. Some studies suggest that directly targeted killing of CAF may be a way to reduce CAF infiltration in tumors. But due to the lack of specific cell surface markers, it is hard to precisely target CAFs. The reversal of the functional state of CAF provides a new idea for the development of new anticancer therapies. At present, limiting the function of CAF by targeting stromal CAF signals and effectors has become an important supplement to tumor therapy, but further mechanism and function studies are still needed.</p></sec></sec><sec id=\"S5.SS2\"><title>Anti-angiogenesis Therapy</title><p>VEGF is the most typical activator of angiogenesis. Currently, anti-angiogenesis therapy is divided into three types (<xref rid=\"B55\" ref-type=\"bibr\">Cortez et al., 2018</xref>; <xref rid=\"B114\" ref-type=\"bibr\">Hironaka, 2019</xref>): (1) inhibiting VEGF; (2) inhibiting its receptor, VEGFR; and (3) inhibiting Angs (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Clinical trials of therapies that target angiogenesis in ovarian cancer.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Immunotherapy agents</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Target</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Trial type</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Disease status</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>N</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Trial number</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">References</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"7\" rowspan=\"1\"><bold>VEGF inhibitors</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VEGF</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Stage III or stage IV epithelial ovarian cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1873</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT00262847</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B36\" ref-type=\"bibr\">Burger et al., 2011</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ovarian cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1528</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ISRCTN91273375</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B192\" ref-type=\"bibr\">Perren et al., 2011</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ovarian cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1528</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ISRCTN91273375</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B188\" ref-type=\"bibr\">Oza et al., 2015</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Platinum-resistance EOC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">44</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT00097019</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B188\" ref-type=\"bibr\">Oza et al., 2015</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Platinum-sensitive relapsed epithelial ovarian cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">484</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT00434642</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B1\" ref-type=\"bibr\">Aghajanian et al., 2012</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recurrent platinum-sensitive ROC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">484</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT00434642</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B2\" ref-type=\"bibr\">Aghajanian et al., 2015</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Persistent or relapse epithelial ovarian cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">62</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B37\" ref-type=\"bibr\">Burger et al., 2007</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Relapsed platinum-sensitive epithelial ovarian cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">674</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT00565851</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B52\" ref-type=\"bibr\">Coleman et al., 2017</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Relapsed platinum-resistant ovarian cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">361</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT00976911</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B196\" ref-type=\"bibr\">Pujade-Lauraine et al., 2014</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Relapsed ovarian cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">70</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT00072566</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B93\" ref-type=\"bibr\">Garcia et al., 2008</xref></td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"7\" rowspan=\"1\"><bold>VEGF receptors inhibitors</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sorafenib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VEGFR</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Platinum-resistant ovarian cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">174</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT01047891</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B46\" ref-type=\"bibr\">Chekerov et al., 2018</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Relapsed ovarian carcinoma</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">71</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT00093626</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B168\" ref-type=\"bibr\">Matei et al., 2011</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sunitinib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VEGFR</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Platinum resistant ovarian cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">73</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT00543049</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B17\" ref-type=\"bibr\">Baumann et al., 2012</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recurrent epithelial ovarian cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">30</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT00388037</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B19\" ref-type=\"bibr\">Biagi et al., 2011</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pazopanib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VEGFR</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ovarian cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">940</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT00866697</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B70\" ref-type=\"bibr\">du Bois et al., 2014</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Platinum-refractory or platinum-resistant advanced ovarian carcinoma</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">74</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT01644825</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B193\" ref-type=\"bibr\">Pignata et al., 2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nintedanib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VEGFR</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Advanced ovarian cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1366</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT01015118</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B71\" ref-type=\"bibr\">du Bois et al., 2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cediranib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VEGFR</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recurrent platinum-sensitive ovarian carcinoma</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">456</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT00532194</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B144\" ref-type=\"bibr\">Ledermann et al., 2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"7\" rowspan=\"1\"><bold>Angs inhibitors</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Trebananib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ang1 and Ang2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recurrent ovarian cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">919</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NCT01204749</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B177\" ref-type=\"bibr\">Monk et al., 2014</xref></td></tr></tbody></table></table-wrap><sec id=\"S5.SS2.SSS1\"><title>Anti-VEGF</title><p>Bevacizumab is a humanized VEGF monoclonal antibody. It can inhibit ECs proliferation and activation by binding and inactivating VEGF (<xref rid=\"B76\" ref-type=\"bibr\">Eskander and Randall, 2011</xref>). Studies have reported that bevacizumab treatment of murine ovarian cancer models could suppress tumor proliferation and prolong survival time (<xref rid=\"B117\" ref-type=\"bibr\">Hu et al., 2002</xref>; <xref rid=\"B176\" ref-type=\"bibr\">Monk et al., 2006</xref>; <xref rid=\"B121\" ref-type=\"bibr\">Huynh et al., 2007</xref>; <xref rid=\"B158\" ref-type=\"bibr\">Mabuchi et al., 2008</xref>). Currently, using bevacizumab alone or combined with other therapeutics has had successful outcomes. Bevacizumab was quickly used in the clinic due to its amazing therapeutic effects in animal models. Bevacizumab, as the first FDA-approved anti-angiogenesis antibody, was applied to the treatment of ovarian cancer. <xref rid=\"B36\" ref-type=\"bibr\">Burger et al. (2011)</xref> designed a phase 3 trial (GOG-218) to explore the antitumor efficiency of bevacizumab in ovarian cancer. They enrolled 1873 women with newly diagnosed stage III or IV EOC. They randomly divided patients into three groups: control treatment, bevacizumab-initiation treatment, and bevacizumab-throughout treatment. The results demonstrated that the median PFS with the control treatment was 10.3 months, while with bevacizumab-initiation treatment and bevacizumab-throughout treatment, it was 11.2 and 14.1 months, respectively. Additionally, the median OS in the control treatment, bevacizumab-initiation treatment and bevacizumab-throughout treatment was 39.3, 38.7, and 39.7 months, respectively. Thereby, ovarian cancer patients treated with bevacizumab was supposed to have a longer PFS.</p><p>Subsequently, the ICON7 trial reported the similarity results in ovarian cancer patients. In the ICON7 trial, they enrolled 1528 patients with ovarian carcinoma and randomly allocated them into the standard-therapy group and bevacizumab group. The data indicated that when the follow-up was 19.4 months, patients in the bevacizumab group had a longer median PFS (restricted mean) than that of the standard-therapy group (21.8 months vs. 20.3 months). Moreover, when prolonging the follow-up to 42 months, the PFS (restricted mean) was 24.1 months in the bevacizumab group and 22.4 months in the standard-therapy group. Additionally, patients in the bevacizumab group had a median OS of 36.6 months compared with a median survival of 28.8 months in the standard-therapy group (<xref rid=\"B192\" ref-type=\"bibr\">Perren et al., 2011</xref>). Then, <xref rid=\"B188\" ref-type=\"bibr\">Oza et al. (2015)</xref> reported the final OS results of ICON7. They found no significant difference in restricted mean OS between the two groups (45.5 months in the bevacizumab group versus 44.6 months in the standard-therapy group). However, for patients with high risk, the restricted mean OS was longer in the bevacizumab group compared to that in the standard-therapy group (39.9 months in the bevacizumab group vs 34.5 months in the standard-therapy group). For patients with non-high risk, there was no significant difference in the restricted mean OS between the bevacizumab group and the standard-therapy group (49.7 months vs. 48.4 months). Overall, both the GOG-218 and ICON7 trial proved that chemotherapy combined with bevacizumab could not prolong the overall survival in ovarian cancer. However, high risk patients treated with chemotherapy plus bevacizumab show an OS benefit compared to those treated chemotherapy alone.</p><p>Bevacizumab also showed antitumor activity in platinum-resistant EOC. <xref rid=\"B40\" ref-type=\"bibr\">Cannistra et al. (2007)</xref> designed a phase 2 trial for bevacizumab in patients with platinum-resistant EOC or peritoneal serous cancer. They enrolled 44 patients and treated them with intravenous bevacizumab 15 mg/kg every 3 weeks. In the research, seven patients had a partial response. Patients had a median PFS of 4.4 months. So in platinum-resistant cancer, bevacizumab also had antitumor activity.</p><p>Bevacizumab also has antitumor activity in recurrent ovarian cancer. <xref rid=\"B1\" ref-type=\"bibr\">Aghajanian et al. (2012)</xref> reported a phase 3 clinical trial (OCEANS) in platinum-sensitive relapsed ovarian cancer. In total, 484 patients were enrolled and randomly assigned to the bevacizumab arm (<italic>n</italic> = 242) or placebo arm (<italic>n</italic> = 242). All patients were treated with carboplatin. The results showed that patients in the bevacizumab arm had a longer PFS compared to that of patients in the placebo arm (12.4 months vs. 8.4 months, respectively). Additionally, patients in the bevacizumab arm also showed an enhanced objective response rate (78.5% versus 57.4%) and duration of response (10.4 months versus 7.4 months) compared with those of patients in the placebo arm. They also analyzed the OS of the two arms. However, the results showed no significant difference in OS between the bevacizumab arm and the placebo arm (33.6 months in the bevacizumab arm vs 32.9 months in the placebo arm) (<xref rid=\"B2\" ref-type=\"bibr\">Aghajanian et al., 2015</xref>). Later, <xref rid=\"B52\" ref-type=\"bibr\">Coleman et al. (2017)</xref> reported a phase III clinical trial of bevacizumab combined with chemotherapy in patients with relapsed platinum-sensitive EOC. The data showed that the median OS was significantly longer in the chemotherapy plus bevacizumab group compared to that of the chemotherapy group (42.2 months in the chemotherapy plus bevacizumab group versus 37.3 months in the chemotherapy group). Additionally, the median PFS in patients with chemotherapy was 10.4 months, while in chemotherapy plus bevacizumab, the median PFS was 13.8 months (<xref rid=\"B52\" ref-type=\"bibr\">Coleman et al., 2017</xref>). Above studies implied that bevacizumab could improve the PFS and median OS in recurrent platinum-sensitive recurrent ovarian cancer, while it had no significant effect for OS.</p><p>There have also been studies using bevacizumab-treated relapsed platinum-resistant ovarian cancer. In a phase III clinical trial (AURELA) the results demonstrated that patients in the bevacizumab with chemotherapy group had a significantly increased PFS rate than that of the chemotherapy group. The median PFS in the bevacizumab with chemotherapy group and the chemotherapy group was 6.7 and 3.4 months, respectively. However, there was no meaningful OS increase in the bevacizumab with chemotherapy group. Therefore, they thought that the combination of bevacizumab with chemotherapy could improve the PFS in patients with relapsed platinum-resistant ovarian cancer (<xref rid=\"B196\" ref-type=\"bibr\">Pujade-Lauraine et al., 2014</xref>).</p><p>Bevacizumab combined with cyclophosphamide also showed its effect in recurrent ovarian cancer. In a phase 2 clinical trial, seventy patients with relapsed ovarian carcinoma were treated intravenously with bevacizumab and cyclophosphamide. The results showed that 17 patients had a partial response. Moreover, patients had a median progression time of 7.2 months and a median survival time of 16.9 months (<xref rid=\"B93\" ref-type=\"bibr\">Garcia et al., 2008</xref>).</p><p>VEGF plays a crucial role in angiogenesis. Nowadays, due to the pro-tumor effect in tumor progress, targeting VEGF became an attractive therapeutic method. As the first FDA-approved anti-angiogenesis antibody, bevacizumab was applied to the treatment of cancer. Studies demonstrated that bevacizumab combined with chemotherapy increases the PFS in newly prognosis ovarian cancer, platinum-resist ovarian cancer and recurrent ovarian cancer. Nevertheless, bevacizumab added to chemotherapy could not enhance the OS. Further investigated was needed.</p></sec><sec id=\"S5.SS2.SSS2\"><title>Inhibitors of VEGF Receptors</title><p>Sorafenib (also called BAY 43-9006) is a double inhibitor of VEGF and RAF kinase that can promote tumor angiogenesis by targeting RTKs and the PAF/MEK/ERK pathway (<xref rid=\"B260\" ref-type=\"bibr\">Wilhelm et al., 2004</xref>). <xref rid=\"B46\" ref-type=\"bibr\">Chekerov et al. (2018)</xref> reported a randomized, double-blind, placebo-controlled, phase II study of the effect of sorafenib in combination with topotecan in women with platinum-resistant ovarian carcinoma. They randomly assigned patients into the topotecan combined sorafenib (oral 400 mg bid) group (<italic>n</italic> = 85) of the topotecan plus placebo group (<italic>n</italic> = 89). The results showed that patients in the topotecan plus sorafenib group had improved PFS compared with that of the topotecan plus placebo group (6.7 months vs. 4.4 months). There were the same results for OS (17.1 months in the topotecan plus sorafenib group vs. 10.1 months in the topotecan plus placebo group). Subsequently, <xref rid=\"B168\" ref-type=\"bibr\">Matei et al. (2011)</xref> described a phase 2 clinical trial of sorafenib in relapsed ovarian carcinoma. They treated patients with oral 400 mg sorafenib bid. The results suggested that only 2 women had partial response and 20 patients had stable disease. In brief, sorafenib could increase the survival time in ovarian cancer.</p><p>Sunitinib is a multi-targeted tyrosine kinase inhibitor that acts through targeting PDGF receptor (PDGFRs), VEGFR-1-2-3, KIT (a stem cell factor) and Flt3 (a tyrosine protein receptor) (<xref rid=\"B16\" ref-type=\"bibr\">Bauerschlag et al., 2010</xref>). <xref rid=\"B17\" ref-type=\"bibr\">Baumann et al. (2012)</xref> reported a phase II trial of sunitinib in patients with platinum-resistant ovarian cancer. Patients were divided into a non-continuous sunitinib group and a continuous sunitinib group. Six patients in the non-continuous group and two patients in the continuous group had a complete response or partial response. Additionally, the median PFS times for the non-continuous group and continuous group were 4.8 and 2.9 months, respectively. The median OS was 13.6 months vs. 13.7 months for the non-continuous group and continuous group. Additionally, <xref rid=\"B19\" ref-type=\"bibr\">Biagi et al. (2011)</xref> reported the effect of sunitinib in relapsed ovarian cancer. In patients who received oral sunitinib treatment, there was a median PFS of 4.1 months. Hence, using sunitinib in recurrent ovarian cancer patients could increase the survival time.</p><p>Pazopanib was an anti-angiogenic drug that targets VEGF receptor, FGF receptors (FGFRs) 1–3, and PDGFRs α and β. A phase 2 enrolled 940 patients with ovarian cancer and randomly divided into a pazopanib group (800 mg once daily for 24 months) (<italic>n</italic> = 472) and a placebo group (<italic>n</italic> = 478). The results revealed that patients in the pazopanib group had an improved median PFS compared with that of the placebo group (mean PFS 17.9 months versus 12.3 months). Conversely, there was no difference in OS between the two groups. Additionally, <xref rid=\"B193\" ref-type=\"bibr\">Pignata et al. (2015)</xref> reported a phase 2 trial to assess the effect of combined pazopanib and paclitaxel in patients with platinum-refractory or platinum-resistant advanced ovarian carcinoma. They enrolled 74 patients who were randomly assigned to the paclitaxel and pazopanib group (<italic>n</italic> = 37) or the paclitaxel group (<italic>n</italic> = 37). The data indicated that patients in the paclitaxel and pazopanib group had significantly longer PFS than that of patients in the paclitaxel group (mean PFS 6.35 vs 3.49 months). The median OS was 19.1 months in the paclitaxel and pazopanib group and 13.7 months in the paclitaxel group. Above researches indicated that pazopanib treatment had an improve PFS in ovarian cancer.</p><p>Nintedanib is also an oral angiokinase inhibitor, which can inhibit the effect of VEGFR1-3, FGRs 1-3 and PDGFRs α and β. A phase 3 trial was performed to elucidate the effect of nintedanib combined with first-line chemotherapy in advanced ovarian cancer. Of the 1366 patients enrolled, 911 were assigned to the nintedanib group, and 455 were assigned to the placebo group. The data suggested the mean PFS was 17.2 months in the nintedanib group compared with 16.6 months in the placebo group. Therefore, they suggested that the combination of nintedanib and first-line chemotherapy could significantly increase the PFS in advanced ovarian cancer.</p><p>Cediranib is a VEGF receptor (VEGFR1-3) inhibitor. In a randomized phase 3 trial (ICON6) the investigators randomly assigned 456 patients with relapsed platinum-sensitive ovarian carcinoma into three groups: group A received placebo with chemotherapy and then placebo maintenance, group B received cediranib 20 mg with chemotherapy and then placebo maintenance, and group C received cediranib once daily with chemotherapy and then cediranib once daily maintenance. The results showed that 90% of patients (410 of 456) had disease progression, including 96% of patients in group A (113 of 118), 90% of patients in group B (156 of 174) and 86% of patients in group C (141 of 164). Moreover, patients in group C had a longer median PFS compared to that of groups A and B (mean PFS = 8.7, 9.9, and 11.0 months in groups A, B, and C, respectively) (<xref rid=\"B144\" ref-type=\"bibr\">Ledermann et al., 2016</xref>). Thus, they considered cediranib with chemotherapy could significantly improve the PFS in patients with relapsed ovarian cancer.</p><p>VEGFR inhibition is an important component of anti-angiogenic therapy. Several VEGFR inhibitors have been introduced into clinical studies. The research results suggest that treatment of VEGFR inhibitors could improve the PFS in ovarian cancer. These provide a novel therapeutic option for ovarian cancer.</p></sec><sec id=\"S5.SS2.SSS3\"><title>Angs Inhibitor</title><p>Ang1 and Ang2, expressed on ECs, were connected with the TIE2 receptor. They could mediate vascular remodeling by a signaling pathway, which was different from the VEGF pathway. Trebananib (also called AMG386) is a peptide-Fc. Through binding with Ang1 and Ang2, it prevents the connection of the TIE2 receptor and Angs. Thus, trebananib showed antitumor activity in ovarian cancer. A double blind phase 3 study detected the antitumor activity of trebananib in patients with recurrent ovarian cancer. A total of 919 patients were enrolled, and they were randomly divided into the trebananib group (<italic>n</italic> = 461) and the placebo group (<italic>n</italic> = 458). In the placebo group, patients were treated with intravenous paclitaxel 80 mg/m2 and placebo weekly. The results showed that patients with trebananib treatment had a significantly longer median PFS compared to patients with placebo treatment (7.2 months compared to 5.4 months). However, there were no significant differences between the trebananib group and the placebo group in the OS analysis (17.3 months in the paclitaxel group and 19.0 months in the placebo group) (<xref rid=\"B177\" ref-type=\"bibr\">Monk et al., 2014</xref>).</p><p>Trebananib inhibited Angs 1 and 2 and improved the PFS in ovarian cancer. However, the role of Angs inhibitors in recurrent ovarian cancer remains to be further studied.</p></sec></sec><sec id=\"S5.SS3\"><title>TAM-Targeted Antitumor Strategies</title><p>Tumor-associated macrophages are a crucial part of the TME. It is known that TAMs have a significant association with the proliferation, invasion, migration and clinical outcomes of ovarian carcinoma. In recent years, significant progress has been made in research on TAM-targeted strategies. Based on previous research, TAM-targeted strategies can be divided into four types: (1) suppressing macrophage recruitment; (2) inhibiting TAM survival; (3) increasing the tumouricidal activity of M1 macrophages; and (4) limiting the tumor-promoting activity of M2 macrophages (<xref rid=\"B241\" ref-type=\"bibr\">Tang et al., 2013</xref>; <xref rid=\"B44\" ref-type=\"bibr\">Cassetta and Pollard, 2018</xref>).</p><sec id=\"S5.SS3.SSS1\"><title>Suppressing Macrophage Recruitment</title><p>Various chemokines and cytokines promote macrophage recruitment to tumor tissues, such as C-C motif chemokine ligand 2 (CCL2), macrophage colony-stimulating factor (GM-CSF), VEGF, CXCL-12 and hypoxia-inducible factors (HIFs) (<xref rid=\"B241\" ref-type=\"bibr\">Tang et al., 2013</xref>; <xref rid=\"B44\" ref-type=\"bibr\">Cassetta and Pollard, 2018</xref>). Thus, regulating the relevant chemoattractants is a promising approach for suppressing macrophage recruitment and tumor therapy.</p><p>CCL2 (also called monocyte chemotactic protein-1 [MCP-1]) is a member of the MCP chemokine family that is produced by tumor cells and stromal cells such as myeloid cells, ECs and fibroblasts and acts as a chemoattractant for T cells, NK cells and monocytes (<xref rid=\"B251\" ref-type=\"bibr\">Ueno et al., 2000</xref>; <xref rid=\"B54\" ref-type=\"bibr\">Conti and Rollins, 2004</xref>). CCR2 is a receptor of CCL2, including CCR2A and CCR2B. Among them, CCA2B is the main isoform of CCR2 that is highly expressed on NK cells and monocytes. CCR2A is expressed on smooth muscle cells and a portion of monocytes. It has been reported that CCL2-CCR2 signaling is involved in tumor metastasis (<xref rid=\"B149\" ref-type=\"bibr\">Lim et al., 2016</xref>). In the initial stage of metastasis, tumor cells breakdown ECM and travel to blood vessels. During this stage, CCL2 guides the migration of cancer cells through linking with CCR2. In addition, CCL2 promotes the migration of cancer cells by inducing the expression of MMP2 as well as MMP9 (<xref rid=\"B239\" ref-type=\"bibr\">Tang and Tsai, 2012</xref>). Then, cancer cells invade into blood vessels for metastatic dissemination, which requires TAMs. CCL2 promotes cancer cell intravasation and extravasation because it is a chemoattractant for TAMs. Interestingly, CCL2-CCR2 signaling stimulates angiogenic switching via recruiting myeloid cells and suppresses immune-mediated killing by recruiting MDSCs (<xref rid=\"B118\" ref-type=\"bibr\">Huang et al., 2007</xref>; <xref rid=\"B153\" ref-type=\"bibr\">Low-Marchelli et al., 2013</xref>).</p><p>CCL2 was highly expressed on paclitaxel-resistant ovarian cancer cells and showed an antitumor effect in ovarian cancer. Moisan et al. reported C1142 (a mouse CCL2 inhibitor) combined with carboplatin in the treatment of ovarian cancer mouse model could improve the efficacy of carboplatin (<xref rid=\"B174\" ref-type=\"bibr\">Moisan et al., 2014</xref>). Additionally, <xref rid=\"B218\" ref-type=\"bibr\">Sandhu et al. (2013)</xref> reported a phase I trial investigating the effect of carlumab in solid tumors. They enrolled forty-four patients in total, including eight ovarian cancer patients. All of them received different doses of carlumab (also called CNTO 888), which is a human anti-CCL2 monoclonal antibody. The results showed that patients with advanced ovarian carcinoma achieved a more than 50% decrease in CA125 and achieved 10.5 months of stabilized disease.</p><p>M-CSF (also called CSF-1) is also a chemokine for macrophages and is secreted by a variety of stromal cells and epithelial cells (<xref rid=\"B262\" ref-type=\"bibr\">Wyckoff et al., 2004</xref>). In addition, its receptor, named M-CSFR, CSF-1R or CD115, is a receptor tyrosine kinase and is restricted to mononuclear phagocytes (<xref rid=\"B28\" ref-type=\"bibr\">Bonelli et al., 2018</xref>). It is known that the binding of CSF-1 and CSF-1R regulates the differentiation, function and survival of macrophages through inducing tyrosine kinase (TK)–mediated autophosphorylation in the cytoplasm and the production of intracellular cascade signals (<xref rid=\"B48\" ref-type=\"bibr\">Chitu and Stanley, 2006</xref>; <xref rid=\"B119\" ref-type=\"bibr\">Hume and MacDonald, 2012</xref>). Hence, the CSF-1/CSF-1R axis can be blocked by anti-CSF antibodies, anti-CSF-1R antibodies and some molecule inhibitors that suppress the function of tyrosine kinases (<xref rid=\"B119\" ref-type=\"bibr\">Hume and MacDonald, 2012</xref>; <xref rid=\"B207\" ref-type=\"bibr\">Ries et al., 2015</xref>).</p><p>Studies implied that CSF-1R inhibitors suppressed the proliferation and metastasis in ovarian cancer via blocking macrophages. <xref rid=\"B178\" ref-type=\"bibr\">Moughon et al. (2015)</xref> reported using CSF-1R inhibitors in advanced ovarian carcinoma. They found that during the late stage of ovarian cancer, mice treated with GW2580, a CSF-1R kinase inhibitor, had markedly reduced ascites volume and infiltration of M2 macrophages. Thus, they thought that CSF-1R inhibitors decreased the ascites volume by blocking macrophages. Subsequently, <xref rid=\"B274\" ref-type=\"bibr\">Yu et al. (2018)</xref> reported the antitumor effect of CSF-1R inhibitors in cisplatin-resistant ovarian cancer. They found that CSF-1R was highly expressed in cisplatin-resistant SKOV3 and CaoV-3 cells (human ovarian cancer cell lines). When cancer cells were treated with pexidartinib, a CSF-1R inhibitor, with or without cisplatin, the combination of pexidartinib and cisplatin significantly inhibited ovarian cancer cell proliferation and induced apoptosis in cancer cells. They also found that the combination of pexidartinib and cisplatin treatment more efficiently suppressed tumor growth compared to using cisplatin alone in mouse models. Thus, they confirmed that CSF-1R inhibitors could inhibit the proliferation of cisplatin-resistant ovarian cancer. Recently, <xref rid=\"B155\" ref-type=\"bibr\">Lu and Meng (2019)</xref> reported the function of BLZ945 in ovarian cancer. They established an ovarian cancer model and treated them with docetaxel with or without BLZ945, which is an inhibitor of the CSF1CSF1R pathway. The data showed that both the docetaxel group and BLZ945 group had decreased tumor growth. Similarly, the docetaxel plus BLZ945 group had a significant decrease in tumor growth compared with that of the docetaxel or BLZ945 alone group. Additionally, docetaxel increased TAM infiltration, while the BLZ945 group showed decreased TAM abundance. They also found that the BLZ945 group had decreased VEGF and MMP9 expression levels, which were closely related to metastasis. Correspondingly, the DTX and BLZ945 combination group had significantly deregulated VEGF and MMP9. Moreover, the docetaxel plus BLZ945 group had less lung metastasis than those observed in the other groups. Therefore, they confirmed that BLZ945 inhibited the proliferation and metastasis of ovarian cancer by suppressing TAMs.</p><p>CCL2, M-CSF, VEGF, CXcl-12, and HIFs are molecules that can improve macrophage recruitment. As mentioned above, targeted related chemical attractants enhance the anti-tumor activity of ovarian cancer. Therefore, studies suggested that inhibition of macrophage recruitment can be used as a complement to treatment strategies.</p></sec><sec id=\"S5.SS3.SSS2\"><title>Inhibiting TAM Survival</title><p>As a part of TAM-targeted strategies for cancer, the inhibition of TAM survival may be realized by attenuated bacteria, immunotoxin-conjugated mAbs and chemical reagents that induce macrophage apoptosis or by activating immune cells such as T lymphocytes to kill TAMs.</p><p>Bisphosphonate is an important drug for depleting macrophages. Kobayashi et al. reported that incubating SKOV3 and OVCAR5 cells (human ovarian cancer cell lines) with alendronate a second-generation bisphosphonate, they found that alendronate exerted concentration-dependent growth inhibition on these cells. Then, they treated the mogp-TAg transgenic mouse with alendronate and discovered a significant decrease in tumor mass in the reproductive tract. Additionally, mice that received alendronate treatment tolerated it well and showed no influence on body weight. Therefore, they suggested that alendronate inhibited the proliferation of ovarian cancer <italic>in vivo</italic> and <italic>in vitro</italic> (<xref rid=\"B138\" ref-type=\"bibr\">Kobayashi et al., 2017</xref>).</p><p>Apart from inducing macrophage apoptosis, TAMs can be suppressed by the activation of an adapted immune response, especially cytotoxic T lymphocytes. However, we did not find studies that applied the inhibition of the adapted immune response to ovarian cancer treatment.</p></sec><sec id=\"S5.SS3.SSS3\"><title>Increasing the Tumouricidal Activity of M1 Macrophages</title><p>In the TME, immunosuppressive M2 macrophages comprise mainly TAMs (<xref rid=\"B205\" ref-type=\"bibr\">Ren et al., 2014</xref>). However, due to the plasticity of polarization, TAMs still maintain the potential to repolarize from tumor-promoting M2 macrophages to tumor-resisting M1 macrophages (<xref rid=\"B22\" ref-type=\"bibr\">Biswas and Mantovani, 2010</xref>). As mentioned before, the polarization of macrophages relies on cytokines. When exposed to IFN-γ, LPS, GM-CSF and IL-12, macrophages mainly polarize to M1 macrophages. In contrast, when exposed to IL-4, IL-10, and IL-13, macrophages polarize to M2 macrophages. Therefore, regulating these cytokines could contribute to TAM-targeted antitumor strategies.</p><p>It was reported that the NF-κB signaling pathway was associated with TAM polarization (<xref rid=\"B104\" ref-type=\"bibr\">Hagemann et al., 2009</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Biswas and Lewis, 2010</xref>; <xref rid=\"B163\" ref-type=\"bibr\">Mancino and Lawrence, 2010</xref>; <xref rid=\"B241\" ref-type=\"bibr\">Tang et al., 2013</xref>).</p><p>The NF-κB family includes five transcription factors: RelA/p65, RelB, c-Rel, NF-κB1 (precursor proteins p50/p105) and NF-κB2 (p100/p52) (<xref rid=\"B104\" ref-type=\"bibr\">Hagemann et al., 2009</xref>). Several agents, such as TLR (Toll-like receptors) agonists, anti-IL-10R mAb and anti-CD40 mAb, can activate NF-κB through classical or non-classical pathways (<xref rid=\"B164\" ref-type=\"bibr\">Mantovani et al., 2004</xref>; <xref rid=\"B230\" ref-type=\"bibr\">Sica and Bronte, 2007</xref>; <xref rid=\"B163\" ref-type=\"bibr\">Mancino and Lawrence, 2010</xref>). In the NF-κB signaling pathway, NF-κB modulates many crucial genes in macrophages and many tumor-promoting genes, such as VEGF, IL-6, TNF-α, and COX2 (<xref rid=\"B104\" ref-type=\"bibr\">Hagemann et al., 2009</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Biswas and Lewis, 2010</xref>). In addition, the inactivation of NF-κB mediates polarization of TAMs to immunosuppressive M2 macrophages, while NF-κB reactivation adjusts TAMs to tumouricidal M1 macrophages (<xref rid=\"B21\" ref-type=\"bibr\">Biswas and Lewis, 2010</xref>). The NF-κB signaling pathway also regulates the development and proliferation of T and B lymphocytes (<xref rid=\"B128\" ref-type=\"bibr\">Jost and Ruland, 2007</xref>), modules angiogenesis and plays a vital role in tumorigenesis (<xref rid=\"B195\" ref-type=\"bibr\">Pramanik et al., 2018</xref>).</p><p>Recently, a TLR-7 agonist, which can activate NF-κB, was applied to cancer treatment. <xref rid=\"B94\" ref-type=\"bibr\">Geller et al. (2010)</xref> reported a clinical trial on the antitumor activity of a TLR-7 agonist in relapsed ovarian, cervix and breast cancer. They enrolled fifteen patients, including ten patients with recurrent ovarian cancer. All of the patients received 852A (a TLR-7 agonist). The results showed that only one patient with stage IIIc serous ovarian cancer had stable disease after treatment with 24 doses of 852A, while she did not continue the trial because of disease progression.</p><p>IL-12 is also a factor that can enhance macrophage polarization to M1 macrophages. IL-12 can also promote Th1 response, which polarizes macrophage to M1 macrophages (<xref rid=\"B20\" ref-type=\"bibr\">Biswas et al., 2012</xref>; <xref rid=\"B154\" ref-type=\"bibr\">Lu, 2017</xref>; <xref rid=\"B225\" ref-type=\"bibr\">Shapouri-Moghaddam et al., 2018</xref>). <xref rid=\"B231\" ref-type=\"bibr\">Silver et al. (1999)</xref> reported that in an animal model of ovarian cancer, mice treated with IL-12 had decreased tumor growth and even tumor regression. Therefore, they suggested that IL-12 had an antitumor effect on ovarian cancer. Subsequently, a phase I trial reported the treatment of relapsed chemotherapy-resistant ovarian cancer with the IL-12 plasmid/lipopolymer complex. The results showed that 31% of the patients treated with IL-12 treatment had stable disease, while 69% of patients had progressive disease. In addition, patients with high IL-12 treatment had longer survival than that of patients with low IL-12 treatment (<xref rid=\"B7\" ref-type=\"bibr\">Anwer et al., 2010</xref>). Similarly, <xref rid=\"B8\" ref-type=\"bibr\">Anwer et al. (2013)</xref> reported that 17% of patients with platinum-sensitive relapsed ovarian cancer treated with IL-12 had completed response, 33% had a partial response, 42% had stable disease, and 8% had progressive disease. Later, <xref rid=\"B6\" ref-type=\"bibr\">Alvarez et al. (2014)</xref> reported that treated platinum-resistant relapsed ovarian cancer patients with EGEN-001 (an IL-12-based immunotherapeutic agent) no patients had complete or partial responses, 35% (7 of 16 patients) had stable disease, and 45% (9 of 16 patients) had progressive disease. Moreover, the median OS and PFS of EGEN-001-treated patients were 9.17 and 2.89 months, respectively. Therefore, they suggested the limited activity of EGEN-001 in platinum-resistant relapsed ovarian cancer.</p><p>Tumor-associated macrophages have the plasticity for polarization, which means that TAMs could repolarization from tumor promoting M2-type to tumor-killing M1-type. Several molecular are reported associated with TAM re-polarization, such as NF-κB and IL-12. Recent years, several researches implied that targeted NF-κB and IL-12 enhance macrophage polarization to M1 macrophages in ovarian cancer. However, the specific signaling pathways are not fully clear. Hence, further studies are needed.</p></sec><sec id=\"S5.SS3.SSS4\"><title>Limiting the Tumor-Promoting Activity of M2 Macrophages</title><p>Apart from the above TAM-targeted antitumor strategies, limiting the tumor-promoting activity of M2 macrophages is also a promising strategy. STAT3, a member of the STATs (Signal Transducers and Activators of Transcription) family, is generally inactivated and located in the cytoplasm (<xref rid=\"B143\" ref-type=\"bibr\">Laudisi et al., 2018</xref>). It can be activated by various receptors, including cytokines such as IL-6 and IL-10, as well as IL-11 and growth factors such as VEGF, EGF and FGF (<xref rid=\"B45\" ref-type=\"bibr\">Chai et al., 2016</xref>; <xref rid=\"B87\" ref-type=\"bibr\">Furtek et al., 2016</xref>). Once binding to their ligand, the conformation of the receptors changes, which promotes signal propagation and leads to the activation of JAKs. Then, activated JAKs, including JAK1 and JAK2, induce the phosphorylation of STAT3 at Tyr705 and lead to the translocation of activated STAT3 dimers to the nucleus, where it binds to DNA and enhances gene transcription (<xref rid=\"B272\" ref-type=\"bibr\">Yu H. et al., 2014</xref>). It has been reported that STAT3 could modulate tumorigenesis by regulating associated gene expression. For instance, STAT3 regulates the expression of c-Myc and cyclin D1, which are linked to the cell cycle (<xref rid=\"B157\" ref-type=\"bibr\">Luwor et al., 2013</xref>). STAT3 also modulates angiogenesis via the gene expression of VEGF and IL-8 and regulates migration by MMP-2 and MMP-9 gene expression (<xref rid=\"B281\" ref-type=\"bibr\">Zhang et al., 2010</xref>). More recently, studies found that STAT3 is associated with the polarization of TAMs (<xref rid=\"B241\" ref-type=\"bibr\">Tang et al., 2013</xref>; <xref rid=\"B259\" ref-type=\"bibr\">Wang et al., 2018</xref>). Additionally, the inhibition of STAT3 decreased TAM polarization to M2 macrophages (<xref rid=\"B86\" ref-type=\"bibr\">Fujiwara et al., 2011</xref>).</p><p>HO-3867 is an inhibitor of STAT3 and has a significant antitumor effect on ovarian cancer (<xref rid=\"B223\" ref-type=\"bibr\">Selvendiran et al., 2010</xref>; <xref rid=\"B246\" ref-type=\"bibr\">Tierney et al., 2012</xref>; <xref rid=\"B202\" ref-type=\"bibr\">Rath et al., 2014</xref>; <xref rid=\"B242\" ref-type=\"bibr\">Tang et al., 2015</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Bixel et al., 2017</xref>; <xref rid=\"B214\" ref-type=\"bibr\">Saini et al., 2017</xref>; <xref rid=\"B271\" ref-type=\"bibr\">Yoshikawa et al., 2018</xref>). <xref rid=\"B223\" ref-type=\"bibr\">Selvendiran et al. (2010)</xref> found that HO-3867 induced the apoptosis of A2780 cells through activating caspase-3 and caspase-8 and promoted G2/M cell-cycle arrest via regulating cell-cycle regulatory molecules such as cyclin, p21, p27, p53 and cdk2. Additionally, they observed a dose-dependent reduction of tumor volume in mice with ovarian cancer. Then, <xref rid=\"B202\" ref-type=\"bibr\">Rath et al. (2014)</xref> reported that HO-3867 reduced tumor growth in a chemotherapy-resistant ovarian cancer model in a dose-dependent manner. Subsequently, <xref rid=\"B214\" ref-type=\"bibr\">Saini et al. (2017)</xref> found that HO-3867 inhibited tumor size and tumor metastasis in ovarian cancer.</p><p>WP1066 is also a STAT3 inhibitor with antitumor activity. <xref rid=\"B242\" ref-type=\"bibr\">Tang et al. (2015)</xref> found that WP1066 markedly suppressed the clonogenicity and invasion activity of SKOV3 and SKOV3/DDP cells (a cisplatin-resistant ovarian cancer cell line). However, it increased the apoptosis of SKOV3 and SKOV3/DDP cells. When treated with WP1066 and cisplatin in combination, the inhibition of proliferation and apoptosis increased compared to that of cisplatin alone. Thus, they suggested that WP1066 inhibited proliferation and metastasis and increased the apoptosis and chemosensitivity of ovarian cancer cells.</p><p>M2 macrophages show tumor-promoting activity. Above studies provide that STAT3 is involved in increased M2 macrophages. The inhibition of STAT3 decreased the proliferation and metastasis of cancer cells.</p><p>In summary, TAMs-targeted treatment strategies include suppressing macrophage recruitment and TAM survival and increasing the transformation of M2 macrophages to M1 macrophages. TAMs participate in tumor progression through fairly complex mechanisms. However, our understanding of TAMs and its interaction with the tumor microenvironment are not deep enough. Therefore, more researches are needed to facilitate the development and clinical application of TAM-targeted antitumor strategies.</p></sec></sec><sec id=\"S5.SS4\"><title>Immune Checkpoint Inhibitors</title><p>Immune checkpoints play important roles in modulating T cell function in the TME (<xref rid=\"B286\" ref-type=\"bibr\">Zhao and Subramanian, 2017</xref>). Immune checkpoint therapy limits inhibitory pathways in T cells, thereby enhancing antitumor immune responses (<xref rid=\"B226\" ref-type=\"bibr\">Sharma and Allison, 2015</xref>). Hence, it changes cancer treatment. Among them, CTLA-4 and PD1/PD-L1 are important immune checkpoints for ovarian cancer (<xref rid=\"B173\" ref-type=\"bibr\">Mittica et al., 2016</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Bose, 2017</xref>) (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>).</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Immune checkpoint inhibitors in ovarian cancer.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Immunotherapy agents</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Target</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Disease status</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>N</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Trial number</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">References</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"7\" rowspan=\"1\"><bold>CTLA-4 inhibitors</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ipilimumab</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">CTLA-4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ovarian cancer</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B116\" ref-type=\"bibr\">Hodi et al., 2003</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Stage IV ovarian cancer</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B115\" ref-type=\"bibr\">Hodi et al., 2008</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recurrent platinum-sensitive ovarian carcinoma</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01611558</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"7\" rowspan=\"1\"><bold>PD-1 inhibitors</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nivolumab</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PD-1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Platinum-resistant ovarian cancer</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">UMIN000005714</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B105\" ref-type=\"bibr\">Hamanishi et al., 2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 1b</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Advanced ovarian cancer</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02054806</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B253\" ref-type=\"bibr\">Varga et al., 2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"7\" rowspan=\"1\"><bold>PD-L1 inhibitors</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BMS-936559</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PD-L1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ovarian cancer</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">200</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT00729664</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B30\" ref-type=\"bibr\">Brahmer et al., 2012</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Avelumab</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phase 1b</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Refractory or relapsed ovarian cancer</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">125</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01772004</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B68\" ref-type=\"bibr\">Disis et al., 2019</xref></td></tr></tbody></table></table-wrap><sec id=\"S5.SS4.SSS1\"><title>Anti-CTLA-4 Antibody</title><p>CTLA-4 is a transmembrane glycoprotein that is mainly expressed on activated T cells, such as regulatory T cells and memory T cells (<xref rid=\"B244\" ref-type=\"bibr\">Tarhini and Iqbal, 2010</xref>). It is widely known that CTLA-4 inhibits the T lymphocyte-mediated antitumor immune response by intrinsic and extrinsic cell pathways (<xref rid=\"B244\" ref-type=\"bibr\">Tarhini and Iqbal, 2010</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Brunner-Weinzierl and Rudd, 2018</xref>). The intrinsic cell pathways include suppressing cytokine receptor signaling and protein translation, activating ubiquitin ligases and recruiting phosphatases (<xref rid=\"B244\" ref-type=\"bibr\">Tarhini and Iqbal, 2010</xref>). In contrast, in the extrinsic cell pathways, CTLA-4 competitively binds to members of the B7 family and transmits inhibitory signals, thereby reducing the activation of T cells (<xref rid=\"B74\" ref-type=\"bibr\">Egen et al., 2002</xref>; <xref rid=\"B285\" ref-type=\"bibr\">Zhao Y. et al., 2018</xref>). In addition, CTLA-4 produces a reversing signal via B7 and induces the secretion of IDO, resulting in the decomposition of tryptophan and inhibition of the proliferation of T cells (<xref rid=\"B24\" ref-type=\"bibr\">Boasso et al., 2005</xref>).</p><p>Anti-CTLA-4 antibodies have immune suppression by limiting CTLA-4 to bind with members of the B7 family, thereby suppressing the recognition of antigen-specific T cells. <xref rid=\"B116\" ref-type=\"bibr\">Hodi et al. (2003)</xref> reported a trial investigating the activity of MDX-CTLA-4 (a CTLA-4 inhibitor, also called ipilimumab) in patients with metastatic ovarian cancer. One patient with ovarian carcinoma had a stable CA-125 level 1 month after antibody injection. Meanwhile, she experienced a reduction in ascites and pain. The other patient had a 43% decrease in CA-125 level in the initial 2 months. Subsequently, <xref rid=\"B115\" ref-type=\"bibr\">Hodi et al. (2008)</xref> treated ovarian cancer patients with ipilimumab. The data showed that one patient had obvious antitumor effects. There is also a phase II clinical trial underway studying the antitumor effect of ipilimumab in patients with relapsed platinum-sensitive ovarian carcinoma.</p></sec><sec id=\"S5.SS4.SSS2\"><title>Anti-PD1/PD-L1 Antibodies</title><p>PD-1 (also called CD279) is a type I transmembrane protein that is expressed on a variety of immune cells, including activated T cells and B cells, NK cells, monocytes and DCs (<xref rid=\"B85\" ref-type=\"bibr\">Frydenlund and Mahalingam, 2017</xref>). PD-L1 and PD-L2 are receptors for PD-1, which all belong to the B7 family (<xref rid=\"B179\" ref-type=\"bibr\">Moy et al., 2017</xref>). Among them, PD-L1 shows a wide range of expression on hematopoietic cells and non-hematopoietic cells, while PD-L2 is only expressed on APCs (antigen presenting cells) (<xref rid=\"B179\" ref-type=\"bibr\">Moy et al., 2017</xref>). PD-L1 and PD-L2 can be induced by extrinsic proinflammatory signals, such as TNF-α, IFN-γ, ILs and GM-CSF (<xref rid=\"B258\" ref-type=\"bibr\">Wang X. et al., 2017</xref>; <xref rid=\"B282\" ref-type=\"bibr\">Zhang et al., 2017</xref>; <xref rid=\"B172\" ref-type=\"bibr\">Mimura et al., 2018</xref>). Apart from this, PD-L1 is also induced by intrinsic signaling pathways, including the PI3K-AKT pathway and the JAK/STAT pathway (<xref rid=\"B125\" ref-type=\"bibr\">Jiang et al., 2019</xref>). It is known that PD-1 has an immune-suppressive ability through binding with PD-1 receptors (<xref rid=\"B33\" ref-type=\"bibr\">Brunner-Weinzierl and Rudd, 2018</xref>). After binding with PD-1 receptors, PD-1 stimulates intracellular signaling pathways and suppresses immune cell activation, which inhibits the production of cytokines and antibodies from immune cells, which in turn exhausts the immune cells and maintains immune system homeostasis (<xref rid=\"B170\" ref-type=\"bibr\">McDermott and Atkins, 2013</xref>). Additionally, PD-1 can not only inhibit T lymphocyte activation and accelerate T lymphocyte apoptosis but can also be regulated by molecules in the TME, such as TGF-β, IL-7, IL-15, IL-21 and IFN-α (<xref rid=\"B135\" ref-type=\"bibr\">Kinter et al., 2008</xref>; <xref rid=\"B245\" ref-type=\"bibr\">Terawaki et al., 2011</xref>; <xref rid=\"B204\" ref-type=\"bibr\">Rekik et al., 2015</xref>; <xref rid=\"B125\" ref-type=\"bibr\">Jiang et al., 2019</xref>). PD-1 inhibitors, including anti-PD-1 antibodies and anti-PD-L1 antibodies, reverse the suppression of antigen-specific T cells through blocking PD-1 or PD-1 ligands (<xref rid=\"B107\" ref-type=\"bibr\">Hamanishi et al., 2016</xref>).</p><p>As precision medicine develops, patient selection tends to be a social economic trend, which also goes for PD-L1/PD-1 blockade therapy. In ovarian cancer, the expression of PD-L1 on DCs and macrophages correlates with clinical efficacy of PD-L1 and PD-1 blockade, suggesting that the APC PD-L1 expression may be a predicting factor of therapeutic benefit and patient selection indicator (<xref rid=\"B150\" ref-type=\"bibr\">Lin et al., 2018</xref>).</p><sec id=\"S5.SS4.SSS2.Px1\"><title>Anti-PD-1 antibodies</title><p>Nivolumab is an anti-PD-1 monoclonal antibody that inhibits the combination of PD-1 ligands. There was a clinical trial exploring the effect of Nivolumab in patients with platinum-resistant ovarian cancer. It divided twenty patients with platinum-resistant ovarian cancer into a high-dose cohort and a low-dose cohort. The results showed that in the low-dose group, one patient had a partial response, and four patients had stable disease. However, in the high-dose group, two patients had a complete response, and two patients had stable disease. Additionally, for all 20 patients, the median PFS time and median overall time were 3.5 and 20.0 months, respectively (<xref rid=\"B105\" ref-type=\"bibr\">Hamanishi et al., 2015</xref>).</p><p>Pembrolizumab is also a humanized anti-PD-1 monoclonal antibody. Andrea and his colleagues reported that treated advanced ovarian cancer patients with pembrolizumab, One patient with complete response, two patients with partial response, seven patients with stable disease and sixteen patients with disease progression. The median OS and PFS were 13.8 and 1.9 months (<xref rid=\"B253\" ref-type=\"bibr\">Varga et al., 2019</xref>), respectively.</p></sec><sec id=\"S5.SS4.SSS2.Px2\"><title>Anti-PD-L1 antibodies</title><p>BMS-936559 is a humanized high affinity anti-PD-L1 monoclonal antibody that blocks PD-L1 binding to PD-1 and CD80. <xref rid=\"B30\" ref-type=\"bibr\">Brahmer et al. (2012)</xref> reported a clinical trial regarding the activity of BMS-936559 in advanced cancer. A total of 200 patients were enrolled, 17 of whom were ovarian cancer patients. The results demonstrated that at a 10 mg/kg dose, 1 ovarian cancer patient achieved a partial response, and 3 ovarian cancer patients achieved stable disease. Avelumab, a human anti-PD-L1 antibody, could specifically bind to PD-L1 and block the links with PD-1 (<xref rid=\"B111\" ref-type=\"bibr\">Heery et al., 2017</xref>). <xref rid=\"B68\" ref-type=\"bibr\">Disis et al. (2019)</xref> described a cohort study on the effect of avelumab in refractory or relapsed ovarian cancer. They enrolled 125 patients and treated them with 10 mg/kg avelumab every 2 weeks. The results demonstrated that 12 patients had an objective response. Among them, 1 patient had a complete response. Additionally, the median PFS and OS were 2.6 and 11.2 months, respectively.</p><p>Immune checkpoints are an important part of maintaining self-tolerance. Immune checkpoint therapy enhancing antitumor immune responses via suppressing inhibitory pathways in T cells. Currently researched suggest that immune checkpoint inhibition is a potential way to activate the immune response. But further exploration is needed to improve the cure rate.</p></sec></sec></sec></sec><sec id=\"S6\"><title>Conclusion</title><p>The current standardized treatment for ovarian cancer is optimal cytoreductive surgery plus platinum-based chemotherapy with the carboplatin–paclitaxel regimen (<xref rid=\"B27\" ref-type=\"bibr\">Bolton et al., 2012</xref>). Nevertheless, due to chemotherapy-resistant and refractory diseases, the sensitivity of chemotherapy decreases, thereby decreasing the long-term survival rate and increasing the recurrence rate (<xref rid=\"B148\" ref-type=\"bibr\">Lim and Ledger, 2016</xref>). Currently, the TME is regarded as a possible therapeutic target for ovarian cancer. In this review, we summarized new targeted therapies at the interface between ovarian cancer and the TME (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>): Several ways are adopted to target ovarian TME. CAFs targeting therapy includes direct deletion FAP+ fibroblasts, reverting the activated CAFs into a quiescent state, and targeting CAF-specific pathways; Anti-angiogenesis is an important TME targeting therapeutic strategy. Since VEGF is the most typical activator of angiogenesis, anti-angiogenesis therapy is divided into three types: anti-VEGF, inhibitors of VEGF receptors and Angs inhibitors. Among them, bevacizumab, the first FDA-approved anti-angiogenesis antibody, plays a crucial role in anti-angiogenesis therapy for ovarian cancer. TAM-targeted antitumor strategies also have drawn much research attention. Currently, the present TAM-targeted therapeutics consist of (i) suppressing macrophage recruitment (such as CCL2 inhibitors and CSF-1R inhibitors); (ii) inhibiting TAM survival (e.g., bisphosphonates); (iii) increasing the tumouricidal activity of M1 macrophages (for example, agonists of the NF-κB signaling pathway such as TLR-7 agonist and other agents such as IL-12); and (iv) limiting the tumor-promoting activity of M2 macrophages (inhibitor of STAT3). Finally, increasing evidence of the therapeutic effect of immune checkpoint inhibitors in ovarian cancer has been reported. Immune checkpoint inhibitors limit inhibitory pathways in T cells, thereby enhancing antitumor immune responses. In particular, CTLA-4 and PD1/PD-L1 are important immune checkpoints for ovarian cancer. We also summarized the ongoing clinical trials targeting ovarian cancer tumor microenvironment (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). Although new therapeutic approaches targeting the TME could not cure ovarian cancer, they showed the potential to control the development of ovarian cancer. Besides, with the development of “-omic” technology, scientists are unveiling a more detailed “territory” of ovarian cancer TME; the data drawn from this area is certain to facilitate novel therapy exploration, with the expectation to bring breakthrough discoveries to this deadly disease. We believe that TEM-targeted strategies should be applied in ovarian cancer as a valuable adjuvant therapy.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Tumor microenvironment related therapeutic strategies in ovarian cancer. The graph shows multiple strategies targeted the tumor microenvironment in ovarian cancer. Among them, several strategies are currently in clinical use, while others are at different phases of clinical development. CAFs: Cancer-associated fibroblasts; TGF-β: Transforming growth factor-β; TAMs: Tumor-associated macrophages; CCL2: chemokine (C-C motif) ligand 2; CSF-1R: colony stimulating factor-1 (CSF1) receptor; VEGF: Vascular endothelial growth factors; VEGFR: Vascular endothelial growth factor receptor.</p></caption><graphic xlink:href=\"fcell-08-00758-g003\"/></fig><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>Ongoing clinical trials targeting ovarian cancer tumor microenvironment (TME).</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Drug</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Combination therapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Simple size</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Status</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Trial number</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Phase</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Target VEGF</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paclitaxel; Topotencan</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">48</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03763123</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paclitaxel; Ricolinostat</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Terminated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02661815</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Carboplatin; Paclitaxel</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01219777</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Niraparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">108</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02354131</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Carboplatin; Paclitaxel; Rucaparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">234</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03462212</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Unknown</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02022917</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02884648</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">36</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT00748657</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Not yet recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03611179</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">64</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT00022659</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gemcitabine; Carboplatin; Cisplatin; Oxaliplatin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Terminated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01936974</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paclitaxel; Cisplatin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT00511992</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Irinotecan</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01091259</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Topotecan</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT00343044</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gemcitabine; Carboplatin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">45</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT00267696</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RAD001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01031381</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tocotrienol</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT04175470</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Erlotinib; Paclitaxel; Carboplatin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT00520013</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paclitaxel; Carboplatin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">62</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT00129727</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Anetumab Ravtansine; paclitaxel</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">96</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03587311</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Niraparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">106</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03326193</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fosbretabulin Tromethamine</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">107</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01305213</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Everolimus</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">150</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Unknown</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT00886691</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paclitaxel; Carboplatin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">190</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT00937560</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Temsirolimus</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">252</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01010126</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cyclophosphamide</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT00856180</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Capecitabine; Carboplatin; Oxaliplatin; Paclitaxel</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01081262</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Carboplatin; Paclitaxel</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03635489</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rucaparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">190</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Not yet recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT04227522</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paclitaxel; Carboplatin; PLD; Gemcitabine</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">406</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Unknown</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01802749</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Carboplatin; Paclitaxel</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1021</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01239732</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paclitaxel; Carboplatin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">400</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Unknown</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01706120</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Target VEGFR</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Apatinib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fluzoparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">98</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03075462</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chiauranib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03166891</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BIBF 1120</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01669798</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JI-101</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01853644</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Regorafenib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">43</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02736305</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tivozanib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01853644</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Apatinib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Albumin-bound paclitaxel</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Not yet recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03942068</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Apatinib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PLD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">150</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT04348032</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cediranib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Olaparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02340611</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pazopanib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paclitaxel</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">118</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02383251</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Regorafenib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tamoxifen</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">68</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02584465</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cediranib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Laparib; Paclitaxel; Pegylated Liposomal Doxorubicin Hydrochloride; Topotecan</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">680</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02502266</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cediranib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Olaparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">618</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03278717</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Target CTLA-4</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tremelimumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Olaparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02571725</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tremelimumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Olaparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">170</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT04034927</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Target PD-1</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ABBV-181</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SC-003</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">74</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Terminated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02539719</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PDR001</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ribociclib; Fulvestrant</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03294694</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nivolumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">COM701</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">140</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03667716</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Modified Vaccinia Virus Ankara Vaccine Expressing p53</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03113487</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AMG386</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03239145</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nivolumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Varlilumab</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">175</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02335918</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Carboplatin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03029598</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PLX3397</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">78</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Terminated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02452424</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Galinpepimut-S</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">90</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03761914</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Niraparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">122</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02657889</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ENB003</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">130</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Not yet recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT04205227</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sintilimab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Manganese Chloride; nab-paclitaxel; Platinum chemotherapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03989336</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TSR042</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Niraparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">150</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03955471</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nivolumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rucaparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03824704</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02644369</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">376</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02674061</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gemcitabine; Cisplatin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02608684</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Carboplatin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Not yet recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT04387227</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Carboplatin; Paclitaxel</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02766582</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DPX-Survivac; Cyclophosphamide</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">42</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03029403</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DPX-Survivac; Cyclophosphamide</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">184</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03836352</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SHR-1210</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Famitinib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">265</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03827837</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nivolumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TSR-042; Chemotherapy Drugs</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">196</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Not yet recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03651206</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nivolumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rucaparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1012</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03522246</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TSR-042</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Niraparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1228</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03602859</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Target PD-L1</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Atezolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Carbplatin, Cyclophophamide</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02914470</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Atezolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RO6870810</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">36</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Terminated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03292172</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Atezolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Carboplatin; Paclitaxel; Niraparib; Gemcitabine; PLD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">414</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03598270</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MEDI4736</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Eribulin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03430518</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MEDI4736</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Focal radiotherapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03283943</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Atezolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DEC-205/NY-ESO-1 Fusion Protein CDX-1401; Guadecitabine; Poly ICLC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">75</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Suspended</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03206047</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Avelumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Entinostat</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">140</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02915523</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MEDI4736</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PLD; Motolimod;</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02431559</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MEDI4736</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ONCOS-102</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">78</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02963831</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TQB2450</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Anlotinib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Not yet recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT04236362</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Atezolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Vigil</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03073525</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Avelumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Terminated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03312114</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MEDI4736</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Azacitidine</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02811497</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MEDI4736</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TPIV200</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02764333</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Atezolizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Carboplatin; Paclitaxel; Niraparib; Gemcitabine; PLD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">414</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03598270</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Avelumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PLD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">566</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02580058</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Target VEGF combined with VEGFR</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BIBF 1120</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02835833</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sorafenib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">55</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT00436215</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Target VEGF combined with PD-1</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Not yet recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03596281</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TSR042; Niraparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03574779</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab; Cyclophosphamide</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02853318</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab; Olaparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">44</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Not yet recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT04361370</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab; Carboplatin; Paclitaxel</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">45</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Not yet recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03275506</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nivolumab; Rucaparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">76</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02873962</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab; Carboplatin; Paclitaxel</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1086</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03740165</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TSR042; Niraparib; Carboplatin; Paclitaxel</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">337</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Not yet recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03806049</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Target VEGF combined with PD-L1</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MEDI4736; Olaparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">427</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02734004</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Atezolizumab; Carboplatin; Paclitaxel</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03394885</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Avelumab; M6620; Carboplatin; Paclitaxel; Gemcitabine; PLD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03704467</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Atezolizumab; Cobimetinib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03363867</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MEDI4736; Olaparib</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">74</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT04015739</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Atezolizumab; Acetylsalicylic acid</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">160</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02659384</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Atezolizumab; Pegylated Liposomal Doxorubicin Hydrochloride</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">488</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Suspended</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02839707</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Avelumab; Chemotherapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">79</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03642132</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Atezolizumab; Platinum-based chemotherapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">614</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02891824</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Atezolizumab; Chemotherapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">664</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03353831</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MEDI4736; Olaparib; Carboplatin; Paclitaxel</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1056</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03737643</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Atezolizumab; Paclitaxel; Carboplatin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1300</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03038100</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Target VEGFR combined with PD-1</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Apatinib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SHR-1210</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Not yet recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT04068974</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lenvatinib</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">180</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03797326</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Target Angs combined with PD-1</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AMG386</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pembrolizumab</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03239145</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Target Angs combined with VEGF</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MEDI3617</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bevacizumab; Paclitaxel; Carboplatin</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">162</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Completed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT01248949</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Target CTLA-4 combined with PD-1</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ipilimumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nivolumab</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">48</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03508570</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ipilimumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nivolumab</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Terminated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03342417</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ipilimumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nivolumab</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">62</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03355976</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tremelimumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nivolumab</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Active, not recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02498600</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Target CTLA-4 combined with PD-L1</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tremelimumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MEDI4736; Chemotherapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">61</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03249142</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tremelimumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Olaparib; MEDI4736</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">36</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT02953457</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tremelimumab</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MEDI4736</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recruiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NCT03026062</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr></tbody></table><table-wrap-foot><attrib><italic>PLD, pegylated liposomal doxorubicin.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S7\"><title>Author Contributions</title><p>YFY and JY drafted the manuscript. YY designed all figures and made substantial revision to the original manuscript. XZ and XW checked and modified the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by the Key Research Projects of Science and Technology Department Foundation of Sichuan Province (2017SZ0002), a Pilot demonstration project for the transfer and transformation of new drug innovation results (2018ZX09201018-013), the Innovative research group projects (81821002), and the National Major Scientific and Technological Special Project for “Significant New Drugs Development” (No. 2018ZX09733001).</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aghajanian</surname><given-names>C.</given-names></name><name><surname>Blank</surname><given-names>S. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Pharmacol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Pharmacol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Pharmacol.</journal-id><journal-title-group><journal-title>Frontiers in Pharmacology</journal-title></journal-title-group><issn pub-type=\"epub\">1663-9812</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32848803</article-id><article-id pub-id-type=\"pmc\">PMC7431691</article-id><article-id pub-id-type=\"doi\">10.3389/fphar.2020.01218</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Pharmacology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>\n<italic>CYP3A5</italic> Gene-Guided Tacrolimus Treatment of Living-Donor Egyptian Kidney Transplanted Patients</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Mendrinou</surname><given-names>Effrosyni</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"author-notes\" rid=\"fn002\">\n<sup>†</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/991048\"/></contrib><contrib contrib-type=\"author\"><name><surname>Mashaly</surname><given-names>Mohamed Elsayed</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><xref ref-type=\"author-notes\" rid=\"fn002\">\n<sup>†</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/1009513\"/></contrib><contrib contrib-type=\"author\"><name><surname>Al Okily</surname><given-names>Amir Mohamed</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mohamed</surname><given-names>Mohamed Elsayed</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Refaie</surname><given-names>Ayman Fathi</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/1043330\"/></contrib><contrib contrib-type=\"author\"><name><surname>Elsawy</surname><given-names>Essam Mahmoud</given-names></name><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Saleh</surname><given-names>Hazem Hamed</given-names></name><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/1042125\"/></contrib><contrib contrib-type=\"author\"><name><surname>Sheashaa</surname><given-names>Hussein</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><xref ref-type=\"author-notes\" rid=\"fn003\">\n<sup>‡</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/62091\"/></contrib><contrib contrib-type=\"author\"><name><surname>Patrinos</surname><given-names>George P.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff5\">\n<sup>5</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff6\">\n<sup>6</sup>\n</xref><xref ref-type=\"author-notes\" rid=\"fn001\">\n<sup>*</sup>\n</xref><xref ref-type=\"author-notes\" rid=\"fn003\">\n<sup>‡</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/43424\"/></contrib></contrib-group><aff id=\"aff1\">\n<sup>1</sup>\n<institution>Department of Pharmacy, School of Health Sciences, University of Patras</institution>, <addr-line>Patras</addr-line>, <country>Greece</country>\n</aff><aff id=\"aff2\">\n<sup>2</sup>\n<institution>The Urology-Nephrology Center, Department of Dialysis and Transplantation, Mansoura University</institution>, <addr-line>Mansoura</addr-line>, <country>Egypt</country>\n</aff><aff id=\"aff3\">\n<sup>3</sup>\n<institution>Department of Nephrology, Zagazig University</institution>, <addr-line>Zagazig</addr-line>, <country>Egypt</country>\n</aff><aff id=\"aff4\">\n<sup>4</sup>\n<institution>Urology and Nephrology Center, Department of Laboratories, Mansoura University</institution>, <addr-line>Mansoura</addr-line>, <country>Egypt</country>\n</aff><aff id=\"aff5\">\n<sup>5</sup>\n<institution>Zayed Center of Health Sciences, United Arab Emirates University</institution>, <addr-line>Al-Ain</addr-line>, <country>United Arab Emirates</country>\n</aff><aff id=\"aff6\">\n<sup>6</sup>\n<institution>Department of Pathology, College of Medicine and Health Sciences, United Arab Emirates University</institution>, <addr-line>Al-Ain</addr-line>, <country>United Arab Emirates</country>\n</aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Luis Abel Quiñones, University of Chile, Chile</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Carla Baan, Erasmus University Rotterdam, Netherlands; Dorothea Lesche, Peter MacCallum Cancer Centre, Australia</p></fn><corresp id=\"fn001\">*Correspondence: George P. Patrinos, <email xlink:href=\"mailto:gpatrinos@upatras.gr\" xlink:type=\"simple\">gpatrinos@upatras.gr</email>\n</corresp><fn fn-type=\"equal\" id=\"fn002\"><p>†These authors share first authorship</p></fn><fn fn-type=\"equal\" id=\"fn003\"><p>‡ These authors share last authorship</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Pharmacogenetics and Pharmacogenomics, a section of the journal Frontiers in Pharmacology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1218</elocation-id><history><date date-type=\"received\"><day>28</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>27</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Mendrinou, Mashaly, Al Okily, Mohamed, Refaie, Elsawy, Saleh, Sheashaa and Patrinos</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Mendrinou, Mashaly, Al Okily, Mohamed, Refaie, Elsawy, Saleh, Sheashaa and Patrinos</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><sec><title>Background</title><p>Tacrolimus is an approved first-line immunosuppressive agent for kidney transplantations. Part of interindividual and interethnic differences in the response of patients to tacrolimus is attributed to polymorphisms at CYP3A5 metabolic enzyme. <italic>CYP3A5</italic> gene expression status is associated with tacrolimus dose requirement in renal transplant recipients.</p></sec><sec><title>Materials and Methods</title><p>In this study, we determined the allelic frequency of <italic>CYP3A5*3</italic> in 76 renal transplanted patients of Egyptian descent. Secondly, we evaluated the influence of the <italic>CYP3A5</italic> gene variant on tacrolimus doses required for these patients as well on dose-adjusted tacrolimus trough-concentrations.</p></sec><sec><title>Results</title><p>The <italic>CYP3A5*3</italic> variant was the most frequent allele detected at 85.53%. Additionally, our results showed that, mean tacrolimus daily requirements for heterozygous patients (<italic>CYP3A5*1/*3</italic>) were significantly higher compared to homozygous patients (<italic>CYP3A5*3/*3</italic>) during the first year after kidney transplantation.</p></sec><sec><title>Conclusion</title><p>This is the first study in Egypt contributing to the individualization of tacrolimus dosing in Egyptian patients, informed by the <italic>CYP3A5</italic> genotype.</p></sec></abstract><kwd-group><kwd>CYP3A5</kwd><kwd>kidney transplantation</kwd><kwd>living donor</kwd><kwd>tacrolimus</kwd><kwd>Egyptian population</kwd><kwd>dose requirements</kwd><kwd>C/D ratio</kwd><kwd>tacrolimus blood levels</kwd></kwd-group><counts><fig-count count=\"4\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"24\"/><page-count count=\"7\"/><word-count count=\"2654\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Chronic Kidney Disease (CKD) is a long-term, progressive, and irreversible condition characterized by functional and structural kidney damages lasting for at least 3 months (<xref rid=\"B12\" ref-type=\"bibr\">Levin et al., 2013</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Webster et al., 2017</xref>). Kidney transplantation is the optimal kidney replacement therapy for patients who have reached end-stage renal disease (ESRD) (<xref rid=\"B19\" ref-type=\"bibr\">Thongprayoon et al., 2020</xref>). Transplant recipients require life-long immunosuppression to prevent allograft rejection. Tacrolimus, a calcineurin inhibitor, is the most frequently used drug in kidney transplantation recipients. The impressive results of tacrolimus treatment, however, are offset by its side effects, narrow therapeutic index and variable and unpredictable pharmacokinetics (<xref rid=\"B17\" ref-type=\"bibr\">Tang et al., 2016</xref>). For this reason, therapeutic drug monitoring (TDM) is crucial in daily practice. Renal transplant recipients usually receive standard weight-based dose which is then adjusted according to TDM to maintain tacrolimus blood concentrations within the therapeutic range. However, using TDM do not guarantee optimal treatment efficacy or lack of rejections and adverse reactions (<xref rid=\"B3\" ref-type=\"bibr\">Birdwell et al., 2015</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Yanik et al., 2019</xref>). Genetic factors are considered to play important role in the interindividual and interethnic variability in pharmacokinetics of tacrolimus (<xref rid=\"B10\" ref-type=\"bibr\">Ghafari et al., 2019</xref>).</p><p>CYP3A5 is an enzyme responsible for the metabolism of tacrolimus. Single nucleotide polymorphisms in <italic>CYP3A5</italic> gene explain 40–50% of the variability in tacrolimus metabolism and clearance (<xref rid=\"B21\" ref-type=\"bibr\">Woillard et al., 2017</xref>). The A to G transition at position 6986 in intron 3 of the <italic>CYP3A5</italic> gene is the most well-studied genomic variant which contributes to dose requirement of tacrolimus (<xref rid=\"B15\" ref-type=\"bibr\">Prasad et al., 2020</xref>). <italic>CYP3A5*3</italic> allele results in alternative spicing of the mRNA which leads to absence of CYP3A5 protein activity and is associated with reduced tacrolimus dose requirement (<xref rid=\"B9\" ref-type=\"bibr\">Ferraris et al., 2011</xref>). The presence of the wild-type allele (<italic>CYP3A5*1</italic>) contributes significantly to the increase of CYP3A activity associated with recovery of renal function after transplantation (<xref rid=\"B16\" ref-type=\"bibr\">Suzuki et al., 2015</xref>). Two more variant alleles, <italic>CYP3A5*6</italic> and <italic>CYP3A5*7</italic>, result also at loss of expression of the functional protein in homozygotes (<xref rid=\"B3\" ref-type=\"bibr\">Birdwell et al., 2015</xref>).</p><p>Several studies in different populations have shown that <italic>CYP3A5</italic> expressors, who carry at least one <italic>CYP3A5*1</italic> allele require 50% (1.5–2-fold) higher tacrolimus doses compared to <italic>CYP3A5</italic> non-expressors those who are homozygous for the variant alleles (<italic>CYP3A5*3</italic>, <italic>CYP3A5*6</italic>, or <italic>CYP3A5*7</italic>) (<xref rid=\"B3\" ref-type=\"bibr\">Birdwell et al., 2015</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Chen and Prasad, 2018</xref>). However, this association between <italic>CYP3A5</italic> genotypes and tacrolimus dose requirement has not yet been studied in Egyptian kidney transplantation recipients.</p><p>In this study, we aimed to determine the allelic frequency of <italic>CYP3A5*3</italic> among Egyptian patients that have undergone transplantation and to evaluate the influence of this polymorphism on tacrolimus daily dose and on metabolism rate in adult patients during the first year after kidney transplantation.</p></sec><sec sec-type=\"materials|methods\" id=\"s2\"><title>Materials and Methods</title><sec id=\"s2_1\"><title>Study Population</title><p>For the present study, 76 unrelated kidney transplanted adult patients were enrolled in Urology and Nephrology Center at Mansoura University Hospital in Egypt. All patients underwent renal transplantation from living donors and were under tacrolimus immunosuppressive treatment for at least one year. Recipients received a standard bodyweight-based tacrolimus initial dose (day -1 before transplantation) of 0.1 mg/kg twice per day. Blood samples were collected into EDTA tubes and stored at -80°C till analyzed. Therapeutic drug monitoring was applied to all samples for dose adjustment. The target whole-blood concentration in early period after transplantation is 10–20 ng/ml and in the maintenance period (after 3 months) 5–10 ng/ml. Tacrolimus daily dose, tacrolimus blood levels, demographic, and clinical data were obtained from medical files of the patients at the beginning of the post-transplant period and at 12 months after transplantation. Patients with diarrhea or vomiting, liver disease, advanced renal dysfunction, or other disorders that could have altered the absorption of tacrolimus or patients that will be co-prescribed drugs that affect the pharmacokinetics of tacrolimus and its pharmacological effect (antifungals, antiepileptics, macrolide antibiotics) were excluded from the study.</p><p>The study was conducted in compliance with the declaration of Helsinki and was approved by the Ethics Committee of the Mansoura University Hospital and written informed consent was obtained from all subjects.</p></sec><sec id=\"s2_2\"><title>DNA Extraction and Genotyping</title><p>Total genomic DNA was extracted from the peripheral blood, followed by determination of its concentration and purity. The <italic>CYP3A5</italic> single nucleotide polymorphism (SNP) – <italic>CYP3A5*3</italic> (rs776746) was genotyped by PCR-restriction fragment length polymorphism (RFLP), using the SspI restriction endonuclease as previously described (<xref rid=\"B13\" ref-type=\"bibr\">Mendrinou et al., 2015</xref>).</p></sec><sec id=\"s2_3\"><title>Statistical Analysis</title><p>Estimation of allele and genotype frequencies was performed using gene counting method and their deviation from Hardy-Weinberg equilibrium was assessed by Pearson’s goodness of fit chi-square test (degree of freedom = 1). Continuous variables are shown as mean and standard deviation and qualitative data are expressed as frequency and percentage.</p><p>Continuous data were tested for normality using Kolmogorov-Smirnov and Shapiro-Wilk tests (p = 0.05) and visualized with Q-Q plots. Depending on the distribution, comparisons for variables between two groups were performed with two-tailed test or Wilcoxon test for related samples and with unpaired t-test or Mann-Whitney test for independent samples. The categorical data were analyzed using two-tailed Fisher’s exact test.</p><p>In the present study patients were divided into two groups according to their genotype [CYP3A5 expressors (*1/*1 or *1/*3) and CYP3A5 non-expressors (*3/*3)]. Both groups were examined for statistically significant difference in dose requirements, tacrolimus blood levels, and C/D ratio (dose corrected trough concentration of Tac). These data were compared at different time points among related samples (patients with the same genotype) and at the same time points among independent samples (patients with different genotype).</p><p>Statistical analysis was performed using SPSS Statistics 25.0 (IBM SPSS software) and GraphPad Prism 8.0. The significance level was set at p<0.05.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec id=\"s3_1\"><title>Demographic Characteristics of the Patients</title><p>A total of 76 kidney transplant recipients were included in this study and they all were adults and self-reported Egyptians. According to the date of the transplantation, there were missing data for 17 of the patients regarding tacrolimus dose. The characteristics of 59 recipients according to their CYP3A5 genotype are shown in <xref rid=\"T1\" ref-type=\"table\">\n<bold>Table 1</bold>\n</xref>. There were no statistically significant differences between the two groups with respect to sex, family history, age of CKD, age at transplantation, time waiting for transplantation, incidence rejection, or donor type.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Comparison of the clinical characteristics, tacrolimus daily dose, tacrolimus blood levels, and C/D ratio of the study population between <italic>CYP3A5</italic> expressors and non-expressors.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristics</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Non-expressors (*3/*3) n = 41</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Expressors (*1/*3, *1/*1) n = 18</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">P value</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gender, n (%)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Male</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35 (85.37%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13 (72.2%)</td><td valign=\"top\" rowspan=\"2\" align=\"center\" colspan=\"1\">0.2841</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6 (14.63%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5 (27.8%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Onset of CKD, years, mean (range) (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.2 (9–55)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.2 (14–65)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.2403</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Onset at transplantation, years, mean (range) (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.2 (10–55)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.8 (14–67)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.2983</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Time waiting for transplant, years, mean (range) (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (0–6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.56 (0–4)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.1965</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Graft rejection, n (%)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Yes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8 (19.5%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6 (33.3%)</td><td valign=\"top\" rowspan=\"2\" align=\"center\" colspan=\"1\">0.3224</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33 (80.5%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12 (66.7%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Family history, n (%)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Yes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3 (7.3%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (11.1%)</td><td valign=\"top\" rowspan=\"2\" align=\"center\" colspan=\"1\">0.6359</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">38 (92.7%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16 (88.9%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Donor type, n (%)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Living Related</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33 (80.5%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14 (77.8%)</td><td valign=\"top\" rowspan=\"2\" align=\"center\" colspan=\"1\">1.0000</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Living unrelated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8 (19.5%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (22.2%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Initial Tac D, mg/day, mean (range) (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.76 (2–11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.86 (6–14)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><0.0001</bold>\n</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1-year Tac D, mg/day, mean (range) (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.21 (1.5–10.5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.81 (2.5–13)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><0.0001</bold>\n</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Initial Tac C, ng/ml, mean (range) (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.09 (2–22.6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.89 (2–13.5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.3035</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1-year Tac C, ng/mL, mean (range) (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.39 (3.3–11.7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.15 (4.9–9.9)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.6373</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Initial C/D ratio, ng/ml per mg/day, mean (range) (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.50 (0.2–9.4)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.64 (0.18–1.5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0586</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1-year C/D ratio, ng/ml per mg/day, mean (range) (SD)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.10 (0.6–5.8)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.10 (0.63–2.84)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.0003</bold>\n</td></tr></tbody></table><table-wrap-foot><p>D, tacrolimus daily dose; C, tacrolimus blood concentration; SD, standard deviation.</p><p>Bolded data are those which are statistically significant. </p></table-wrap-foot></table-wrap></sec><sec id=\"s3_2\"><title>Frequency of the <italic>CYP3A5*3</italic> Variant in Kidney Transplant Recipients</title><p>Of the 76 kidney transplant recipients, the <italic>CYP3A5*3/*3</italic> genotype was observed in 55 (72.37%) cases, <italic>CYP3A5*1/*3</italic> in 20 (26.32%) cases, and <italic>CYP3A5*1/*1</italic> in 1 (1.32%) case. Total allelic frequency was 85.53% for <italic>CYP3A5*3</italic> and 14.47% for <italic>CYP3A5*1</italic> (<xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figure 1</bold>\n</xref>). No deviation from Hardy-Weinberg equilibrium was observed for the genotype frequencies (χ<sup>2</sup> = 0.58323 < 3.841).</p><fig id=\"f1\" position=\"float\"><label>Figure 1</label><caption><p>Genotype and allelic frequencies of 76 renal transplant recipients for <italic>CYP3A5</italic> gene.</p></caption><graphic xlink:href=\"fphar-11-01218-g001\"/></fig></sec><sec id=\"s3_3\"><title>Association of the <italic>CYP3A5</italic> Genotype With Tacrolimus Dose, Tacrolimus Blood Levels, and C/D Ratio</title><p>For the 59 patients, tacrolimus initial doses (mean ± standard deviation) for <italic>CYP3A5*1</italic> carriers and <italic>CYP3A5*3/*3</italic> groups were 9.861 ± 2.182 (range: 6.0–14.0) and 6.756 ± 2.478 mg/day (range: 2.0–11.0), while doses one year after transplantation were 7.806 ± 3.158 (range: 2.5–13.0) and 4.207 ± 2.083 mg/day (range: 1.5–10.5), respectively. This shows a significant reduction of the dosage for both genotypic groups, 20.84% for expressors (<italic>CYP3A5</italic>*1/*3 or *1/*1) (P = 0.0017) and 37.73% for non-expressors (<italic>CYP3A5*3/*3</italic>) (P < 0.0001). Differences between initial and first-year doses are shown in <xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figure 2</bold>\n</xref>.</p><fig id=\"f2\" position=\"float\"><label>Figure 2</label><caption><p>Differences between initial and first-year doses as stratified by <italic>CYP3A5</italic> genotype.</p></caption><graphic xlink:href=\"fphar-11-01218-g002\"/></fig><p>Comparing the starting daily dose between <italic>CYP3A5*3/*3</italic> and <italic>CYP3A5*1</italic> carriers, mean dose for <italic>CYP3A5*1</italic> carriers was significantly higher (45.96%) than for <italic>CYP3A5*3/*3</italic> (P < 0.0001). One-year mean tacrolimus dose for <italic>CYP3A5*1</italic> carriers was 85.55% higher than for <italic>CYP3A5*3/*3</italic> (P < 0.0001) (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figure 3</bold>\n</xref>).</p><fig id=\"f3\" position=\"float\"><label>Figure 3</label><caption><p>Tacrolimus dose for <italic>CYP3A5</italic> genotypes. Each genotype appears as paired blots (first blot for initial dose-second blot for first-year dose). *p < 0.05.</p></caption><graphic xlink:href=\"fphar-11-01218-g003\"/></fig><p>Average tacrolimus blood concentrations in <italic>CYP3A5</italic> non-expressors was higher in both time points compared with <italic>CYP3A5</italic> expressors. However, there was no significant differences between the two groups neither at the early post-transplant period (p = 0.3035) nor at the maintenance period (p = 0.6373).</p><p>\n<italic>CYP3A5*1</italic> recipients exhibited significantly lower C/D ratios (47.89% lower) than those homozygous for the variant allele (*3/*3) at one year of treatment (1.097 ± 0.5829 and 2.105 ± 1.030 ng/ml per mg/day, respectively, p = 0.0003). However, there was no significant difference between the two groups at the early post-transplant period (p = 0.0586). Significant increase was observed at C/D ratios comparing the two time points among <italic>CYP3A5*1</italic> carriers (p = 0.0003) and among <italic>CYP3A5*3/*3</italic> recipients (p = 0.0123) <bold>(</bold>\n<xref ref-type=\"fig\" rid=\"f4\">\n<bold>Figure 4</bold>\n</xref>).</p><fig id=\"f4\" position=\"float\"><label>Figure 4</label><caption><p>Dose-Adjusted Tacrolimus Trough-Concentrations for <italic>CYP3A5</italic> genotypes. Each genotype appears as paired blots (first blot for initial dose-second blot for first-year dose). *p < 0.05.</p></caption><graphic xlink:href=\"fphar-11-01218-g004\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>The biggest challenge for clinicians is the long-term maintenance of renal grafts after a kidney transplantation. Tacrolimus is one of the currently used immunosuppressive therapies, but its administration may be the causative factor of many side effects and graft rejection (<xref rid=\"B18\" ref-type=\"bibr\">Thishya et al., 2018</xref>). In addition to the highly variable oral bioavailability, pharmacokinetics of tacrolimus is characterized by diversity among individuals in the first-pass metabolism and systemic clearance. These differences are largely due to <italic>CYP3A5</italic> polymorphisms and their effect on the metabolism of tacrolimus.</p><p>Pharmacogenomics studies have reported significant association between the <italic>CYP3A5</italic> genotype and the daily doses required for kidney transplant recipients. Most of them noticed that tacrolimus doses were significantly higher in patients carrying *1 allele (<italic>CYP3A5*1/*1</italic> + <italic>CYP3A5*1/*3</italic>) compared to recipients homozygous for *3 allele (<italic>CYP3A5*3/*3</italic>) (<xref rid=\"B17\" ref-type=\"bibr\">Tang et al., 2016</xref>). Our study aimed to analyze the distribution of <italic>CYP3A5</italic> allele frequency in the Egyptian population. In the study population (n = 76) the three genotypic groups, <italic>CYP3A5*1/*1</italic>, <italic>CYP3A5*1/*3</italic>, and <italic>CYP3A5*3/*3</italic> were observed in 1.32, 26.32, and 72.37% respectively. The distribution of <italic>CYP3A5</italic> gene showed that the <italic>CYP3A5*3</italic> allele was 85.53%. In previous studies in the Egyptian population, different frequencies were reported for the <italic>CYP3A5*3</italic> allele, ranging from as low as 11% to as high as 78% (<xref rid=\"B23\" ref-type=\"bibr\">Zayed and Mehaney, 2015</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Abo El Fotoh et al., 2016</xref>; <xref rid=\"B5\" ref-type=\"bibr\">El Wahab et al., 2017</xref>). Studies published in other North African populations (Algerians, Morocco, Tunisians, Libyans) showed that the <italic>CYP3A5*3</italic> allele was the most prevalent with a frequency that reaches even 90% (<xref rid=\"B14\" ref-type=\"bibr\">Novillo et al., 2015</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Fernández-Santander et al., 2016</xref>), whereas in the African population as a whole is observed great diversity from 4 to 95% (<xref rid=\"B24\" ref-type=\"bibr\">Zhou et al., 2017</xref>).</p><p>Several studies have been conducted in North Africans in order to evaluate the effect of <italic>CYP3A5</italic> variants on tacrolimus dosage and on tacrolimus blood concentrations normalized by the dose and proved that there is significant difference between renal transplant patients with the <italic>CYP3A5*1</italic> allele compared to homozygotes for the <italic>CYP3A5*3</italic> allele, especially during the early post-transplant phase (<xref rid=\"B6\" ref-type=\"bibr\">Elmachad et al., 2012</xref>; <xref rid=\"B2\" ref-type=\"bibr\">Aouam et al., 2015</xref>). To our knowledge, this is the first study to examine the association of the <italic>CYP3A5*3</italic> allele with tacrolimus dose requirements and C/D ratios in Egyptian kidney transplant recipients. To date, in the Egyptian population, some studies have been conducted examining the correlation of the <italic>CYP3A5</italic> genotype but in liver transplant patients (<xref rid=\"B7\" ref-type=\"bibr\">Fathy et al., 2016</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Helal et al., 2017</xref>). Our results showed that tacrolimus doses were reduced between the first administration and one year after transplantation, regardless of genotype. Additionally, individuals homozygous for the <italic>CYP3A5*3</italic> allele need significantly lower tacrolimus daily dose than those carrying *1 allele (p < 0.05). Concentration/dose ratio was significantly lower in <italic>CYP3A5*1</italic> expressors. All these indicate that <italic>CYP3A5</italic> expressors require a larger tacrolimus dose in order to maintain the same blood concentration.</p><p>Although there are minor limitations in our study, single center and small cohort, our results showed that frequency of the <italic>CYP3A5*3</italic> variant seems to be higher as compared with previous studies in the Egyptian population and in agreement to that reported prevalence of this allele for other North African or Caucasian populations. Furthermore, comparison of tacrolimus dose requirement for renal transplant patients showed statistically significant difference among genotypes. It is important to draw up different treatment plan for different recipients. As <italic>CYP3A5</italic> shows great heterogeneity in African population, there is a need for pharmacogenomic testing prior to tacrolimus administration after kidney transplantation to achieve genotype-guided dose and contribute to a better-individualized immunosuppressive therapy.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by Mansoura University Hospital. The patients/participants provided their written informed consent to participate in this study.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>EM, MMa, and GP conceded the study. MMa, AA, MMo, AR, EE, HHS, and HS provided samples and clinical data. EM performed the analysis. EM and GP compiled the draft manuscript. GP and HS provided funding. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><title>Acknowledgments</title><p>This project was partly funded by the National Research Infrastructure on Integrated Structural Biology, Drug Screening Efforts and Drug target functional characterization (INSPIRED; Grant number MIS 5002550).</p></ack><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\">\n<person-group person-group-type=\"author\"><name><surname>Abo El Fotoh</surname><given-names>W. M. M.</given-names></name><name><surname>Abd el naby</surname><given-names>S.a. A.</given-names></name><name><surname>Habib</surname><given-names>M. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Oncol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Oncol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Oncol.</journal-id><journal-title-group><journal-title>Frontiers in Oncology</journal-title></journal-title-group><issn pub-type=\"epub\">2234-943X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850465</article-id><article-id pub-id-type=\"pmc\">PMC7431692</article-id><article-id pub-id-type=\"doi\">10.3389/fonc.2020.01611</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Oncology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Computed Tomography Features and Clinicopathological Characteristics of Gastric Sarcomatoid Carcinoma</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Liu</surname><given-names>Yi-yang</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/969836/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Liang</surname><given-names>Pan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Feng</surname><given-names>Kai-xiang</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Kui-sheng</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Yue</surname><given-names>Song-wei</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ji</surname><given-names>Jiang</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Wei-wei</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhao</surname><given-names>Xi-tong</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Gao</surname><given-names>Jian-bo</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Radiology, The First Affiliated Hospital of Zhengzhou University</institution>, <addr-line>Zhengzhou</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Thyroid Surgery, The First Affiliated Hospital of Zhengzhou University</institution>, <addr-line>Zhengzhou</addr-line>, <country>China</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Pathology, The First Affiliated Hospital of Zhengzhou University</institution>, <addr-line>Zhengzhou</addr-line>, <country>China</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Radiology, General Hospital, Ningxia Medical University</institution>, <addr-line>Yinchuan</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Ziwen Liu, Peking Union Medical College Hospital (CAMS), China</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Savio George Barreto, Medanta The Medicity, India; Haruhiko Sugimura, Hamamatsu University School of Medicine, Japan; Xiuying Xiao, Shanghai Jiao Tong University, China</p></fn><corresp id=\"c001\">*Correspondence: Jian-bo Gao, <email>fccyisunshine@gs.zzu.edu.cn</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Gastrointestinal Cancers, a section of the journal Frontiers in Oncology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>1611</elocation-id><history><date date-type=\"received\"><day>06</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>24</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Liu, Liang, Feng, Chen, Yue, Ji, Li, Zhao and Gao.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Liu, Liang, Feng, Chen, Yue, Ji, Li, Zhao and Gao</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><sec><title>Purpose</title><p>Gastric sarcomatoid carcinoma (GSC) is a very rare malignant tumor. The purpose of this study is to describe the clinical, computed tomography (CT), and pathologic features of GSC to increase awareness of this entity.</p></sec><sec><title>Methods</title><p>The CT features and clinical data of five patients with pathologically documented GSC were retrospectively analyzed and compared with the corresponding data of gastric adenocarcinoma and lymphoma.</p></sec><sec><title>Results</title><p>Among the 5 patients, 4 were male, and 1 was female. The median age was 59 years. Of the 5 cases of GSC, 3 were in the gastric fundus and cardia, 1 was in the gastric body, and 1 was in the gastric fundus. The gastric wall had local thickening in 4 cases and mass formation in 1 case, with stenosis and deformation of the adjacent gastric cavity. The long-axis diameter of the lesions ranged from 1.4 to 10.2 cm (mean, 4.97 cm) and was <10 cm in 4 cases and >10 cm in 1 case. The tumor showed predominantly inhomogeneous density, with radiodensity values ranging from 30 to 53 HU. In addition, ulcers with an irregular base and slightly raised borders were observed in 4 of 5 cases. After an injection of contrast material, heterogeneous (<italic>n</italic> = 4) or homogeneous (<italic>n</italic> = 1) enhancement was observed. After contrast medium injection, obvious enhancement was seen in 2 cases, and moderate enhancement was seen in 3 cases; the peak tumor signal was observed in the portal phase. Two of the patients demonstrated evidence of lymph node involvement, and in one patient, the boundary between the lesion and the left lobe of the liver was unclear, with low attenuation in the right lobe of the liver with circular enhancement. The remaining two patients showed no evidence of metastasis.</p></sec><sec><title>Conclusion</title><p>Although GSC is extremely rare, it should be considered in the differential diagnosis of gastric adenocarcinoma and lymphoma. CT findings, combined with patient age and sex, can provide support for the diagnosis of GSC. However, the final diagnosis must be confirmed with histopathology.</p></sec></abstract><kwd-group><kwd>sarcomatoid carcinoma</kwd><kwd>stomach</kwd><kwd>gastric cancer</kwd><kwd>tomography</kwd><kwd>X-ray computed</kwd><kwd>diagnosis</kwd></kwd-group><counts><fig-count count=\"3\"/><table-count count=\"5\"/><equation-count count=\"0\"/><ref-count count=\"26\"/><page-count count=\"9\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Sarcomatoid carcinomas (SCs) are extremely rare aggressive malignant tumors characterized by distinct cellular morphology (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). The features of this tumor were first described in 1982 by Snover et al. (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). SCs can occur in a wide variety of sites, including the respiratory tract, digestive tract, genitourinary tract, breast and thyroid glands (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>). However, these tumors are rare in the digestive tract, especially in the stomach. As of April 2020, there are only six cases of gastric sarcomatoid carcinoma (GSC) reported in the English medical literature. These previous reports focused on the pathological and clinical manifestations; them have not systematically described the radiologic appearance of the tumor. Due to the more invasive nature and poorer prognosis of GSC than pure gastric adenocarcinoma (GAC) and gastric lymphoma (GL), it is clinically beneficial to narrow down the differential diagnoses by understanding the computed tomography (CT) characteristics of GSC. The present study analyzed our experience in diagnosing five patients with GSC in terms of the imaging findings and clinical features. To the best of our knowledge, our study represents the largest series of GSCs to date.</p><p>In addition, due to the rarity of GSC, the differential diagnosis between GSC and other types of malignant gastric tumors has not received much attention, so we also initially explored the differential diagnosis of GSC from GAC and GL.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><p>The protocol was approved by the Medical Ethics Committee of Zhengzhou University. Informed consent was obtained from all patients.</p><sec id=\"S2.SS1\"><title>Patient Selection</title><p>From August 2010 to January 2020, we searched the pathology records and the Picture Archiving and Communication Systems (PACS) of our hospital. The search terms included (stomach) and (sarcomatoid carcinomas). A total of five patients were identified as having SC and were enrolled in the present study. We retrospectively reviewed all clinical data (demographic features, laboratory findings, clinical interventions) and the histologic findings of the five biopsy or operation specimens.</p></sec><sec id=\"S2.SS2\"><title>CT Evaluation</title><p>Five GSC patients underwent CT examinations. The CT scans were acquired with a 64-row multidetector device (DiscoveryCT750HD, GE Healthcare, Waukesha, WI, United States). Conventional axial scanning was performed before and after an intravenous (i.v.) injection of nonionic iohexol (iopromide, 370 mg/mL, GE Medical Systems, 1.5 mL/kg and 3 mL/s) through a dual-head pump injector (Medrad, Warrendale, PA, United States). The imaging parameters were as follows: tube voltage, 120 kV; tube current, 350 mA; field of view (FOV), 500 mm; matrix, 512 × 512 mm; and section thickness, 0.75 mm. Finally, a 20-mL saline flush was performed at a rate of 3 mL/s.</p><p>Contrast-enhanced CT scans were acquired with scanning delays of 30 s (arterial phase, AP) and 70 s (portal venous phase, PP) after the i.v. injection of the contrast agent started. The CT dose index volume for the three phases was 15 mSv.</p></sec><sec id=\"S2.SS3\"><title>Image Analysis</title><p>Two experienced radiologists, 14 and 30 years of abdominal CT experience, performed a retrospective analysis of the CT images. All analyses were performed with an AW4.7 workstation (GE Healthcare), and the radiologists were blinded to the clinical information of the patients. The evaluated parameters included the tumor location (gastric cardia, gastric fundus, gastric body, gastric angle, and gastric antrum), long-axis diameter, shape, growth pattern, serosa condition, attenuation, and enhancement characteristics, such as the enhancement pattern and degree of enhancement. The enhancement pattern of the tumor was classified as homogeneous or heterogeneous based on the AP image. The degree of enhancement of the tumor was based on dynamic CT imaging using HU attenuation, where “obvious enhancement” was defined as >40 HU, “moderate enhancement” as >20 HU and “mildly enhancement” as <20 HU.</p><p>The GSCs were staged with the Union for International Cancer Control (UICC) TNM staging standard. All imaging findings were compared with the postoperative pathological findings. The accuracy rate = the number of CTs coincident with the pathological diagnosis/the number of actual pathological diagnoses × 100%.</p></sec><sec id=\"S2.SS4\"><title>Pathological Evaluation</title><p>Three patients underwent gastrectomy, and two underwent endoscopic biopsy. The three gastrectomy specimens measured 23 cm × 14.5 cm × 1.8 cm, 14.0 cm × 7.5 cm × 1.0 cm, and 23 cm × 8 cm × 4 cm, respectively; in two of these tumors, the mucosal surface of the excised specimen showed ulcerative masses of approximately 7.0 cm × 6.0 cm × 1.0 cm and 5 cm × 3 cm. The remaining specimen was a soft mass measuring 13 cm × 10 cm × 2 cm. For biopsy, multiple samples were acquired, and the diameter of each sample was 0.3 cm. According to the relevant literature, the diagnostic criteria for GSC were generally as follows: (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>) the tumor originated from the stomach; and (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>) the tumor consisted of both carcinomatous and sarcomatoid components, and the sarcomatoid component accounted for more than 50% of the tissue. In addition, if biopsy was performed, the sarcomatoid component can be seen in every sample. Furthermore, sarcomatoid regions express epithelial markers such as CK or EMA.</p><p>The specimens were fully stretched, fixed and soaked in 3.7% formaldehyde solution for 24 h. All biopsy specimens were analyzed. The specimens underwent routine dehydration, paraffin embedding, sectioning into 4 μm thick sections, and hematoxylin eosin (HE) staining. Immunohistochemical staining was performed using a Roche BenchMark XT automatic immunohistochemical detector. The antibodies used in this study included AE1/AE3, CK(L), CK8/18, epithelial membrane antigen (EMA), vimentin, P40, P63, and antigen KI67 (Ki-67). All antibodies listed above were purchased from DAKO (Dako, Glostrup, Denmark).</p></sec><sec id=\"S2.SS5\"><title>Comprehensive Comparative Analysis</title><p>Each patient with GSC was matched by age (±3 years), year of diagnosis, and sex to four patients with GAC, GL; 20 patients with each disease were retrieved from PACS. Patients with GSC were compared with those with GAC, GL in terms of demographic, clinical and CT characteristics (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Comparison between GSC and GAC, GL.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GSC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GAC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">GL</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Age (median age, range)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(59, 53–65 years)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(54, 49–67 years)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(52.5, 48–66 years)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Main symptoms</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Epigastric discomfort/pain</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3 (60%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13 (65%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14 (70%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Intermittent vomiting</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (20%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (10%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (10%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Acute hematemesis/Bloody stool</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (20%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3 (15%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (20%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Dysphagia</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (10%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Location</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Cardia and Fundus</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (80%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8 (40%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (10%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Body</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (20%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6 (30%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10 (50%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Antrum</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (100%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6 (30%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8 (40%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">The long-axis diameter (median size, range)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(4.2, 1.4–10.2 cm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(3.4, 1.3–7.7 cm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(6.6, 1.2–19.2 cm)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Shape</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Focal thickening</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (80%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15 (75%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12 (60%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Diffuse thickening</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (10%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7 (35%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Mass</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (20%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3 (15%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (5%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Serosal surface/bare area</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Clear</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (20%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15 (70%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15 (75%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Unclear</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (80%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5 (30%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5 (25%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ulcers</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Yes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (80%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16 (80%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8 (40%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (20%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (20%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12 (60%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Density characteristics</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Heterogeneous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (80%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (20%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5 (25%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Homogeneous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (20%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16 (80%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15 (75%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Enhancement patter</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Heterogeneous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (80%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12 (60%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13 (65%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Homogeneous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (20%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8 (40%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7 (35%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lymph node involvement*</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Yes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (40%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9 (45%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8 (40%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3 (60%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11 (55%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12 (60%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Liver involvement*</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Yes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (20%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (80%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20 (100%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20 (100%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Therapy</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Resection</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (67%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17 (85%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (10%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Chemotherapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (10%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16 (80%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Resection and Chemotherapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (33%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Neoadjuvant chemotherapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (5%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Radiation therapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (5%)</td></tr></tbody></table></table-wrap></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Patient Characteristics</title><p>The patients included four men and one woman ranging in age from 53 to 65 years, with a median age of 59 years. The clinical and CT features of these patients are summarized in <xref rid=\"T2\" ref-type=\"table\">Tables 2</xref>, <xref rid=\"T3\" ref-type=\"table\">3</xref>. All patients had nonspecific symptoms, including abdominal discomfort, epigastric discomfort, nausea or vomiting. The other presenting symptoms included hematemesis or weight loss. Three patients underwent radical resection, in which only one patient was treated with adjuvant chemotherapy after surgery. And two patients chose to deny treatment. In addition, we also reviewed the upper gastrointestinal radiography results (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Clinical and pathological factors of the five GSC patients.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Case</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Sex</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Age (years)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Complaint</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Location</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maximum diameter</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tumor marker (cm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Anemia</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Therapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Metastasis</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">65</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sudden hematemesis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lesser curvature</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5.0</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Normal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">59</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Epigastric discomfort, Intermittent vomiting</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Remnant stomach (Cardia and Fundus)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">10.2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TAP (+)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Rn</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">62</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Epigastric pain</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cardia and Fundus</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4.2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CA125 (+)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">None</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Epigastric pain</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fundus</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Normal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">None</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">54</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Epigastric pain</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cardia and Fundus</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4.0</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R&C</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr></tbody></table><table-wrap-foot><attrib><italic>“+” yes/present/positive, “–” no/absent/negative; F = female, M = male, age in years; R = Radical gastrectomy; Rn = Remnant gastrectomy; C = chemotherapy; NA = not available.</italic></attrib></table-wrap-foot></table-wrap><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>Computed tomography features of the five GSC patients.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Case</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Gross features of the tumor</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Ulcers</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Growth mode</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Density characteristics</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Enhancement patter</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Margin</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Focal thickening</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Intracavity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Heterogeneous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Heterogeneous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Unclear</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Focal thickening</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Intracavity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Heterogeneous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Heterogeneous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Unclear</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mass</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Intracavity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Homogeneous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Homogeneous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Unclear</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Focal thickening</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Intracavity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Heterogeneous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Heterogeneous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Clear</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Focal thickening</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Intracavity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Heterogeneous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Heterogeneous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Unclear</td></tr></tbody></table><table-wrap-foot><attrib><italic>“+” yes/present/positive, “–” no/absent/negative.</italic></attrib></table-wrap-foot></table-wrap><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Characteristics of X-ray examinations of a 65-year-old male patient with GSC. <bold>(A,B)</bold> Reveals that there is a huge niche with irregular shapes at the small curvature of the stomach; the niche is located inside the outline of the stomach; the niche is surrounded by transparent bands with different widths, that is, ring embankments, with irregular outlines. The surrounding mucosa is thickened, interrupted, and the local gastric cavity is narrowed.</p></caption><graphic xlink:href=\"fonc-10-01611-g001\"/></fig><p>The laboratory findings revealed that patient 2 was positive for tumor abnormal protein (TAP) and patient 3 was positive for carbohydrate antigen 125 (CA125). Before treatment, hemoglobin and erythrocyte count decreased in three patients (patients 1, 2, and 3), and platelet count was elevated in four patients (patients 1, 2, 3 and 4).</p></sec><sec id=\"S3.SS2\"><title>Pathological Features</title><p>Micropathologically, the gastric tumor cells showed infiltrative growth. The cytological characteristics of the tumor cells showed obvious malignant characteristics. Microscopically, the spindle cell structure and the nucleus were obviously atypical, pleomorphic and enlarged. Mitotic figures were visible (<xref ref-type=\"fig\" rid=\"F2\">Figures 2A,B</xref>). On immunohistochemical examination, the tumor cells showed positive staining for AE1/AE3, CK(L), CK8/18, EMA, P40, vimentin. The Ki-67 index was higher than 50% (<xref ref-type=\"fig\" rid=\"F2\">Figures 2C–I</xref>). All five tumors were diagnosed as GSC. In addition, the sarcomatoid component showed spindle cell sarcomatoid morphology.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Histological and immunohistochemical features of GSC. <bold>(A,B</bold>) Hematoxylin-eosin (HE) staining showing tumor cells demonstrated spindle-shaped structures, significant atypical nuclei, pleomorphic nuclei and giant nuclei; Mitotic figures visible. Tumor cells showed infiltrative growth. Cells were stained with hematoxylin and eosin stain (magnification, A × 200; B × 50). By immunohistochemistry, the tumor cells were positive for AE1/AE3 <bold>(C)</bold>, CK(L) <bold>(D)</bold>, CK8/18 <bold>(E)</bold>, EMA <bold>(F)</bold>, P40 <bold>(G)</bold>, and vimentin <bold>(H)</bold>. Moreover, 50% of them were positive for Ki-67 <bold>(I)</bold>. The final diagnosis was SC [magnification <bold>(C–I)</bold> ×200].</p></caption><graphic xlink:href=\"fonc-10-01611-g002\"/></fig></sec><sec id=\"S3.SS3\"><title>CT Findings</title><p>Of the 5 cases of GSC, 3 were in the gastric fundus and cardia (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>), 1 was in the gastric body, and 1 was in the gastric fundus; of these tumors, one was a recurrence in the remnant stomach. The CT manifestations of this tumor included local thickening (<italic>n</italic> = 4), mass formation (<italic>n</italic> = 1). The long-axis diameter of the lesions ranged from 1.4 to 10.2 cm (mean size, 4.97 cm). In addition, ulcers with an irregular base and slightly raised borders were observed in 4 of 5 cases. Among the three patients who underwent surgery, two lesions invaded the gastric serosa, and the remaining lesion invaded the gastric bare area. Among the two patients with biopsy-proven GSC, one patient exhibited tumor invasion of the gastric bare area. The major changes in the CT imaging characteristics were an irregular outer layer of the gastric wall and obscuration of the perigastric fat. Initially, the CT findings were interpreted as GAC in four cases and GL in 1.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Sarcomatoid carcinoma of the stomach in 62-year-old women. <bold>(A)</bold> Unenhanced CT image of stomach reveals an intraluminal mass of homogeneous attenuation, with an irregular surface, at the gastric fundus and cardiac region. <bold>(B–D)</bold> Contrast-enhanced CT image shows obvious homogeneous enhancement of mass, with the peak value of the tumor on the portal phase. In perigastric lymph nodes, an enlarged and significantly enhancement lymph node can be seen. <bold>(B)</bold> Arterial phase of contrast enhancement image. <bold>(C)</bold> Portal phase of contrast enhancement image. <bold>(D)</bold> Portal phase of contrast enhancement coronal image.</p></caption><graphic xlink:href=\"fonc-10-01611-g003\"/></fig><p>The tumor showed predominantly inhomogeneous density, and the radiodensity values were 30–53 HU in the noncontrast phase. Heterogeneous enhancement was seen in four cases due to necrotic or cystic areas, and the other tumor revealed homogeneous enhancement. The radiodensity values on the AP images ranged from 41 to 92 HU and 60 to 96 HU in the venous phase. After contrast medium injection, two tumors showed obvious enhancement, and moderate enhancement was seen in the other three tumors; the peak tumor value was observed in the portal phase. One of the three patients who underwent surgery demonstrated evidence of lymph node involvement; in one patient, the boundary between the lesion and the left lobe of the liver was unclear, and the area with low attenuation was confirmed by pathology as a metastatic lesion in the right lobe of the liver with circular enhancement. The remaining patient showed no evidence of metastasis. Among the two patients with biopsy specimens, one patient was identified as having lymph node metastasis on CT.</p></sec><sec id=\"S3.SS4\"><title>CT Staging Versus Pathological Staging of GSC</title><p>None of the GSCs were staged as T1-T2 by CT or pathology. The accuracy of CT staging T3 and T4 GSC was 100% (1/1) and 100% (2/2), respectively. The overall diagnostic accuracy of CT for determining the T stage of GSC was 100% (3/3).</p><p>None of the GSCs were staged as N2-N3 by CT or pathology. The accuracy of CT in staging N3 and N4 GSC was 50% (1/2) and 0% (0/1), respectively. The overall diagnostic accuracy of CT for determining the N stage of GSC was 33.3% (1/3).</p><p>The comparison of TN staging based on CT and pathology is shown in <xref rid=\"T4\" ref-type=\"table\">Table 4</xref>.</p><table-wrap id=\"T4\" position=\"float\"><label>TABLE 4</label><caption><p>CT and pathological TN staging for comparison.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Case</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">CT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Pathological stage</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NO. 1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">T4aN0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">T4aN1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NO. 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">T3N0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">T3N0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NO. 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">T3N1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NO. 4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">T3N0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NO. 5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">T4aN1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">T4aN0</td></tr></tbody></table><table-wrap-foot><attrib><italic>NA = not available. T = tumor; N = node.</italic></attrib></table-wrap-foot></table-wrap></sec></sec><sec id=\"S4\"><title>Discussion</title><p>Sarcomatoid carcinoma is an extremely rare and complicated malignant tumor composed of malignant epithelial components and atypical spindle cells. However, the spindle cells of SCs appear to show evidence of epithelial differentiation, for example, showing epithelial markers or epithelial ultrastructural characteristics instead of a specific line of mesenchymal differentiation. Moreover, some of the current literature emphasizes that the sarcomatous components occupy >50% of the elements involved (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B4\" ref-type=\"bibr\">4</xref>). In the present study, our patients’ tumor cells displayed atypical spindle shapes that expressed the epithelial phenotype.</p><p>Sarcomatoid carcinomas can occur in almost any organ where carcinoma can occur. In the digestive system, the incidences of SCs in the esophagus and liver are relatively high, but SCs are exceedingly rare in the stomach; we could find only six previous reports in the English literature (<xref rid=\"T5\" ref-type=\"table\">Table 5</xref>) (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Between 08/2011 and 4/2020, 753 patients with SC confirmed by pathology were retrospectively analyzed, with only five tumors occurring in the stomach (0.7%). The average age of the reported patients was 62.3 years (range 49–78) and that in our series was 58.6 years (range 53–65). A previous study reported that the sex distribution of male to female GSC patients was 2:1, and the corresponding proportion in our patients was 4:1 (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B5\" ref-type=\"bibr\">5</xref>–<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). It has been noticed that SCs are more common in male patients, and sex is a probable risk factor. GSC patients may present with epigastric pain or discomfort, dysphagia, nausea and vomiting, hematemesis, and emaciation. Due to thickening of the gastric wall, pain or discomfort in the upper abdomen is common. The symptoms can last from a few days to several years without obvious specificity.</p><table-wrap id=\"T5\" position=\"float\"><label>TABLE 5</label><caption><p>Clinical and imaging features of six previously reported cases of GCS.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Case</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Gender</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Age (years)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Location</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Size (cm)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Shape</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Ulcers</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Enhance appearance</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Recurrence/Metastasis?</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Therapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Prognosis</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. (6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">69</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Remnant stomach</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polypoid</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NE</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">None</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA*</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2. (7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">78</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Greater curvature</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polypoid</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NE</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+/–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surgery</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">45 Mo. D</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3. (7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">F</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">57</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lesser curvature</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polypoid</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NE</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+/–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surgery</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5 Mo. D</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4. (7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">F</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">47</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gastroesophageal junction</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ulcerated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NE</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+/+</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surgery</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8 Mo. D</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5. (5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">74</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Remnant stomach</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polypoid</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NE</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–/–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Endoscopy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7 Mo. D</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">6. (1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">49</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Distal stomach</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mass</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Hyper</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–/+</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surgery</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 Mo. D</td></tr></tbody></table><table-wrap-foot><attrib><italic>*The patient succumbed to heart failure before the surgical treatment. An autopsy was performed. “+” yes/present/positive, “–” no/absent/negative. Hyper: hyperdense. NE: no evaluate. Mo = Month, D = Die.</italic></attrib></table-wrap-foot></table-wrap><p>In the present study, 4 of the 5 cases of GSC were recognized in the proximal stomach, and the remaining tumor was found distal to the stomach. Four cases of GSC in the present study had a long-axis diameter less than 10.0 cm, and the remaining tumor had the largest long-axis diameter among our patients (10.2 cm). The location distribution and long-axis diameters of the GSCs in our patients were similar to those in previous reports (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B5\" ref-type=\"bibr\">5</xref>–<xref rid=\"B7\" ref-type=\"bibr\">7</xref>).</p><p>The diagnosis of SC has always been difficult for clinicians and pathologists, especially the differential diagnosis from carcinosarcoma. Carcinosarcomas are regarded as truly biphasic neoplasms composed of distinct malignant epithelial (carcinomatous) and mesenchymal (sarcomatous) components. The sarcoma components show typical specialized differentiation (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). However, in the actual diagnosis process, the terms “sarcomatoid carcinoma” and “carcinosarcoma” have been used interchangeably in some cases. Therefore, the understanding of these tumors has been hampered. Nevertheless, we can try to focus on whether there is a difference between these tumors from a new perspective. The CT finding SC in the stomach have not been previously scientific reported or even detailed description. There are only four simple descriptions. Chun-Chao et al. (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>) reported that a patient with a giant SC presented a mass with a 14 cm diameter in the antrum and body of stomach, which infiltrated the gastric serosa. The hepatic flexure of the colon and gallbladder were also involved on CT. Contrast-enhanced CT images showed obvious enhancement of the two lesions. Sato et al. (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>) reported a patient with SC of the remnant stomach, and the radiographic examination showed an elevated lesion with a large ulcer at the gastric cardiac lesser curvature that measured 6 cm in diameter. The other two reports only described a soft tissue mass or a large tumor in the dilated stomach (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>). On the other hand, within in the upper gastrointestinal tract, although there are fewer reports of carcinosarcoma localized in the stomach, this type of tumor is still more common than SC (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). Gastric carcinosarcoma showed an elevated lesion or thickened gastric walls in 83%–91% of all reviewed cases (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>–<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Tomoaki et al. reported a 79-year-old man with gastric carcinosarcoma, and his veins showed severe invasion. Enhanced abdominal CT showed irregular thickening and slight enhancement of the gastric wall on the side of the lesser curvature, with suspicious bulky lymph nodes (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Yoshiyuki et al. reported a 70-year-old Japanese woman who presented with a soft tissue mass adjacent to the lesser curvature of the stomach that was lobulated, and CT revealed an ulcer on the lesion. The contrast-enhanced CT images showed heterogeneous enhancement of the mass. The final pathological diagnosis was gastric carcinosarcoma (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>). In the present study, we found that GSC showed local thickening of the gastric wall and mass formation, often accompanied by ulcers. The site of the disease was mostly in the proximal part of the stomach, but these tumors can also occur in the remnant stomach. The signal of the tumor was homogeneous or heterogeneous on plain CT scans. After contrast medium injection, 80% (4/5) of tumors demonstrated heterogeneous enhancement on AP images due to cystic areas or necrosis in the lesions. In this study, the enhancement degree of all tumors reached a peak in the PP after contrast enhancement. For these tumors, the enhancement degree in the delayed phase was not significantly reduced. The overall enhancement mode was delayed enhancement. In addition, CT showed that four patients had invasion into the gastric serosal region or gastric bare area, two patients had the characteristics of enlarged perigastric or retroperitoneal lymph nodes and uneven enhancement, and one patient had invasion into the adjacent liver tissue. These findings reflect the metastatic and highly invasive characteristics of GSC. Overall, CT and contrast-enhanced CT can clearly show the primary lesion, infiltration range, lymph node metastasis and distant metastasis of GSC.</p><p>Tomographic diagnosis of GSC has not been attempted because of the rarity of this entity. According to the findings of our study, GSC needs to be differentiated from GAC and GL on CT. Adenocarcinoma is the most common pathological type of gastric tumor and is mainly distributed in the antrum, seldomly in the body and fundus of the stomach. The incidence of GAC is high in men, and the median patient age is 67 years (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). The most common CT signs of GAC are local or extensive thickening of the gastric wall, mass formation (including fungoides-type, polypoid-type masses), rough or smooth serous surfaces, and continuous interruption of the mucosal layer. Tumors involving the mucosal surface can appear enhanced 30–35 s after injecting a contrast agent. The peak value for tumors invading the muscular layer usually appears after 60–70 s and after the mucosal surface is strengthened, the duration is longer (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Primary GL accounts for 1–5% of malignant gastric tumors and is predominantly situated in the gastric antrum, gastric body and gastric fundus. The incidence of GL is high among males, with a median patient age of 55 years. The clinical symptoms included epigastric pain, bleeding, early satiety, and fatigue (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). The most common CT manifestations of GL are diffuse thickening of the gastric wall or a homogeneous soft tissue mass, with slight attenuation or an appearance similar to that of the normal gastric wall. For GL, because of hemorrhage, necrosis, submucosal edema or infarction, the gastric wall may be heterogenous on CT (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). GL originates from a submucosal process, and gastric mucosa is commonly intact in the early stage but shows interruptions or ulceration in the later stage. After contrast medium injection, most GL showed homogeneous and slight enhancement in the delayed phase (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). Lymphoma is considered when distant structures (the mesentery, retroperitoneum, or other parts of the abdomen) have lymph node metastasis (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>).</p><p>The CT findings may only reflect features of GSC but cannot accurately diagnose GSC, let alone explore the origin of the sarcomatous portion. Immunohistochemistry (IHC) also failed to conclusively establish the origin of GSC. Rodrigues et al. used fluorescence <italic>in situ</italic> hybridization (FISH) to confirm that SC and adenocarcinoma have a common origin, that is, the epithelium (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>), while primary GL originated from gastric submucosal lymphoid tissue.</p><p>The main treatment for localized lymphomas is eradication of Helicobacter pylori and surgical treatment, whereas advanced disease often requires radiation or chemotherapy alone (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Surgery is the only treatment option for patients with GAC. Adjuvant chemotherapy and chemoradiotherapy are also often used. Targeted therapy is in the exploration stage (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). However, there are currently no specific National Comprehensive Cancer Network guidelines for the treatment of only GSC because the tumor is relatively rare, although complete surgical resection is the most important treatment method. For example, while chemotherapy is considered in clinical practice, whether chemotherapy can be applied for GSC and the efficacy of chemotherapy remain controversial (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Domblides et al. first evaluated the efficacy of immune checkpoint inhibitors (ICIs) for SC and found that lung SC patients exhibited high response rates and prolonged overall survival (OS) with ICIs (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). This study provides a new idea for the treatment of GSC.</p><p>Because GL tends to be confined to the gastric wall for prolonged periods before tumor spread, its prognosis is better than that of GAC (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). Previous literature has found that SC in the parotid gland, lung, hypopharynx, liver and pancreas have poor prognoses due to metastasis or recurrence, with a survival period of a few months (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B22\" ref-type=\"bibr\">22</xref>–<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Similarly, GSC patients also died or developed metastasis or recurrence within a few months, or it was already in the advanced stage at the first diagnosis. All these clinical manifestations suggest that GSC has a poorer prognosis than GAC and GL (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). In addition, GSC can metastasize through the blood and lymph nodes, and the most common sites of metastasis are the local lymph nodes and liver (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). This conclusion is consistent with our research results.</p></sec><sec id=\"S5\"><title>Conclusion</title><p>The incidence rate of GSC is extremely low, so clinicians and radiologists are not familiar with the features of this tumor. Based on systematic research of this rare tumor and comparisons with common gastric cancers, we found that GSC is more common in men who are approximately 60 years old and is often accompanied by ulcers. The disease is mostly located in the proximal part of the stomach and can also occur in the remnant stomach, with delayed enhancement on contrast-enhanced CT images. These characteristics can provide a reference for further research on GSCs in the future. However, an accurate diagnosis of GSC depends on the combination of clinical, imaging and histopathological features. Due to the aggressive nature and poor prognosis of the tumor, rapid clinical intervention and detailed follow-up with CT are essential.</p></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.</p></sec><sec id=\"S7\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by the Medical Ethical Committee of the Zhengzhou University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.</p></sec><sec id=\"S8\"><title>Author Contributions</title><p>YL: manuscript preparation, literature research, and data analysis. PL: literature research and data analysis. KF: manuscript review and data collection. KC: guidance of pathological knowledge. SY: guidance of imaging knowledge. JJ: imaging data collection and analysis. WL and XZ: manuscript editing. JG: study conception and design, manuscript review and guarantor of integrity of the entire study. All authors have read and approved the final manuscript.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by the National Natural and Science Fund of China (No. 81671682).</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhu</surname><given-names>CC</given-names></name><name><surname>Li</surname><given-names>MR</given-names></name><name><surname>Lin</surname><given-names>TL</given-names></name><name><surname>Zhao</surname><given-names>G.</given-names></name></person-group>\n<article-title>Sarcomatoid carcinoma of the stomach: a case report and literature review.</article-title>\n<source><italic>Oncol Lett.</italic></source> (<year>2015</year>) <volume>10</volume>:<fpage>1385</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.3892/ol.2015.3460</pub-id>\n<pub-id pub-id-type=\"pmid\">26622678</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Snover</surname><given-names>DC</given-names></name><name><surname>Levine</surname><given-names>GD</given-names></name><name><surname>Rosai</surname><given-names>J.</given-names></name></person-group>\n<article-title>Thymic carcinoma. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Psychol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Psychol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Psychol.</journal-id><journal-title-group><journal-title>Frontiers in Psychology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-1078</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849015</article-id><article-id pub-id-type=\"pmc\">PMC7431693</article-id><article-id pub-id-type=\"doi\">10.3389/fpsyg.2020.01666</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Psychology</subject><subj-group><subject>Hypothesis and Theory</subject></subj-group></subj-group></article-categories><title-group><article-title>Levels and Norm-Development: A Phenomenological Approach to Enactive-Ecological Norms of Action and Perception</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Sepúlveda-Pedro</surname><given-names>Miguel A.</given-names></name><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/797701/overview\"/></contrib></contrib-group><aff><institution>Department of Philosophy, University of Montreal</institution>, <addr-line>Montréal, QC</addr-line>, <country>Canada</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Ezequiel A. Di Paolo, IKERBASQUE Basque Foundation for Science, Spain</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Pierre Steiner, Université de Technologie de Compiègne, France; Michael Wheeler, University of Stirling, United Kingdom</p></fn><corresp id=\"c001\">*Correspondence: Miguel A. Sepúlveda-Pedro, <email>miguel.sepulveda.philo@gmail.com</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Cognitive Science, a section of the journal Frontiers in Psychology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1666</elocation-id><history><date date-type=\"received\"><day>03</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>19</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Sepúlveda-Pedro.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Sepúlveda-Pedro</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>The <italic>enactive approach</italic> and <italic>the skilled intentionality framework</italic> are two closely related forms of radical embodied cognition that nonetheless exhibit important differences. In this paper, I focus on a conceptual disparity regarding the normative character of action and perception. Whereas the skilled intentionality framework describes the norms of action and perception as the capacity of embodied agents to become <italic>attuned</italic> (i.e., skilled intentionality) to preestablished <italic>normative frameworks</italic> (i.e., situated normativity), the enactive approach describes the same phenomenon as the <italic>enactment of norms</italic> (i.e., as sense-making) at different levels of organization that go from individual biological agents to linguistic encounters. I will argue that although both accounts accurately recognize important features of the norms of action and perception, they also have significant shortcomings. Norm-attunement accurately sees normative, ecological frameworks as the necessary set of constraints for the existence of norms at play in sociocultural bodily practices, but it fails to acknowledge the temporal and open-ended character of these norms and frameworks. Norm-enactment, by contrast, acknowledges that norms of action and perception are temporally open-ended, but fails to explicitly recognize that environmental normative frameworks are necessary for the enactment and development of all sort of norms in the interactional domain of an agent-environment system. To overcome these problems, I propose an enactive-ecological approach to norms of action and perception. This approach consists in describing norm-enactment as a result of a developmental process I call norm-development. This process describes the enactment of norms from the background of ecological, normative frameworks. These frameworks are norms enacted in the past of the interactional history of the agent-environment system that remain open to new configurations (new norms) in the present. To clarify conceptually norm-development, I appeal to Merleau-Ponty’s descriptions of <italic>norms of perception</italic>, and more particularly to his concept of <italic>spatial levels</italic>. Like the enactive approach, Merleau-Ponty recognizes that perceptual norms emerge in the interactional history of the agent-environment system, but, like the skilled intentionality framework, he also posits that normative frameworks, that he calls levels, enable and constrain the emergence of perceptual norms and its development. Levels are therefore a phenomenological description of ecological normative frameworks that has been temporally constituted and that stay temporally open-ended as a fundamental requisite for the enactment and development of norms of action and perception.</p></abstract><kwd-group><kwd>enactive approach</kwd><kwd>skilled intentionality framework</kwd><kwd>phenomenology</kwd><kwd>normativity</kwd><kwd>affordances</kwd><kwd>Merleau-Ponty</kwd><kwd>embodiment</kwd><kwd>perception</kwd></kwd-group><counts><fig-count count=\"0\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"82\"/><page-count count=\"15\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>The enactive approach and the ecological approach of the skilled intentionality framework are two radical forms of embodied cognition that reject the orthodox conception of cognition as a computational function that is physically implemented in brain processes (e.g., <xref rid=\"B1\" ref-type=\"bibr\">Anderson, 2007</xref>; <xref rid=\"B53\" ref-type=\"bibr\">Metzinger, 2009</xref>). Instead, both the enactive approach and the skilled intentionality framework conceive cognition as an activity rooted in the dynamic sensorimotor coupling of the body and the environment (<xref rid=\"B65\" ref-type=\"bibr\">Rietveld and Kiverstein, 2014</xref>; <xref rid=\"B78\" ref-type=\"bibr\">Varela et al., 2016</xref>). This coupling permits cognitive agents to establish successful cycles of action and perception in bodily practices (<xref rid=\"B23\" ref-type=\"bibr\">Di Paolo et al., 2017</xref>; <xref rid=\"B64\" ref-type=\"bibr\">Rietveld et al., 2018</xref>), and to lay the foundation for other, more complex forms of cognition (<xref rid=\"B24\" ref-type=\"bibr\">Di Paolo et al., 2018</xref>; <xref rid=\"B44\" ref-type=\"bibr\">Kiverstein and Rietveld, 2018</xref>). Despite these shared convictions, there are important differences between these two approaches that prevent their <italic>prima facie</italic> potential complementarity (<xref rid=\"B14\" ref-type=\"bibr\">Chemero, 2009</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Heras-Escribano, 2016</xref>).</p><p>One of the most important discrepancies between the enactive approach and ecological approaches to cognition is Gibson’s claim affirming that ecological information (environmental structures of sensorimotor correlations) exists as a necessary condition for perception (<xref rid=\"B34\" ref-type=\"bibr\">Gibson, 1979/2015</xref>). For the enactive approach, perception depends on the enactment of a normative domain of sensorimotor interactions between an agent and the environment (<xref rid=\"B71\" ref-type=\"bibr\">Thompson, 2007</xref>). This normative domain is enacted in the concrete history of interactions between each individual agent and the environment, and no pregiven norms are given before this concrete sensorimotor history. The existence of ecological information as a necessary condition for perception is thus rejected by the enactive approach (<xref rid=\"B78\" ref-type=\"bibr\">Varela et al., 2016</xref>).</p><p>In this paper, I focus on a more contemporary difference that nonetheless recalls the earlier one. This new divergence arises when the supporters of both approaches claim that cognition is a phenomenon based on norms. Whereas the skilled intentionality framework describes skillful action as a process of <italic>norm-attunement</italic> (skilled intentionality), the enactive approach describes the same phenomenon as <italic>norm-enactment</italic> (sense-making). Norm-attunement implies the existence of normative frameworks (situated normativity) toward which individual subjects become attuned to once they acquire mastery of a bodily skill. Norm-enactment, by contrast, describes the enactment of norms based on the concrete history of interactions of the agent-environment system, but without explicitly acknowledging that normative, ecological sets of constraints are necessary for this process.</p><p>I will argue that both accounts of norms possess accurate descriptions and explanations of norms of action and perception, but that they also have important shortcomings. Norm-attunement accurately describe the existence of normative, ecological frameworks as the necessary set of constraints for the existence of norms of sociocultural bodily practices, but this description fails to acknowledge the temporally open-ended nature of these norms, and their frameworks. The skilled intentionality framework recognizes that norms change over time due to transformations in the environment and as a result of the purposive activity of agents. However, this approach misses a crucial aspect of all embodied practices: the need for a spontaneous transformation of normative frameworks due to the internal dynamics of the interactional space between and agent (or multiple agents) and the environment. Norm-enactment, by contrast, acknowledges that norms of action and perception are temporally open-ended and, consequently, are open to constant changes in light of the complex dynamics of bodily practices, but the enactive approach fails to explicitly recognize that ecological and normative frameworks are necessary for the enactment and development of all sort of norms in the interactional domain of an agent-environment system.</p><p>I propose therefore an “enactive-ecological approach” to norms of action and perception as a way of overcoming these descriptive shortcomings of the skilled intentionality framework and the enactive approach. My proposal is to refine the account of norm-enactment with what I will call <italic>norm-development</italic>. This descriptive model not only conceives of norm-enactment as a temporally open-ended process, but accords with the ecological, normative frameworks that such a process requires.</p><p>To clarify this idea of norm-development, I propose to go back to the phenomenological work of Merleau-Ponty. He recognizes that perceptual norms emerge in the interactional history of the agent-environment system, but he also posits that normative frameworks, that he calls levels, enable and constrain the emergence of perceptual norms and its development. From a phenomenological perspective, the concept of spatial levels designates the ecological frameworks that has been temporally constituted and that stay temporally open-ended, fulfilling thus the description of norm-enactment as norm-development.</p></sec><sec id=\"S2\"><title>Ecological Norms: Normative Frameworks and Norm-Attunement</title><p>In the context of cognitive science, normativity usually refers to the correctness or incorrectness of actions based on activities such as perceiving, remembering, imaging, reasoning and so on<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup>. The subject of norms and normativity has been one of the main axes of the ecological approach of the skilled intentionality framework because this approach has been always concerned about the way that individual cognitive agents acquire the required skills to participate in sociocultural practices (<xref rid=\"B64\" ref-type=\"bibr\">Rietveld et al., 2018</xref>). In this regard, the skilled intentionality framework has two different but interrelated descriptions of norms and normativity: (1) <italic>situated normativity</italic> (<xref rid=\"B62\" ref-type=\"bibr\">Rietveld, 2008</xref>), and (2) <italic>skilled intentionality</italic> (<xref rid=\"B9\" ref-type=\"bibr\">Bruineberg and Rietveld, 2014</xref>). In what follows, I will unpack these two fundamental concepts.</p><sec id=\"S2.SS1\"><title>Situated Normativity</title><p>The notion of situated normativity is motivated by Wittgenstein’s accounts of how a skillful agent is moved to take a particular set of actions to produce a satisfactory outcome of a sociocultural practice. Skillful agents, like tailors and architects, for instance, feel discomfort and discontent if they find the conditions of their practices unsatisfactory (<xref rid=\"B82\" ref-type=\"bibr\">Wittgenstein, 2007</xref>). If they have enough expertise, they can be moved to take action to improve these conditions (<xref rid=\"B64\" ref-type=\"bibr\">Rietveld et al., 2018</xref>). This can happen without the need for conscious reflection, because it is the feeling of dissatisfaction, and the felt demand or solicitation to take a particular set of actions, that actually describes the lived experience of skillful agents in action (<xref rid=\"B62\" ref-type=\"bibr\">Rietveld, 2008</xref>). This description reveals that skillful agents are already attuned to a normative framework that is not individual or private, but social and public. Sociocultural practices like tailoring and architecture have standards that are explicitly or tacitly accepted by a community to which tailors and architects belong. Thus, feelings of dissatisfaction and solicitations of action are grounded on public standards. This description led scholars in the skilled intentionality framework to adopt the idea that situated normativity does not refer to norms enacted by individuals, but to norms that rule the habitual patterns of bodily practices of a sociocultural group. These patterns were called, after Wittgenstein, a <italic>form of life</italic> (<xref rid=\"B65\" ref-type=\"bibr\">Rietveld and Kiverstein, 2014</xref>; <xref rid=\"B64\" ref-type=\"bibr\">Rietveld et al., 2018</xref>).</p><p>A form of life does not occur in a vacuum though. They are entangled with material structures or physical constraints that help to constitute and shape the norms of practices. For this reason, the skilled intentionality framework designates the environmental conditions of a human practice as a <italic>sociomaterial environment</italic> (<xref rid=\"B75\" ref-type=\"bibr\">van Dijk and Rietveld, 2017</xref>). Hence, there is a sociomaterial entanglement in the form of life of human beings. The paradigmatic case of a form of life is a sociocultural human group, but the notion of a form of life, nonetheless, is not exclusive to human beings. Human and non-human animals’ forms of life inhabit spatial regions that can be described as <italic>ecological niches</italic> (<xref rid=\"B65\" ref-type=\"bibr\">Rietveld and Kiverstein, 2014</xref>). These niches are not simply the raw material composition of a spatial region, rather these niches are best seen as the entanglement of this materiality with a form of life; indeed, an ecological niche refers to the whole set of a <italic>landscape of affordances</italic> for a form of life (<xref rid=\"B7\" ref-type=\"bibr\">Bruineberg, 2018</xref>).</p><p>The notion of affordances was originally defined by Gibson as the possibilities for action that the environment affords to an animal, for good or for ill (<xref rid=\"B34\" ref-type=\"bibr\">Gibson, 1979/2015</xref>). Although affordances are perceived in the environment, they cannot be understood without reference to the animal that perceives them, and for this reason it has been argued that affordances are relational properties of the animal-environment system as a whole (<xref rid=\"B34\" ref-type=\"bibr\">Gibson, 1979/2015</xref>; <xref rid=\"B80\" ref-type=\"bibr\">Warren, 1984</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Heft, 1989</xref>). Chemero has argued, nonetheless, that affordances are more than relational properties: they are relations between the bodily skills of an animal and the relevant “features of the environment” (<xref rid=\"B14\" ref-type=\"bibr\">Chemero, 2009</xref>). This is because affordances are only perceived by animals that possess the required bodily skills to exploit the resources the environment affords, and because affordances do not refer to properties of objects but to the contextual conditions of a situation.</p><p>For the skilled intentionality framework, however, affordances should not be understood as relations between an individual animal and the environment, but between a form of life and the material (and in the case of humans, the sociomaterial) environment (<xref rid=\"B64\" ref-type=\"bibr\">Rietveld et al., 2018</xref>). The skilled intentionality framework distinguishes between two different sets of affordances: the first being a <italic>landscape</italic>, and the second being a <italic>field of affordances</italic> (<xref rid=\"B65\" ref-type=\"bibr\">Rietveld and Kiverstein, 2014</xref>). Whereas a landscape of affordances represents all those affordances available to a form of life, a field of affordances refers to a subset of this landscape, composed of affordances relevant for the task of a skillful agent. Such affordances can be seen as the <italic>solicitations</italic> that move an agent to act (<xref rid=\"B64\" ref-type=\"bibr\">Rietveld et al., 2018</xref>). As a result, an ecological niche entails the whole landscape of affordances of a form of life. In the case of non-human animals, the ecological niche is the relation between the patterns of behavior of a species, and the material conditions of their environment. In the case of humans, the ecological niche is the relation of patterns of behavior of a sociocultural group and the broader sociomaterial environment. In both cases, the normative framework is defined in reference to a group of individuals, and not to individuals as such.</p></sec><sec id=\"S2.SS2\"><title>Skilled Intentionality</title><p>The notion of skilled intentionality is built on two main pillars: Merleau-Ponty’s phenomenology and Friston’s free energy principle (<xref rid=\"B9\" ref-type=\"bibr\">Bruineberg and Rietveld, 2014</xref>). Skilled intentionality relates the process of attunement of a skillful agent to the relevant affordances. Phenomenologically, skilled intentionality can be described as the movement of a body toward an optimal equilibrium of the practical situation, or toward what has been called an optimal grip (<xref rid=\"B9\" ref-type=\"bibr\">Bruineberg and Rietveld, 2014</xref>). This tendency was originally defined by Merleau-Ponty in the following paragraph:</p><p>For each object, just as for each painting in an art gallery, there is an optimal distance from which it asks to be seen – an orientation through which it presents more of itself… The distance between me and the object is not a size that increases or decreases, but rather a tension that oscillates around a norm (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 315–316).</p><p>Following Merleau-Ponty, the skilled intentionality framework sees skillful agents as sensitive to the adequate affordances in a situation, and this sensitivity entails the capacity of agents to take the required action to change the equilibrium of a situation, bringing it closer to its optimal state. This supposes that each practical situation entails a norm or an optimal state that is dependent on the goals of individuals, as well as on conditions in the sociomaterial environment (<xref rid=\"B9\" ref-type=\"bibr\">Bruineberg and Rietveld, 2014</xref>).</p><p>The distinction between a field of affordances and a landscape of affordances is crucial, because whereas the landscape of affordances is defined by situated normativity, the field is better defined by skilled intentionality. The field of affordances, contrary to the landscape, is dynamic and can change at multiple temporal scales. At the behavioral scale, for instance, during the execution of a practice, the actions required to reach the optimal grip change constantly, because of the dynamic change of the practice itself (<xref rid=\"B9\" ref-type=\"bibr\">Bruineberg and Rietveld, 2014</xref>). At the developmental scale, the change of interests of an individual and changes in the material conditions of the environment can alter the relevant affordances (<xref rid=\"B14\" ref-type=\"bibr\">Chemero, 2009</xref>). At the sociohistorical scale, the nature of the practices, for example the customs and traditions, can also change, altering the field of affordances (<xref rid=\"B46\" ref-type=\"bibr\">Malafouris and Renfrew, 2013</xref>). Therefore, we can view skilled intentionality as a more flexible and dynamic description of normativity than that found in situated normativity.</p><p>Nevertheless, for the skilled intentionality framework, skilled intentionality and situated normativity are interrelated and complementary. Situated normativity, and the concept of the landscape of affordances, describes subject-independent aspects of norms of cognition. Skilled intentionality and its field of affordances describes the more contingent and subjective aspects of these norms (<xref rid=\"B64\" ref-type=\"bibr\">Rietveld et al., 2018</xref>). However, the dynamic development of a field of affordances can also alter the conditions of the environment, producing a dialectical movement between the agent and the environment that constantly alters the field of affordances (<xref rid=\"B10\" ref-type=\"bibr\">Bruineberg et al., 2018</xref>). Following theories of self-organization, the skilled intentionality framework shows how the conditions of the environment can constrain the self-organization of a system and reveals how the processes of self-organization can alter environmental conditions. This produces an effect of circular causality, where agents and environments become entangled because they are mutually constrained (<xref rid=\"B9\" ref-type=\"bibr\">Bruineberg and Rietveld, 2014</xref>). The result of this dynamic movement is the constant change of the field of affordances.</p><p>The naturalization of skilled intentionality by proponents of the skilled intentionality framework succeeds thanks to Friston’s account of the free energy principle (<xref rid=\"B28\" ref-type=\"bibr\">Friston, 2010</xref>). This principle offers a statistical and dynamical model for understanding how the brain-body-environment system organizes itself to reduce uncertainty, or what is known as <italic>variational free energy<sup><xref ref-type=\"fn\" rid=\"footnote2\">2</xref></sup></italic>.</p><p>Uncertainty, or variational free energy causes an organizational disequilibrium in the brain-body-environment system that is affectively felt by cognitive subjects as a bodily tension that must be reduced (<xref rid=\"B9\" ref-type=\"bibr\">Bruineberg and Rietveld, 2014</xref>). The organizational composition of the body and the brain allows subjects to modulate their coupling with the environment in order to reduce variational free energy by a process called <italic>active inference</italic>. Active inference can produce changes in the system that reorganizes the brain, body, environment system, thanks to processes of motor action (<xref rid=\"B8\" ref-type=\"bibr\">Bruineberg et al., 2016</xref>).</p><p>The tendency to reduce affective tension explains the movement of the body to reach the optimal grip. This activity directed toward the optimum can either occur by changes in the self-organization of the brain-body system or by changes in the structure of the environment. The optimum is thus a norm that tacitly leads agents’ behavior and their perception of affordances.</p><p>In sum, the skilled intentionality framework holds two different accounts of norms and normativity. On the one hand, situated normativity describes normative frameworks of social and biological groups, while on the other hand, skilled intentionality describes the more concrete attunement of individuals to those normative frameworks. I will call the first phenomenon <italic>normative frameworks</italic> and the second <italic>norm-attunement</italic>.</p></sec></sec><sec id=\"S3\"><title>The Norms of Life and Cognition</title><p>The enactive approach rejects the traditional definition of cognition as information processing and proposes instead an understanding of cognition as a form of <italic>sense-making</italic> (<xref rid=\"B25\" ref-type=\"bibr\">Di Paolo and Thompson, 2014</xref>). Sense-making basically implicates the enactment of normative domains of interaction between an agent and the environment. There are four main forms of sense-making related to four different levels of agency: the vital, the sensorimotor, the intercorporeal, and the linguistic levels, In what follows, I shall unpack the main aspects of the different forms of sense-making and the norms of cognition, according to the tenets of the enactive approach.</p><sec id=\"S3.SS1\"><title>Vital Norms</title><p>For the enactive approach, life and cognition share the same type of formal organization (<xref rid=\"B71\" ref-type=\"bibr\">Thompson, 2007</xref>). Cognition is a more complex form of the basic modes of interaction of living organisms and their environments, and it is for this reason that any account of cognition must be derived from the basic descriptions of life (<xref rid=\"B24\" ref-type=\"bibr\">Di Paolo et al., 2018</xref>).</p><p>In this regard, the enactive approach sees living organisms as autonomous systems in precarious conditions with adaptive behavior (<xref rid=\"B25\" ref-type=\"bibr\">Di Paolo and Thompson, 2014</xref>). They are autonomous systems because organisms are systemic wholes of interrelated processes that have <italic>organizational closure</italic> (<xref rid=\"B76\" ref-type=\"bibr\">Varela, 1979</xref>). This means that living systems are composed of a network of processes that are causally interdependent, allowing living systems to constantly produce and maintain networks of processes (<xref rid=\"B25\" ref-type=\"bibr\">Di Paolo and Thompson, 2014</xref>). As with any other physical system, living systems increase entropy with time (<xref rid=\"B67\" ref-type=\"bibr\">Ruiz-Mirazo and Moreno, 2004</xref>), risking the loss of their autonomous organization, and ultimately, death (<xref rid=\"B81\" ref-type=\"bibr\">Weber and Varela, 2002</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Di Paolo, 2005</xref>). To avoid destruction, organisms need to exchange matter and energy with their surroundings through processes of metabolism. As such, organisms should be understood as thermodynamically open systems that constantly renovate their material components to assure the viability of the system (<xref rid=\"B20\" ref-type=\"bibr\">Di Paolo, 2005</xref>). To accomplish these interactional processes with the environment, organisms must adapt or modulate their behavior according to norms that allow the system to remain viable (<xref rid=\"B5\" ref-type=\"bibr\">Barandiaran and Moreno, 2008</xref>). This means that the environment is primarily disclosed to organisms in light of their own fundamental concerns, which can be understood as moving away from destruction (<xref rid=\"B25\" ref-type=\"bibr\">Di Paolo and Thompson, 2014</xref>). Vital norms are thus norms that allow living organisms to satisfy biological needs and maintain <italic>viable</italic> their autonomous organization.</p><p>It should be noted that even this basic form of sense-making is more affective than purely cognitive, because the way the environment is disclosed by an organism is related to how the environment causes affective bodily states in the organism (<xref rid=\"B15\" ref-type=\"bibr\">Colombetti, 2014</xref>). This is to say that it is the body-environment state of organizational disequilibrium, and not the environment as a neutral landscape, that is felt by the living organism. This basic affectivity of life is akin to the affective state of humans described by <xref rid=\"B17\" ref-type=\"bibr\">Damasio (1999)</xref> as the feeling of being alive, which implies all of the brain activity related to the basic regulatory processes of the body. This feeling of being alive is arguably the basic requirement for any kind of sense-making and cognition (<xref rid=\"B30\" ref-type=\"bibr\">Fuchs, 2018</xref>). Therefore, for the enactive approach, the norms of cognition involve fundamentally affective states, and not merely cognitive states.</p></sec><sec id=\"S3.SS2\"><title>Sensorimotor Norms</title><p>At the sensorimotor level, where properly speaking, cognition appears (<xref rid=\"B3\" ref-type=\"bibr\">Barandiaran, 2017</xref>), a new form of sense-making arises thanks to the self-organization of the brain-body-environment system. This process of self-organization allows living agents to interact with the environment to accomplish practical tasks, from fulfilling biological needs, to tasks unrelated to these basic needs (<xref rid=\"B23\" ref-type=\"bibr\">Di Paolo et al., 2017</xref>).</p><p>Sensorimotor interactions are based on patterns of self-movement correlated to changes in the sensorial field. These correlations are known as <italic>sensorimotor contingencies</italic> (<xref rid=\"B60\" ref-type=\"bibr\">O’Regan and Noë, 2001</xref>). When these sensorimotor contingencies involve the coordination of many parts of the body, including brain activity, they are called <italic>sensorimotor coordination</italic> (<xref rid=\"B11\" ref-type=\"bibr\">Buhrmann et al., 2013</xref>). When the coordination of the brain-body-environment system accomplishes a determinate practical task, implicating a normative outcome, sensorimotor contingencies implicate processes of self-organization called <italic>sensorimotor schemes</italic> (<xref rid=\"B23\" ref-type=\"bibr\">Di Paolo et al., 2017</xref>). These schemes are formed and reinforced by the successful realization of tasks, forming clusters of interdependent schemes that create the bodily or sensorimotor <italic>habits</italic> we observe in our everyday tasks. These tasks are given in specific contexts that solicit the enactment of a whole set of interrelated habits, establishing what the supporters of the enactive approach call a <italic>microworld</italic> (<xref rid=\"B77\" ref-type=\"bibr\">Varela, 1999</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Di Paolo et al., 2017</xref>). Thanks to this self-organization of habits, the living body of cognitive agents acquires a new identity, a “sensorimotor self” that becomes different from the self-identity of life, because this new self is constituted by particular <italic>sensorimotor norms</italic> (<xref rid=\"B23\" ref-type=\"bibr\">Di Paolo et al., 2017</xref>, p. 142).</p><p>Although these sensorimotor norms can be rooted in biological needs, such as when human and non-human animals look for food and shelter, they can be also founded on the incorporation of sociocultural practices, such as cooking a dinner or dancing. Nonetheless, even if sensorimotor norms are originated in social frameworks rather than in the biological activity of the body, such norms need to be incorporated by the living body to enact meaning or relevance for the body’s interactions with the environment (<xref rid=\"B23\" ref-type=\"bibr\">Di Paolo et al., 2017</xref>).</p></sec><sec id=\"S3.SS3\"><title>Intersubjective Norms</title><p>The third relevant form of sense-making and normativity articulated by the enactive approach is the enaction of norms that occasionally emerge from the interaction of two or more autonomous systems, called <italic>participatory sense-making</italic> (<xref rid=\"B18\" ref-type=\"bibr\">De Jaegher and Di Paolo, 2007</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Froese and Di Paolo, 2009</xref>). When two or more autonomous systems interact, they often need to coordinate bodily movements in a way that allows each system to adapt its bodily self-organization for the accomplishment of a common goal. On some occasions, these interactions can produce a pattern of coordination that constitutes an emergent form of self-organization that becomes partially autonomous in relation to the purposes of individual participants.</p><p>The effect of this emergent self-organization of the interactive system forces individual participants to modulate their own sensorimotor norms, producing conflict between two different levels of normativity: the individual, and the collective (<xref rid=\"B18\" ref-type=\"bibr\">De Jaegher and Di Paolo, 2007</xref>). Such readjustment of the norms of individuals, caused by the emergent participatory system, allows these individuals to acquire new forms of sense-making, that is, new normative ways of interacting with the environment. These emergent participatory norms cannot be achieved individually, because it is only in the interaction with another participant that such forms of sense-making can be enacted (<xref rid=\"B49\" ref-type=\"bibr\">McGann and De Jaegher, 2009</xref>). However, they can permanently alter the sensorimotor norms of individuals even if they are not actively engaged in a participatory practice (<xref rid=\"B24\" ref-type=\"bibr\">Di Paolo et al., 2018</xref>). Therefore, for the enactive approach, there are norms of sensorimotor interactions that exceed the autonomy of individual living beings because these norms are enacted in a system of coordination that is composed of more than one individual.</p></sec><sec id=\"S3.SS4\"><title>Linguistic Agency and Social Normativity</title><p>The model of participatory sense-making has now moved one step forward and describes the emergence of a new form of agency that can fully account for the normativity at play in sociocultural bodily practices (<xref rid=\"B24\" ref-type=\"bibr\">Di Paolo et al., 2018</xref>). This is a linguistic agency that emerges thanks to the permanent tension at play in the social interactions between individuals and social norms. This primordial tension can produce metastable processes that can function as instruments for the coregulation and meta-coregulation of social coordination, and eventually, to the use of public utterances that open a linguistic dimension for participants in a community. Although this model is too complex to be fully outlined here, it is enough to bring forth its main features to illustrate how, for the enactive approach, different degrees of social normativity emerge in the dialectics of participatory sense-making.</p><p>The original model of participatory sense-making already exhibits a permanent tension between the individual and the social or interactive levels of normativity. In the updated model of participatory sense making, this tension remains constant through different stages of conflict (dissonance) and harmonization (synergy) between the two levels of normativity. This tension initially forces individuals to adjust their own sensorimotor norms (sensorimotor regulation), but eventually such adjustments must be carried out jointly (sensorimotor coregulation). The sensorimotor coregulation of social interactions eventually produces social acts that serve to make these coregulatory acts more efficient. This is a process of meta-coregulation that will be present all across the following stages of the enactive model.</p><p>The efficiency of social acts of coregulation and meta-coregulation in wider social groups lead agents to the mutual recognition of other participants as agents. This becomes explicit in the emergence of a dialogic interaction, where the roles of an active regulator and a passive regulated member are interchangeable. At this dialogical level, agents use utterances to regulate social interactions, and there is a progressive construction of dialogical networks of utterances that are shared by a community in particular contexts of bodily actions, one that <xref rid=\"B24\" ref-type=\"bibr\">Di Paolo et al. (2018)</xref> call <italic>participation genres</italic>.</p><p>Participation genres bears similarities to the notion of micro-worlds at the sensorimotor level of autonomy, although in the former case, the normative structures of interaction involve not only networks of dynamic sensorimotor processes, but also a network of utterances.</p><p>Although these networks are constantly regulated by processes of mutual interpretation between multiple participants, these regulations may also take the form of self-interpretation. This can occur when a user of utterances becomes aware of an impairment between the pragmatic and expressive aspects of her own utterances (e.g., the utterance does not produce in others the responses she is expecting, according to what she is trying to express). This moment is crucial in the enactive model, because a new level of reflective, dialogical dynamics is incorporated to the intersubjective skills of an agent. The successful utterances become regular patterns of dialogical practices for individual participants (either for their interactions with other agents or for their own interactions with the environment). The norms, co-enacted with others and embodied in networks of utterances (participation genres), now play a more explicit role as tools for self-regulation. Utterances are incorporated by individuals as regulatory tools for their expressive and pragmatic goals, and the new dialogical networks, afford the possibility of making explicit and questioning the already existent normativities at play. This gives to agents the opportunity to dialogically reshape and move forward the already existent norms.</p><p>The enactive approach has been criticized for being incapable of explaining social normativity because its account of vital, sensorimotor, and intercorporeal forms of sense-making refers exclusively to the normative domain of individuals (e.g., <xref rid=\"B37\" ref-type=\"bibr\">Heras-Escribano et al., 2015</xref>). Now, however, this approach offers a theoretical sketch of the emergence of social norms as arising from tensions inherent to the social interactions of autonomous agents. Recognizing situated normativity in this long and complex model of the enactive approach is not easy and requires an analysis that exceeds the scope of this paper. However, it is possible to recognize in this model how individual agents progressively acquire new regulatory processes that emerge from social interactions. It is at the final stages that the actions of agents are more explicitly guided by norms that are social and public, but from the early stages, individual agents are constrained by norms that are jointly enacted by more than one individual.</p><p>We can therefore conclude that the most relevant notion of norms found in the enactive approach can be located in the emergence of an interactional domain between one or many agents and its environment. These sense-making norms continue unfolding in time, according to constraints located in the history of interactions of the agent-environment system (<xref rid=\"B71\" ref-type=\"bibr\">Thompson, 2007</xref>; <xref rid=\"B78\" ref-type=\"bibr\">Varela et al., 2016</xref>). This descriptive account of norms is what I shall call <italic>norm-enactment</italic>.</p></sec></sec><sec id=\"S4\"><title>Enacting Norms or Following Rules?</title><p>In traditional cognitive science, perception consists of simply retrieving information from the environment, thanks to the brain’s capacity to produce internal representations or models from sensorial stimuli. It is assumed in these models that the facts of the world are independent of the subject, and the role of cognitive systems is simply that of accessing this ready-made reality. From this perspective, if cognition and meaning are normative, it is only because the contents of internal representations are more or less accurately correlated to the facts of a ready-made world (e.g., <xref rid=\"B54\" ref-type=\"bibr\">Millikan, 1984</xref>).</p><p>Neither the enactive approach, nor the skilled intentionality framework assumes that cognition consists in the production of internal representations of a ready-made world (<xref rid=\"B23\" ref-type=\"bibr\">Di Paolo et al., 2017</xref>; <xref rid=\"B64\" ref-type=\"bibr\">Rietveld et al., 2018</xref>). Rather, for these approaches, it is the active engagement of embodied agents that makes possible the experience of a meaningful world. Sensorimotor interactions establish the primordial link between agents and the environment, and it is in this domain that fundamental norms of interaction and perceptual meaning emerge. The primordial layer of perceptual meanings is better understood as the opportunities for action that the environment provides for the accomplishment of sensorimotor tasks. However, these perceptual meanings depend on the bodily skills of agents. Although this basic picture works for the enactive approach and for the skilled intentionality framework, there is nevertheless one fundamental divergence in their claims that should call our attention. Whereas the enactive approach describes the constitution of cycles of action and perception in individuals as <italic>the enactment of norms</italic>, the skilled intentionality framework describes them as <italic>the attunement of individuals to pregiven normative frameworks</italic>. In my view, both accounts of norms have relevant shortcomings on this account.</p><p>Norm-attunement sees normative frameworks as pregiven sets of constraints that shape the embodiment of skillful agents, but these constraints are not themselves reshaped by dynamic processes of embodiment. The normative frameworks described by situated normativity are not eternal nor unmovable; they change as a result of transformations in the material structure of the environment and in the body (<xref rid=\"B9\" ref-type=\"bibr\">Bruineberg and Rietveld, 2014</xref>). Ultimately, they can change as a result of the intervention of agents possessing higher forms of cognition (<xref rid=\"B66\" ref-type=\"bibr\">Rietveld et al., 2017</xref>, <xref rid=\"B64\" ref-type=\"bibr\">2018</xref>). Significantly, these descriptions leave out, however, one crucial aspect of all bodily practices (social or not), and it is this: the incessant transformation of norms due to the dynamical interaction of agents and the environment.</p><p>Norm-enactment, by contrast, fails to explicitly acknowledge the central role that ecological (normative) structures play in constraining the interactional domain of an agent(s)-environment system. These ecological structures, we must be clear, are not raw physical structures, but material structures impregnated with meaning. These environmental structures are not only necessary for explaining the enactment of norms, but also for understanding their progressive development.</p><p>Ecological and normative frameworks, however, are temporally open-ended structures that may constantly change due to tensions and ever-present movement within bodily practices. I will describe later how these sorts of temporally open-ended and ecological structures are necessary for the enactment of norms. I will call the descriptive account of the enactment of norms that includes these type of structures <italic>norm-development</italic>.</p><sec id=\"S4.SS1\"><title>Breaking the Rules: Creativity and Improvisation</title><p>As we’ve seen, the norm-attunement element of the skilled intentionality framework offers a well-grounded theory of how, from a third-person perspective, individual agents incorporate the normative standards of a community for the realization of a bodily practice. This approach accurately recognizes that to explain the normative regulation of bodily sociocultural practices, normative frameworks, based on social conventions, are required. Norm-attunement, however, does not acknowledge that the constitution and progressive development of social norms are not extrinsic to the active participation of individuals. The process of incorporation of sensorimotor norms (social or not) actually implies an internal dynamic movement that causes a self-movement, or a natural development of norms. Let me illustrate this phenomenon with the paradigmatic case of jazz improvisation.</p><p>Jazz improvisation requires some normative frameworks that jazz musicians respect, such as harmonic shifts and progressions (<xref rid=\"B79\" ref-type=\"bibr\">Walton et al., 2015</xref>). Jazz standards also provide a framework for improvisation. The personal style of each musician embodies their own normative way of playing jazz (<xref rid=\"B68\" ref-type=\"bibr\">Sawyer, 1992</xref>). Nonetheless, the improvisation – a good one at least – entails a dimension where all these norms are structures allowing agents to engender new ways of expression (<xref rid=\"B55\" ref-type=\"bibr\">Montuori, 2003</xref>), that is, new norms. This is true first of all because jazz improvisation consists in renewing the normative framework of jazz standards according to the current conditions of the environment. Such an environment may include the emotional states of participants, their interactions (<xref rid=\"B45\" ref-type=\"bibr\">Linson and Clarke, 2017</xref>), as well as the public (<xref rid=\"B68\" ref-type=\"bibr\">Sawyer, 1992</xref>; <xref rid=\"B79\" ref-type=\"bibr\">Walton et al., 2015</xref>). However, it is important to see that the success of an improvisation (or acting according to a norm) consists in doing the same thing, but always in a new way (<xref rid=\"B69\" ref-type=\"bibr\">Schiavio and Cummins, 2015</xref>), i.e., in a way that breaks pre-established rules (<xref rid=\"B6\" ref-type=\"bibr\">Barron, 1963</xref>). Successfully establishing a new norm in an improvisation (a new way to do things right) is more often than not a pre-planned action. Rather, success is the result of enacting a new “sense” when agents are immersed in the dynamics of the practice (<xref rid=\"B79\" ref-type=\"bibr\">Walton et al., 2015</xref>). This does not mean that jazz improvisation consists in unreflective action, since reflective and unreflective actions can be at play (<xref rid=\"B68\" ref-type=\"bibr\">Sawyer, 1992</xref>) at the same time. Instead, committing errors or breaking rules in unpredictable ways allows agents to reshape the pre-established normative framework and thereby enact new norms (<xref rid=\"B55\" ref-type=\"bibr\">Montuori, 2003</xref>; <xref rid=\"B79\" ref-type=\"bibr\">Walton et al., 2015</xref>).</p><p>In jam sessions, improvisation already exhibits an open-endedness in its interactional norms, but the dynamic “self-movement” of this practice goes further. In these sessions, musicians constantly and collectively create new structures of sense (new melodic patterns, licks, riffs, etc.) from previously given structures (standards, personal styles, musical rules). Some of these new melodic patterns are successful and may become part of the habitual repertoire of one or more of the practitioners. These new musical patterns are also open-ended structures, because even as repetitive musical phrases, they nonetheless vary all the time. The accumulation of new musical patterns can eventually transform and update the current personal norms of each musician (a personal style), as well as the norms of the particular collectivity (a group’s style). The personal style of a musician, or the collective style of an ensemble can acquire a level of success that influences musicians in a wider community, and create a whole new style (e.g., Miles Davis and the birth of Cool Jazz). Once again, all along this process, reflective and unreflective actions take place, but the transformation of an old social norm (let’s say Bebop) into a new one (e.g., cool jazz) cannot happen if musicians do not break (intentionally and unintentionally) previously given norms.</p><p>Jazz improvisation is a paradigmatic example of interactional dynamics moving forward the development of norms because, for jazz practitioners, spontaneous creativity and novelty is an explicit command for the right accomplishment of this practice. This creativity nonetheless is not an aesthetic luxury for other bodily practices, it is a necessity.</p><p>The use of recurrent and historically acquired patterns needing constant adjustments to deal with new circumstances takes place in all sorts of bodily practices. <xref rid=\"B2\" ref-type=\"bibr\">Baber et al. (2019)</xref> for instance, describe how goldsmiths need to constantly adjust and adapt previously acquired techniques, in light of the type of “responses” the material sends back to their bodily actions. This dynamic process of re-adaptation involves a continual re-interpretation of the space of available affordances, one that keeps changing insofar as the process of making jewelry continues. The expertise of a goldsmith actually consists in being capable to make the proper adjustments of their habitual patterns of interaction to suit contextual demands.</p><p>Likewise, <xref rid=\"B41\" ref-type=\"bibr\">Ingold (2010)</xref> claims that many social practices consist in the constant adaptation of action to the constant flow of both material and forces. For Ingold, the paradigmatic example of bodily practices is textile weaving, whereby weavers use available material to design a unique path of becoming, one that embodies the context of the weavers. Ingold contrast how weavers deal with the dynamical flow of materials with modern architecture. Ingold claims that modern architects are distant from the process of building, and instead of dealing with the flow of materials, they reflectively imagine and plan the form and shape that materials will acquire. This modern practice is in sharp contrast with that followed by medieval architects. The architect responsible for the cathedral of Chartres, for instance, was the master of builders, and, as such, he stayed on site in the building process to deal with the contingencies of his endeavor. In this case, there was actually no plan in advance, and the final outcome was the result of the process of dealing with both the available material, as well as contextual demands (<xref rid=\"B41\" ref-type=\"bibr\">Ingold, 2010</xref>).</p><p>The status of architecture has been a major concern for supporters of the skilled intentionality framework. Contrary to Ingold, I do not think contemporary architecting is a disembodied practice as he describes it. The skilled intentionality framework has convincingly argued that architecture is tightly connected to affective bodily sensitivities (<xref rid=\"B63\" ref-type=\"bibr\">Rietveld and Brouwers, 2017</xref>; <xref rid=\"B66\" ref-type=\"bibr\">Rietveld et al., 2017</xref>). This argument is helpful in closing the gap between basic and complex forms of cognition (<xref rid=\"B64\" ref-type=\"bibr\">Rietveld et al., 2018</xref>). For the practice of architecture, the complex entanglement of different bodily actions involving the use of many cognitive and technological resources needs further analysis, so that we may understand how a contemporary architect deals direct or indirectly with the material flow. Be this as it may, I am convinced we should not describe architecture as a disembodied practice, but rather as a very complex form of embodied and enculturated practice.</p><p>It is important to understand that the dynamic development of norms is not restricted to social practices. Social interactions between agents and technological artifacts such as tools make the internal dynamics of bodily practices more complex, but in the direct relation between a solitary agent and the environment, there is already a need for constant adjustment of sensorimotor norms. I will describe these processes in section “Sensorimotor Development.”</p><p>In sum, whether social or not, bodily practices involve a dynamic encounter between the habitual past and the unexpected demands of the environment in the present. For this reason, norms of action and perception are always subject to a continuous development. How practitioner adapt to the contingencies of the present does not always entail significant change, so we can assume that a static set of norms are at play in many practices over long periods of time. This does not mean that the permanent dynamics of bodily practices are not at work. It is precisely for this reason that norms of action and perception should be seen as temporally open-ended norms, subject to changes in the endogenous interactions of a bodily practice. This is an aspect that the skilled intentionality framework fails to acknowledge in their descriptions of norm-attunement.</p></sec><sec id=\"S4.SS2\"><title>The Self-Movement of Norms</title><p>The enactive approach offers a more accurate description of the temporally open-ended nature of norms than the skilled intentionality framework. Norm-enactment not only acknowledges the constant dynamic adjustment of norms in light of environmental contingencies, it also sees the constitution of social norms as a dynamic process of tensions and coregulations between autonomous agents that involves the constant movement and development of norms. This does not save the enactive approach from an important shortcoming. This is the neglect of the role that ecological structures can play in the constitution and development of norms (cf. <xref rid=\"B48\" ref-type=\"bibr\">McGann, 2014</xref>).</p><p>Sense-making is the enactment of norms at the biological, sensorimotor, and social scales. At the biological level, in the paradigmatic descriptions of vital norms, it is clear that these norms are constrained by the physical conditions of the environment. At the same time, it is not equally clear if they are constrained by precedent normative frameworks as well. In the classical example of the enactive approach, where <italic>E. coli</italic> bacteria respond behaviorally to the presence of glucose, it is argued that bacteria make sense of this chemical compound according to their metabolic needs (<xref rid=\"B71\" ref-type=\"bibr\">Thompson, 2007</xref>, p. 74), thus bacteria make sense of the glucose as food.</p><p>This description suggests that, for the enactive approach, meaningless physical matter acquires meaning thanks to the interests (teleology) of the organism. In this regard, <xref rid=\"B19\" ref-type=\"bibr\">De Jesus (2018</xref>, p. 873) criticizes the enactive approach for what he names an “epistemic perspectivalism” of this approach. He argues that sense-making involves the description of the world “in itself” that appears differently (as meaningful worlds) to subjects with different bodies. Such a picture described by De Jesus, however, presupposes a distance between the environment and the agent that is surmounted epistemologically. This description of sense-making is inaccurate.</p><p>For the enactive approach, the world described by physics and chemistry (e.g., glucose) is not a description of the world-in-itself, or an objective reality independent of any agent as it is for mainstream scientific approaches. Enactivists see the descriptions of science as part of the meaningful world of humans, and as the result of an embodied and enculturated practice. For the supporters of the enactive approach, scientific descriptions are not descriptions of an objective world (<xref rid=\"B72\" ref-type=\"bibr\">Thompson, 2016</xref>). Therefore, the meaning “food” for bacteria, and what a scientist conceives of as “glucose” are not two epistemic perspectives of the same object. Instead, they are two different forms of an agent-environment entanglement.</p><p>We need to be clear that the birth of norms is not the result of putting in connection two alien objects, but a reorganization, a new sense of a pregiven form of an entanglement already at play. When we describe the emergence of a vital norm, like <italic>E. coli</italic> bacteria perceiving glucose as food, we cannot state that an organism projects meaning on a raw physical substance. It is more proper to say that an organism <italic>incorporates</italic> an aspect of the environment into its interactional domain (<italic>Umwelt</italic>). An incorporation, in this case, means a reorganization of the agent-environment system, the acquisition of a new sense or a new norm for the sort of interactions this system maintains. For instance, <xref rid=\"B5\" ref-type=\"bibr\">Barandiaran and Moreno (2008)</xref> posits that normativity, at the level of life, entails two different kind of processes: constructive processes and interactive processes. The first set of processes consists in the network of processes needed to maintain the autonomous organization of a living organism. They are topologically localized into the boundaries of the organizational closure of the system. The second set comprises the processes of interaction between the agent and the environment that are needed to maintain the viability of the system. It is important to note that both set of processes are necessary to preserve the viability of the system; i.e., both constructive and interactional processes are constitutive of the vital norms of a living organism.</p><p>As part of one single system, any change in the norm of interactions means a reconfiguration of the whole system (Gestalt). This is precisely what happens when <italic>E. coli</italic> bacteria find lower levels of glucose and high levels of lactose. The bacteria change its constructive processes (its gene expression) to metabolize lactose instead of glucose, adapting their interactional processes to the current conditions of the environment (<xref rid=\"B5\" ref-type=\"bibr\">Barandiaran and Moreno, 2008</xref>). In this case, the adoption of a new norm consists in a reconfiguration of the whole Gestalt, and not simply on the way the agent makes sense of the environment. Since the acquisition of a new norm implies changes in the body of the living agent, or in its constructive processes, the adaptive behavior of an agent also entails some sort of incorporation.</p><p>It is common to speak about incorporations in the literature of the enactive approach when human agents change their sense-making capacities through the habitual use of tools (<xref rid=\"B21\" ref-type=\"bibr\">Di Paolo, 2009</xref>; <xref rid=\"B73\" ref-type=\"bibr\">Thompson and Stapleton, 2009</xref>). This is called <italic>tool-incorporation</italic> (<xref rid=\"B31\" ref-type=\"bibr\">Fuchs and De Jaegher, 2009</xref>). There is, however, another sort of incorporation that occurs when other living agents transform our sense-making. This is called <italic>mutual incorporation</italic> (<xref rid=\"B31\" ref-type=\"bibr\">Fuchs and De Jaegher, 2009</xref>). There is a third form of incorporation that is not the integration of an environmental aspect into the boundaries of the body, but the incorporation of aspects of the environment into the perceptual field of agents and that are not necessarily affordances. This is something I will call <italic>excorporations</italic>, and I will explain their relevance in the section that follows.</p></sec><sec id=\"S4.SS3\"><title>Excorporations and Norm-Development</title><p>The term excorporation was coined by Merleau-Ponty scholar David Morris to describe, from a phenomenological stance, those aspects of the environment that are vital for the body, but that remain external to the body (<xref rid=\"B56\" ref-type=\"bibr\">Morris, 2004</xref>, p. 131). He describes excorporations as the counterpart of bodily habits topologically situated in the environment. The idea of excorporation is similar to that of affordances in the language of ecological approaches, but different because the term does not refer to specific practical meanings of things, but to anchorage points of the environment that allow agents to become situated in place (to reside or to inhabit it).</p><p>Contrary to incorporations, which are portable aspects of the environment (e.g., the cane of a blind person) excorporations are not portable and remain situated in places (e.g., the door frame of my bedroom). As a result, they appear to be subject independent, but they are not. They are the counterpart of bodily habits, or, better yet, bodily habits are the counterpart of the places a body inhabits. As an example, Morris describes how Earth excorporations are constitutive aspects of the way we inhabit as bodily agents of our planet (<xref rid=\"B56\" ref-type=\"bibr\">Morris, 2004</xref>). A further analysis of excorporations can be revealed by examining the notion of spatial levels in Merleau-Ponty’s phenomenology. I will come back to this subject in the last section. For the time being, we need to understand that the idea of excorporation shows us that the environment is entangled to the body in such a way that we must stop thinking of the agent and the environment as two separated objects that become linked only when a sense-making norm arises. The agent-environment entanglement always precedes the enactment of a norm. This enactment is a reorganization or a reconfiguration of the agent-environment entanglement, and the actualization of a pregiven norm.</p><p>For this reason, the analysis of sense-making should show us that the enactment of a norm is the result of the actualization of the historical past (a pre-given norm) of the agent-environment system in the current flow of the present. In this case, actualization does not mean a mere adaptation or a transformation of the historical past into the present conditions, as when we change our old-fashioned clothes for the latest fashion designs. Actualization means the conflict arisen from the encounter of bodily habits and the unexpected conditions of the present. This encounter produces a disparity or a tension between the past and the present, making the agent-environment entanglement move forward while engendering concrete living acts that constantly reorganizes the agent-environment entanglement (see <xref rid=\"B57\" ref-type=\"bibr\">Morris, 2017</xref> for a phenomenological interpretation of this phenomenon).</p><p>Somehow, every action and perception cycle are the enactment of a new norm, or at least, its actualization (you could not step in the same river twice). But the tendency of agents to reduce the tension created by the disparity between the habitual past and the unexpected present produces a stabilization or a balance that normalizes agent-environment interactions. However, when the disparity creates an important amount of tension, a major reconfiguration of the entanglement is needed, and the enactment of a fully-fledge new norm occurs, which can be understood as a new normalization of the interactional agent-environment domain. Piaget’s theory of equilibration, evoked by the enactive approach to explain the development of sensorimotor norm (<xref rid=\"B22\" ref-type=\"bibr\">Di Paolo et al., 2014</xref>, <xref rid=\"B23\" ref-type=\"bibr\">2017</xref>), resonates with this conception of norm-development.</p></sec><sec id=\"S4.SS4\"><title>Sensorimotor Development</title><p>For the enactive approach to sensorimotor norms, <italic>Piaget’s theory of equilibration</italic> illustrates the adaptation and transformation of sensorimotor schemes (see section “Skilled Intentionality”) for generating new ways for these schemes to function, when agents find new challenges in the environment (<xref rid=\"B23\" ref-type=\"bibr\">Di Paolo et al., 2017</xref>). Two processes are crucial here: <italic>assimilation</italic> and <italic>accommodation</italic>.</p><p>Assimilation refers to the integration of an environmental aspect into the physiological or cognitive/behavioral structure of the agent (<xref rid=\"B23\" ref-type=\"bibr\">Di Paolo et al., 2017</xref>), and, in the words of scholars of the enactive approach, this is “one way of saying that the agent and environmental sides of a sensorimotor scheme are in agreement according to the relevant norm” (<xref rid=\"B23\" ref-type=\"bibr\">Di Paolo et al., 2017</xref>, p. 84). This resonates with what I have called an incorporation of the agent-environment entanglement.</p><p>Accommodation, on the other hand, describes the processes by means of which an agent modulates its physiological and/or behavioral structures to facilitate the assimilation of an aspect of the environment that is not yet assimilated. Equilibration, thereby, is the process by which a sensorimotor organization reaches a new stability, reducing the tension and the disparity caused by the encounter of the novel. The result is thus a dialectical process that transforms the past into a new present, reducing the tension between the two, and engendering a new norm. This is what I mean by norm-development.</p><p>The only aspect that needs to be reconsidered in this theory is the idea that the enactment of a new norm involves a modulation or an adaptation of the body of an agent and its pattern of behavior, without considering that changes in excorporations also occur. These changes transform the sense of a situation and, consequently, change the specific meaningful aspects of the environment. For instance, learning to swim can be understood as the acquisition of a new skill that comprises multiple sensorimotor schemes (e.g., kicking, stroking, and breathing). In this example, the water of the pool excorporates a sort of place where I can find affordances for floating, diving, toppling, etc. This new agent-place entanglement becomes pregnant with a new realm of possibilities for learning different swimming styles, explorations, dancing, etc. Before the basic swimming-norm was acquired, the water of the pool was not a place of residence, nor it was imbued with a rich landscape of affordances and solicitations. Instead, the pool was a place where doing things in-the-water were senseless.</p><p>Norm-development is thus the result of enacting norms, and not a process of following static rules. For this reason, the notion of sense-making is more adequate for understanding the norms of perception than notions of situated normativity and skilled intentionality<sup><xref ref-type=\"fn\" rid=\"footnote3\">3</xref></sup>. The descriptions of the skilled intentionality framework and ecological approaches are nonetheless quite useful for explaining the nature of what I’ve been calling excorporations. A field and a landscape of affordances are useful concepts for understanding the counterpart of bodily habits which has been the focus of the enactive approach. Nonetheless, a truly enactive interpretation of these concepts is needed. As a first step to understanding the ecological realm from an enactive perceptive, I will describe the account of norms and spatial levels found in Merleau-Ponty’s phenomenology. This should help us to understand the logic of norm-development from a full-fledged enactive-ecological approach.</p></sec></sec><sec id=\"S5\"><title>Norm-Development and the Dialectic Movement of Levels</title><p>In this last section, I specify the characteristics of norm-development, in light of the normative account of perception described by Merleau-Ponty (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, <xref rid=\"B52\" ref-type=\"bibr\">2013</xref>), with a special focus on the notions of <italic>spatial levels</italic> and <italic>levels shift</italic> (<xref rid=\"B70\" ref-type=\"bibr\">Talero, 2005</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Marratto, 2012</xref>; <xref rid=\"B57\" ref-type=\"bibr\">Morris, 2017</xref>, <xref rid=\"B58\" ref-type=\"bibr\">2018</xref>). In the first section, I will introduce the context of perceptual norms from the standpoint of phenomenology. In the second section, I will refer explicitly to the notion of levels and, in the last one, how levels shift involves a phenomenological description of norm-development.</p><sec id=\"S5.SS1\"><title>Horizons and Virtual Fields</title><p>Phenomenology comes with its own conception of norms that must be clarified before we put forward a phenomenological account of norms of perception that can productively dialogue with the enactive approach and the skilled intentionality framework. I will start by sketching out the normative character of experience in the context of phenomenology.</p><p>Phenomenology describes and analyzes subjective experience, but phenomenology is not a description of the contents of our subjective experience. Instead, phenomenology aims to describe and analyze the structural aspects, or the invariants of experiences (<xref rid=\"B32\" ref-type=\"bibr\">Gallagher, 1997</xref>). In this regard, phenomenology is a <italic>transcendental philosophy</italic> because it is concerned with the conditions of possibility for having experiences, i.e., for those necessary structures that constitute our perception, remembering, thinking, and so on.</p><p>To accomplish a transcendental analysis of experience, Husserl applied a strategy known as the phenomenological <italic>epoché</italic> (<xref rid=\"B38\" ref-type=\"bibr\">Husserl, 1982</xref>, p. 61). This epoché puts aside any judgment about the positive existence of the objects we experience, something we spontaneously do in our everyday lives and even in our scientific claims. Husserl called this the <italic>natural attitude</italic> of experience (<xref rid=\"B38\" ref-type=\"bibr\">Husserl, 1982</xref>). By utilizing the epoché, we can shift our attention from <italic>what</italic> things are given in our experience, to <italic>how</italic> these things are given in experience, thereby adopting a <italic>phenomenological attitude</italic>.</p><p>From this transcendental standpoint, Husserl, similarly to Brentano, holds that acts of consciousness (i.e., perception, memory, imagination, etc.) are usually directed at something (perceptual objects, memories, expectations, etc.). This relation between acts of consciousness and objects of experience is named <italic>intentionality</italic>. This intentional relation between acts and objects is normative mainly because it implies what Husserl called a <italic>structure of fulfillment</italic> (<xref rid=\"B39\" ref-type=\"bibr\">Husserl, 2001</xref>, p. 280–283), which simply refers to how the intention of an act can be fulfilled by the intended object (<xref rid=\"B16\" ref-type=\"bibr\">Crowell, 2013</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Doyon, 2015</xref>).</p><p>The intentional structure of fulfillment is particularly important for describing perceptual experiences, because the intention of perception is always directed toward a real and concrete object, and not merely to an imaginary or an abstract one. My visual perception of a tree intends the actual tree, not the concept or the re-presentation of a tree. However, perceptual objects will never fully fulfill my perceptual intentions because perceptual objects are always presented only partially (cf. <xref rid=\"B40\" ref-type=\"bibr\">Husserl, 2013</xref>). For instance, my perception of a tree from the window of my house presents only one sensorial profile of the tree (e.g., a couple of branches), whereas many other profiles remain hidden to my view (the backside of the branches, the trunk, the roots, etc.).</p><p>Despite this incomplete fulfillment, my perception is about the whole tree, not about one profile of the tree. This is nowadays called the problem of perceptual presence (<xref rid=\"B59\" ref-type=\"bibr\">Noë, 2004</xref>). This problem raises the question of (1) what conditions make possible that the sensorial givenness of only one profile of the tree evokes my experience of the tree as a unified whole; and also the question of (2) what makes one profile match with the anticipation of my intentional act that perceptually intends a tree. To respond to these questions, we need to clarify what the constitutive aspects of my perceptual experience are, as well as the character of the norm that relates the profile and the object.</p><p>The response of Merleau-Ponty to these questions originated in Husserl’s works<sup><xref ref-type=\"fn\" rid=\"footnote4\">4</xref></sup> is essentially that (1) the presence of perceptual objects as we perceive them is given thanks to a fundamental link between the bodily motor skills of a subject, and the motor significances of things. Merleau-Ponty called this link <italic>motor intentionality</italic> (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 113). For Merleau-Ponty, the lived body of a subject implies an articulated unity that he called body schema (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 100–103). This articulation is performed according to the needs of practical task. That is to say that the body schema is basically the self-organization of the body according to sensorimotor norms (cf. <xref rid=\"B33\" ref-type=\"bibr\">Gallagher, 2005</xref>). The lived thing, by contrast, is a unity of motor significations correlated with the motor skills of the body schema. We can interpret motor significations here as affordances. Hence the lived thing is the unity of affordances correlated with the motor skills of the body unified in the body schema (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 334).</p><p>The synthesis of the thing (as a unity of affordances) is nonetheless a temporal synthesis or a synthesis of transition (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 344). Only one profile of a thing is given at the present moment, however, the profiles non-viewed of the thing are lived as anticipations for motor actions. For instance, I cannot see the backside of my computer, but I can anticipate that if I turn it around, I will see its backside. This synthesis depends nonetheless on the synthesis of the body which is also temporal because the lived body is articulated thanks to its acquisition of bodily habits. These habits anticipate the encountering of perceived things in the way our body is familiarized to do it. The synthesis is, however, unfinished because both the body and the thing remain open to unexpected encounters, to failures in the norm that coordinate the movements of the body and the constraints of things (cf. <xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 476).</p><p>Returning to the second of our earlier questions, (2) what allows for the disclosure of a thing, as a whole, from the sensorial givenness of only one of its profiles are the motor significations that such a profile affords to the body. Motor significations are invitations that the thing manifests or presents to my body from the current sensorial presence for exploring and manipulating it (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>). The tree is given as a whole not because I imagine or represent the whole tree from my partial view of it, but because the tree itself affords further explorations to my motor skills, and its profiles, even those that remain invisible, are not really absent but <italic>present</italic> as correlates of my motor skills (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>).</p><p>However, since things are never given as fully present, the possibility remains that my anticipations mismatch the actual conditions of things. Maybe if I get closer to the window, I can realize that the tree is not a real tree but a hologram of a tree, and then I won’t be able to touch it, to climb it, or to see its back, as I anticipate it. It was just an illusion. Perception, therefore, rests on anticipations that are never completely fulfilled. Nevertheless, there are still some angles or perspectives from where things are disclosed optimally. In this context, Merleau-Ponty claims that the optimum of perception (the optimal grip) is the way that an object present itself more clearly (<xref rid=\"B42\" ref-type=\"bibr\">Kelly, 2005</xref>), that is to say to find the right bodily articulation that better disclose the affordances of things (see section one). However, for Merleau-Ponty, the optimum (or the norm of perception) is not constituted by the characteristics of the thing itself, nor even by the relation between the body and the thing, but by the whole horizonal structure within which the body-thing correlation is enveloped (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>).</p><p>A horizon is a phenomenological description of many structural aspects of experience that accompany the intended objects. For Husserl horizonal aspects or experiences are co-intended or co-given (<xref rid=\"B38\" ref-type=\"bibr\">Husserl, 1982</xref>, p. 94). Hence, horizons, roughly speaking, are those aspects of the perceptual field that play the role of a background for those objects I’m focusing my attention. However, horizons are more than a mere accompaniment to focused objects; they are a constitutive part of my lived experience of them. For this reason, Merleau-Ponty holds that the optimum of perception involves the equilibrium of internal and external horizons (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, 316). Husserl defines the internal horizons of perceptual objects as those aspects that are not directly intended, but are still part of the intended object, as with the unseen profiles of the tree. The external horizons, by contrast, are those elements that surrounds the object, like the garden where the tree is rooted, the blue sky that contrasts with the green of its leaves, etc. (<xref rid=\"B38\" ref-type=\"bibr\">Husserl, 1982</xref>).</p><p>Horizonal aspects of experience are not only those elements implicit in the perceptual field, but also the motor skills that correlate the motor significations of things. That is why Merleau-Ponty claims that the body is the third element implicit in the figure-ground couple of perception (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 103). However, these bodily skills are constrained by the whole relation of forces present in the field. As Merleau-Ponty claimed in <italic>The Structure of Behavior</italic> (<xref rid=\"B50\" ref-type=\"bibr\">Merleau-Ponty, 1963</xref>), the soccer field, for the player, is not an object but a field of forces where consciousness consists in the dialectics between the milieu and the body. Moreover, this field constantly changes considering the actions accomplished by the body, establishing new lines of forces (<xref rid=\"B50\" ref-type=\"bibr\">Merleau-Ponty, 1963</xref>, p. 168–169). Therefore, the norms of perception are not constituted by the characteristics of things as such, so much as these characteristics are implicitly correlated to the abilities and skills of the body, and all those horizonal aspects that structure the whole condition of the phenomenal field where any focused aspect is always embedded.</p><p>The whole normative framework of perceptual experiences is thus ecological because such a framework depends on structures present in the environment that constitute the way an object can be optimally disclosed by a perceiver. The adjective ecological, in this case, does not only implicate the relation between an agent and the environment, as ecological approaches affirm, but also the subjective engagement of an agent into its environment. That is, how the environment appears for the agent according to its embodied subjectivity.</p><p>The optimum is thus the norm of a whole situation that can involve many worldly aspects that constitute the forces of the field, but that can be also altered, changing the orientation of these forces, manifested in a new sense of perceptual experiences. To improve our understanding of these perceptual norms, Merleau-Ponty’s scholars have been lately appealing to the notion of spatial levels that I will review in the next section.</p></sec><sec id=\"S5.SS2\"><title>Levels of Perception</title><p>In his <italic>Phenomenology of Perception</italic>, Merleau-Ponty describes the general notion of space from a phenomenological standpoint, including the body as a constitutional aspect of this dimension. Merleau-Ponty highlights that our experience of space, in normal conditions, implies a particular orientation (e.g., up, down, left, right) that is given to us without the need of conscious reflection (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 259). The primordial sense of space, for Merleau-Ponty, is not an abstract geometrical dimension that works as a sort of container for the objects and events that exist in the world, which is what Merleau-Ponty calls <italic>positional spatiality</italic> (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 102). Rather, for Merleau-Ponty, the primordial form of spatiality is a <italic>situational spatiality</italic> that involves the active engagement of the body in the accomplishment of motor tasks (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 102). This general notion of space entails the horizonal domain of all our possible bodily actions (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 260).</p><p>There are more concrete or delimited spatial regions that following <xref rid=\"B12\" ref-type=\"bibr\">Casey (1996</xref>, <xref rid=\"B13\" ref-type=\"bibr\">1998)</xref> we can call <italic>places</italic>. These places possess <italic>anchorage points</italic> that allow our bodies to situate themselves in or inhabit them (<xref rid=\"B13\" ref-type=\"bibr\">Casey, 1998</xref>, 229; <xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 259). The anchorage points grant places a kind of stability, establishing what Merleau-Ponty called <italic>spatial levels</italic> (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 259). These levels are normative aspects of perception because they refer to the habitual or preferential ways our body interacts with the environment, something that presupposes a previous attunement or “a pact,” between the body and world (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 261).</p><p>The kind of normative character of levels thus does not refer to the perception of things, but to the horizonal aspects that accompany it. Talero, for instance, claims that a spatial level “establishes a place or a setting for my actions to range over by inaugurating a preferential perceptual norm within my situational spatiality” (<xref rid=\"B70\" ref-type=\"bibr\">Talero, 2005</xref>, p. 448). That is, <italic>levels are norms of Places</italic>. Unlike things, places are not usually the focus of our attention. Instead, places tend to serve as stable settings that background our everyday activities and aspects of the environment that we find relevant (e.g., things, colors, shapes, etc.). Hence, places, from a phenomenological standpoint, are horizonal aspects of our perceptual intentions that work as the counterpart of our embodied subjectivity.</p><p>The ubiquitous presence of some spatial levels (the more general ones) requires that we alter the normal conditions of our sensorimotor interactions to be able to recognize them. This is what Merleau-Ponty did through his interpretation of a few classical experiments. First, he refers to a Stratton’s experiment where a subject use goggles that invert the visual field for 8 days. The visual field is perceived up-side down at the beginning. After a couple of days of use, however, the subject starts to live the visual field normally but begins to feel that her body is inverted. After 8 days of use, the whole sensorimotor interaction is finally readapted, and the visual field is lived normally (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 255). In the second example, Merleau-Ponty describes Wertheimer’s experiment, where a subject is put in a room, but can only see through a mirror that is tilted at an angle of 45 degrees. The subject initially sees everything obliquely, and even the movement of objects in the visual field is perceived with an oblique deviation. However, after a few minutes, the subject starts to perceive the entire scene vertically once again (<xref rid=\"B51\" ref-type=\"bibr\">Merleau-Ponty, 2012</xref>, p. 259).</p><p>These examples allowed Merleau-Ponty to see that spatial levels exist in our normal sensorimotor coupling, and that some habitual sensorimotor coupling can be altered if we modify the feedback of “normal” sensorimotor loops. Levels thereby are not properties of the environment as such, nor a projection of agents, rather they describe the normative entanglement of both, but more importantly they also describe the open-ended character of the normative frameworks of action and perception that constantly evolve. This is a phenomenon that (<italic>Level shifts</italic> called <xref rid=\"B70\" ref-type=\"bibr\">Talero, 2005</xref>, p. 446).</p><p>In level shifts, it is common that the anchorage points or the structure of the original level is transposed into a new level, just as when we transpose a melody from one tonality to another (cf. <xref rid=\"B50\" ref-type=\"bibr\">Merleau-Ponty, 1963</xref>, p. 87). This kind of transposition of levels explain why our bodies can become geared, in the same manner, to different environments with similar structures. In the two experiments referred by Merleau-Ponty, the sensorimotor loop is altered but the same form is perceived. From a phenomenological standpoint, this happens because our perceptions are based on anticipations of potentialities for motor actions, but critically these anticipations are grounded on the sensorimotor habits previously acquired by the perceiver. These habits are correlated to the motor significations perceived in the environment and are anticipations and motivations for motor action. Therefore, when a level is forced to shift into another level by changes induced in the sensorimotor loop, the body aims to use its habitual sensorimotor coordination, but is forced to reorganize this coordination considering the new circumstances. However, since it is possible to find similar anchorage points in the emergent sensorimotor dynamics, the habitual form can be transposed into the new level.</p><p>The crucial aspect of this description of levels is their open-ended character, which is not only exhibited by the experiments from above, but seems to be a necessary condition for explaining why the interactions between the body and the environment always remain open to continuous readjustments that nonetheless follow predictable paths inherent to the normative frameworks of levels previously enacted (cf. <xref rid=\"B52\" ref-type=\"bibr\">Merleau-Ponty, 2013</xref>, p. 76). The description of levels is ultimately a description of an endogenous developmental process of the agent-environment entanglement. Considering this description of levels, as the normative framework of space and places, our last task is to clarify how levels contribute to our understanding of norm-development.</p></sec><sec id=\"S5.SS3\"><title>The Development of Enactive-Ecological Norms</title><p>The ecological approach of the skilled intentionality framework analyzes the norms of action and perception in terms of what I called normative frameworks (situated normativity) and norm-attunement (skilled intentionality). From this viewpoint, individual agents become attuned to pre-established normative frameworks. The problem with this viewpoint lies in the way it describes normative frameworks as constituted independently of individual agents, and before these agents are engaged in bodily practices.</p><p>The enactive approach, by contrast, is capable of a more adequate account of the continuous development of the norms of practices. Norm-enactment, contrary to norm-attunement, involves the active participation of agents in the constitution and development of norms. However, the enactive approach sometimes reduces its account of norms to a relation between autonomous agents and physical constraints, thereby neglecting the existence of normative frameworks that constrain the enactment of norms.</p><p>I argued above that norm-enactment is not simply the projection of meaning to physical reality, nor can such enactment be understood as the emergence of meaning from mere physical constraints. Rather, norm-enactment entails the constant development of norms from previously given normative frameworks, something I called norm-development. The account of sensorimotor norms of the enactive approach points to the description of this phenomenon. Nonetheless, this account is still insufficient, because it ignores the fact that norm-development does not simply involve the development of bodily habits, but also the development of environmental structures that embody the counterparts of bodily habits.</p><p>To improve our understanding of this dynamic of norm-development, I appealed to the phenomenological account of perceptual norms found in the writings of Merleau-Ponty, and to his concept of spatial levels. From a phenomenological perspective, norms of perception are moments of equilibrium between the whole ecological context (a situation) and the embodied subjectivity of an agent, instituting what Merleau-Ponty called levels. These levels, however, are in constant development (level shift) because the agent-environment entanglement is a temporal and open-ended structure that is in a constant conflict and movement.</p><p>Norm-development, however, is not purely dynamic phenomenon. It also implicates the stability of norms as horizonal normative frameworks. On the side of the agent, this stability is incarnated in bodily habits, while on the side of the environment, stability is expressed as what I described as excorporations. These are anchor points of places that enable and constrain the enactment of more specific aspects of the environment, which we can understand as affordances. Excorporations may relate to the concept of ecological information in the ecological tradition, whereas levels point to the normative frameworks that constrain the enactment of new norms of action and perception.</p></sec></sec><sec id=\"S6\"><title>Conclusion</title><p>The norms of action and perception are not pregiven sets of lawful relations, nor static frameworks that constrain the behavior of agents until we consciously change them to become adapted to the new worldly circumstances. All bodily practices are highly dynamic, our bodies, the environment, our relations with others, are constantly flowing processes that nonetheless find periodical moments of stability. Stability and change are the two crucial features of life and cognition, either from a dynamical systems theory perspective or from a phenomenological analysis. If we failed to acknowledge one of these aspects, we will fail to describe accurately the dynamics of life and cognition. We must therefore construct an approach to norms of action and perception that acknowledge these two central features of norms.</p><p>I proposed an enactive-ecological approach to norms. This approach is based on the process of norm-enactment described by the enactive approach, but that incorporates an account of normative frameworks. Since these frameworks are not accurately described by the skilled intentionality framework, I appealed to Merleau-Ponty’s phenomenology for this task. The notion of spatial levels broadly describes temporally open-ended normative frameworks that constrain bodily practices but also make possible the enactment of new norms for these practices. I named norm-development as the process of norm-enactment that implicates the temporal evolution of normative frameworks. A more detailed descriptions of norm-development is still needed, as well as the way to apply these descriptions to the concrete normative domains of interaction of agent(s)-environment systems.</p><p>These conclusions, in favor of an enactive model over an ecological one, must not let us think that ecological approaches, specially the skilled intentionality framework, are not highly valuable for our study of cognition from a radical embodied cognition perspective. Rather, if my arguments are right, this is a call for ecological approaches to become more truly enactive. Many of the concepts of the skilled intentionality framework and the free energy principle that support this theory are already pointing in this direction (<xref rid=\"B44\" ref-type=\"bibr\">Kiverstein and Rietveld, 2018</xref>).</p><p>Although at a high theoretical level, it is still hard to see a real complementarity between ecological and enactive approaches, their few but crucial discrepancies are currently useful to create a productive dialogue between these two radical forms of embodied cognition. I hope the reader has found in this work a nice example of it.</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>The author confirms being the sole contributor of this work and has approved it for publication.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>I am thankful to Maxime Doyon and David Morris, whose professional guidance and comments on this paper has been quite helpful to elaborate many of the ideas, I’m expressing in here. I also thank to all the professors and colleagues I met at the workshop “Enaction and ecological psychology” on July 2019 at the University of the Basque Country. Many of their public talks and personal comments were quite helpful to shape and refine my ideas. I’m especially thankful to the organizers, Ezequiel Di Paolo and Manuel Heras-Escribano, for their support and welcoming in this workshop. I finally like to acknowledge the great comments I received from the reviewers that made me clarify my ideas and theoretical purposes in this paper.</p></ack><fn-group><fn id=\"footnote1\"><label>1</label><p>In the more specific context of 4e cognition, some philosophers influenced by Wittgenstein’s late philosophy insist that norms and normativity apply only to actions concerning sociocultural practices, because only these practices implicate a criterion of correction that is agreed by a community (<xref rid=\"B37\" ref-type=\"bibr\">Heras-Escribano et al., 2015</xref>). The supporters of the enactive approach hold a wider conception of norms that include multiple aspects of life and sensorimotor interactions between biological agents and the environment (<xref rid=\"B4\" ref-type=\"bibr\">Barandiaran and Egbert, 2014</xref>). I depart from this general definition because later, the more specific conceptions of norms of the skilled intentionality framework, the enactive approach, and Merleau-Ponty’s phenomenology will be defined.</p></fn><fn id=\"footnote2\"><label>2</label><p>In physics, the second law of thermodynamics states that all physical systems have the tendency to increase chaos and disorder, which is analogous to saying that systems have the tendency to reduce (thermodynamical) free energy. Claude Shannon made a similar claim for his theory of information, positing that all informational systems have the tendency to reduce uncertainty in the same lawful manner that physical systems reduce thermodynamical free energy. In the case of informational systems like cognitive systems, however, we are talking about variational, rather than thermodynamical free energy (<xref rid=\"B43\" ref-type=\"bibr\">Kirchhoff and Froese, 2017</xref>).</p></fn><fn id=\"footnote3\"><label>3</label><p>However, the specific notion of active inference, part of the conceptual repertoire of the skilled intentionality framework, may suggest that such development occurs (cf. <xref rid=\"B61\" ref-type=\"bibr\">Ramstead et al., 2019</xref>).</p></fn><fn id=\"footnote4\"><label>4</label><p>The normative condition of experience and more particularly of perception was originated in Husserl’s work (<xref rid=\"B16\" ref-type=\"bibr\">Crowell, 2013</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Doyon, 2015</xref>, <xref rid=\"B27\" ref-type=\"bibr\">2019</xref>). It was Merleau-Ponty, however, who more systematically develop this subject. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Cell Dev. Biol.</journal-id><journal-title-group><journal-title>Frontiers in Cell and Developmental Biology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-634X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850866</article-id><article-id pub-id-type=\"pmc\">PMC7431694</article-id><article-id pub-id-type=\"doi\">10.3389/fcell.2020.00773</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cell and Developmental Biology</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Senescence in Wound Repair: Emerging Strategies to Target Chronic Healing Wounds</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Wilkinson</surname><given-names>Holly N.</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/578400/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Hardman</surname><given-names>Matthew J.</given-names></name><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/503557/overview\"/></contrib></contrib-group><aff><institution>Centre for Atherothrombosis and Metabolic Disease, Hull York Medical School, University of Hull</institution>, <addr-line>Hull</addr-line>, <country>United Kingdom</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Richard George Faragher, University of Brighton, United Kingdom</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Oliver Bischof, Institut Pasteur, France; Suk-Won Jin, Gwangju Institute of Science and Technology, South Korea</p></fn><corresp id=\"c001\">*Correspondence: Matthew J. Hardman, <email>m.hardman@hull.ac.uk</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Cell Growth and Division, a section of the journal Frontiers in Cell and Developmental Biology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>773</elocation-id><history><date date-type=\"received\"><day>21</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>22</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Wilkinson and Hardman.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Wilkinson and Hardman</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Cellular senescence is a fundamental stress response that restrains tumour formation. Yet, senescence cells are also present in non-cancerous states, accumulating exponentially with chronological age and contributing to age- and diabetes-related cellular dysfunction. The identification of hypersecretory and phagocytic behaviours in cells that were once believed to be non-functional has led to a recent explosion of senescence research. Here we discuss the profound, and often opposing, roles identified for short-lived vs. chronic tissue senescence. Transiently induced senescence is required for development, regeneration and acute wound repair, while chronic senescence is widely implicated in tissue pathology. We recently demonstrated that sustained senescence contributes to impaired diabetic healing via the CXCR2 receptor, which when blocked promotes repair. Further studies have highlighted the beneficial effects of targeting a range of senescence-linked processes to fight disease. Collectively, these findings hold promise for developing clinically viable strategies to tackle senescence in chronic wounds and other cutaneous pathologies.</p></abstract><kwd-group><kwd>senescence</kwd><kwd>ageing</kwd><kwd>diabetes</kwd><kwd>wound healing</kwd><kwd>senolytics</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Medical Research Council<named-content content-type=\"fundref-id\">10.13039/501100000265</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"3\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"194\"/><page-count count=\"13\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Senescence, a seminal discovery of <xref rid=\"B61\" ref-type=\"bibr\">Hayflick and Moorhead (1961)</xref>, is a defined process that globally regulates cell fate. Cellular senescence is traditionally described as a terminal stress response, whereby cells are triggered to undergo stable and essentially irreversible cell cycle arrest following initiation by a diverse range of stress-inducing stimuli (<xref rid=\"B64\" ref-type=\"bibr\">Hernandez-Segura et al., 2018</xref>). Indeed, this process acts as an autonomous anti-tumour mechanism, halting incipient neoplastic transformation (<xref rid=\"B47\" ref-type=\"bibr\">Faget et al., 2019</xref>). Yet, senescent cells can be found in non-cancerous tissues, accumulating exponentially with increasing chronological age (<xref rid=\"B71\" ref-type=\"bibr\">Hudgins et al., 2018</xref>; <xref rid=\"B105\" ref-type=\"bibr\">McHugh and Gil, 2018</xref>). These non-proliferative cells retain metabolic capabilities, exhibiting a hypersecretory phenotype (<xref rid=\"B28\" ref-type=\"bibr\">Coppé et al., 2010</xref>). It has recently been shown that some senescent cells may even engulf their neighbouring cells, for a survival advantage (<xref rid=\"B160\" ref-type=\"bibr\">Tonnessen-Murray et al., 2019</xref>). These profound functional behaviours, identified in cells long thought to be non-functional, pose new questions around their tissue roles and consequences. This review will explore emerging roles for cellular senescence in normal and pathological wound repair, highlighting areas of potential therapeutic opportunity.</p></sec><sec id=\"S2\"><title>Senescence as an Anti-Proliferation Mechanism</title><p>It was originally thought that only mitotic cells, which may be highly proliferative, or spend large periods of time in quiescence, undergo senescence (<xref rid=\"B19\" ref-type=\"bibr\">Campisi and di Fagagna, 2007</xref>). This view has since been challenged, as features of senescence are observed in some differentiated cells (<xref rid=\"B78\" ref-type=\"bibr\">Jurk et al., 2012</xref>; <xref rid=\"B166\" ref-type=\"bibr\">von Zglinicki et al., 2020</xref>). The major age- and stress-related processes that induce cellular senescence include replicative exhaustion (<xref rid=\"B61\" ref-type=\"bibr\">Hayflick and Moorhead, 1961</xref>), mitogenic signals (<xref rid=\"B157\" ref-type=\"bibr\">Tchkonia et al., 2013</xref>), oxidative stress (<xref rid=\"B121\" ref-type=\"bibr\">Passos et al., 2010</xref>), DNA breaks (<xref rid=\"B42\" ref-type=\"bibr\">Di Micco et al., 2006</xref>), and epigenomic damage (<xref rid=\"B122\" ref-type=\"bibr\">Pazolli et al., 2012</xref>). These stressors subsequently initiate anti-tumourigenic networks, controlled by transcriptional regulators such as p53 (<xref rid=\"B167\" ref-type=\"bibr\">Vousden and Prives, 2009</xref>). p53 directly transactivates the cyclin dependent kinase (CDK) inhibitor, p21, to inhibit CDK2, CDK4, and CDK6-mediated retinoblastoma protein (pRb) phosphorylation (<xref rid=\"B62\" ref-type=\"bibr\">He et al., 2007</xref>). p16 similarly prevents pRb inactivation, but in a p53-independent manner (<xref rid=\"B22\" ref-type=\"bibr\">Chen et al., 2006</xref>). pRb naturally binds E2F/DP transcription factor complexes to block transcription of E2F target genes, thus failure to phosphorylate pRb halts cell cycle progression from the G1 to S phase (<xref rid=\"B117\" ref-type=\"bibr\">Narita et al., 2003</xref>).</p><p>It is important to note that, while simplified here, the role for p53 in cell survival is complex and somewhat contradictory, as p53 activation can actually suppress senescence, instead causing cell quiescence (<xref rid=\"B38\" ref-type=\"bibr\">Demidenko et al., 2010</xref>) or apoptosis (reviewed in <xref rid=\"B138\" ref-type=\"bibr\">Salminen et al., 2011</xref>). In this regard, a cell’s fate might be decided by the amount of damage sustained, and the expression of other senescence-linked factors. Molecular understanding of senescence is complicated further by the fact that the relative contribution of p21, p16, and other cell cycle regulators is thought to be context dependent (<xref rid=\"B161\" ref-type=\"bibr\">van Deursen, 2014</xref>).</p></sec><sec id=\"S3\"><title>Senescent Cell Characteristics</title><p>Morphologically, senescent cells exhibit flattened, elongated features, and may have multiple nuclei and enlarged vacuoles (<xref rid=\"B129\" ref-type=\"bibr\">Rhinn et al., 2019</xref>). Senescence-associated beta galactosidase is often used as an archetypal senescence biomarker (<xref rid=\"B43\" ref-type=\"bibr\">Dimri et al., 1995</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Debacq-Chainiaux et al., 2009</xref>), yet its specificity has come under criticism (<xref rid=\"B88\" ref-type=\"bibr\">Krishna et al., 1999</xref>; <xref rid=\"B93\" ref-type=\"bibr\">Lee et al., 2006</xref>). For that reason, it is most often used in conjunction with other key biomarkers, such as p16 and p21, to confirm senescence (<xref rid=\"B8\" ref-type=\"bibr\">Baker et al., 2016</xref>; <xref rid=\"B104\" ref-type=\"bibr\">Matjusaitis et al., 2016</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Biran et al., 2017</xref>).</p><p>Senescent cells may also possess regions of highly condensed chromatin (senescence-associated heterochromatic foci; <xref rid=\"B191\" ref-type=\"bibr\">Zhang et al., 2007</xref>) and DNA damage-induced chromatin alterations, including γH2AX and H3K9Me3 (<xref rid=\"B132\" ref-type=\"bibr\">Rodier and Campisi, 2011</xref>). Loss of histones, centrosome aberrations and the breakdown of the nuclear envelope (e.g., degradation of lamin B1) similarly occur in many senescent states to enable rearrangement of heterochromatin (<xref rid=\"B159\" ref-type=\"bibr\">Tigges et al., 2014</xref>; <xref rid=\"B170\" ref-type=\"bibr\">Wang et al., 2017</xref>). These chromatin modifications sequester E2F target genes to potentiate senescence (<xref rid=\"B142\" ref-type=\"bibr\">Shah et al., 2013</xref>). Moreover, senescence is reinforced by microRNA-mediated silencing of E2F target genes (<xref rid=\"B13\" ref-type=\"bibr\">Benhamed et al., 2012</xref>).</p><p>Experimental manipulation of epigenetic marks has demonstrably shown their importance in controlling the molecular induction of cellular senescence. H3K27me3, for example, represses p16 and p14 expression by silencing the INK4a-ARF locus (<xref rid=\"B87\" ref-type=\"bibr\">Kotake et al., 2007</xref>). Removal of H3K27me3, by JMJD3-induced demethylation (<xref rid=\"B3\" ref-type=\"bibr\">Agger et al., 2009</xref>; <xref rid=\"B152\" ref-type=\"bibr\">Sui et al., 2019</xref>) or pharmacological inhibition of the histone lysine methyltransferase, EZH2 (<xref rid=\"B72\" ref-type=\"bibr\">Ito et al., 2018</xref>), promotes p16 expression and senescence. Inhibition of EZH2 also leads to SASP production via enrichment of H3K27ac, and loss of H3K27me3, at SASP-related loci (<xref rid=\"B72\" ref-type=\"bibr\">Ito et al., 2018</xref>). Overexpression of another histone demethylase, UTX, can also silence H3K27me3 to promote cellular senescence (<xref rid=\"B124\" ref-type=\"bibr\">Perrigue et al., 2020</xref>).</p><p>Stressed cells are repressed at the transcriptional level to prevent the expansion of potentially harmful mutations. It is therefore understandable that regulators, such as p53, are not only responsible for initiating senescence, but also decide whether cells should instead enter temporary quiescence or undergo apoptosis (<xref rid=\"B138\" ref-type=\"bibr\">Salminen et al., 2011</xref>). Intriguingly, senescent cells may actually retain heightened resistance to apoptosis, first demonstrated in fibroblasts (<xref rid=\"B172\" ref-type=\"bibr\">Wang, 1995</xref>), possibly due to altered p53 signalling (<xref rid=\"B24\" ref-type=\"bibr\">Childs et al., 2014</xref>) and upregulation of pro-survival pathways (e.g., BCL-2 and ephrins, <xref rid=\"B193\" ref-type=\"bibr\">Zhu et al., 2015</xref>). Indeed, senescent keratinocytes are resistant to ultraviolet radiation-induced apoptosis (<xref rid=\"B21\" ref-type=\"bibr\">Chaturvedi et al., 2004</xref>) and senescent fibroblasts to thapsigargin-induced apoptosis (<xref rid=\"B136\" ref-type=\"bibr\">Ryu et al., 2007</xref>). Senescent endothelial cells, on the other hand, are more likely to undergo apoptosis than their non-senescent counterparts (<xref rid=\"B57\" ref-type=\"bibr\">Hampel et al., 2004</xref>). Clearly, this senescence trait is situational, and not ubiquitous to all cell types.</p><p>The hypersecretory phenotype of senescent cells is most often referred to as the senescence-associated secretory phenotype (SASP), an attribute closely linked to the positive or negative outcomes of tissue senescence that appears to be cell-type and context-dependent. Even though studies have characterised the SASP in multiple cell types, its detailed composition remains elusive. Broadly, the SASP comprises a collection of pro-inflammatory cytokines and chemokines, growth factors, proteases, lipids and extracellular matrix components (<xref rid=\"B49\" ref-type=\"bibr\">Freund et al., 2010</xref>; <xref rid=\"B46\" ref-type=\"bibr\">Elzi et al., 2012</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Acosta et al., 2013</xref>; <xref rid=\"B97\" ref-type=\"bibr\">Lopes-Paciencia et al., 2019</xref>). It is thought to mainly be a feature of senescent cells that have undergone a DNA damage response, as a SASP is not apparent in cells that naturally senesce due to overexpression of p16 and p21 (<xref rid=\"B31\" ref-type=\"bibr\">Coppé et al., 2011</xref>). However, a DNA damage-independent SASP can occur in fibroblasts via p38MAPK phosphorylation, challenging previous preconceptions (<xref rid=\"B50\" ref-type=\"bibr\">Freund et al., 2011</xref>). Collectively, the secretome is the characteristic of senescent cells that confers most of their biological effects, significantly contributing to age-related functional decline (<xref rid=\"B133\" ref-type=\"bibr\">Rodier et al., 2009</xref>) and chronic disease (<xref rid=\"B192\" ref-type=\"bibr\">Zhu et al., 2014</xref>) in autocrine and paracrine manners.</p><p>The SASP is dynamically regulated by a number of factors that mostly converge on the NF-κB complex (<xref rid=\"B154\" ref-type=\"bibr\">Sun et al., 2018</xref>). Inflammatory cytokines, such as IL-1α, can form positive feedback loops with NF-κB and partner cascades to reinforce SASP release and senescence (<xref rid=\"B91\" ref-type=\"bibr\">Kuilman et al., 2008</xref>; <xref rid=\"B118\" ref-type=\"bibr\">Orjalo et al., 2009</xref>). In fact, multiple authors have demonstrated activation of NF-κB gene sets following senescence (<xref rid=\"B90\" ref-type=\"bibr\">Kuilman et al., 2010</xref>; <xref rid=\"B99\" ref-type=\"bibr\">Lujambio et al., 2013</xref>), while p53 and NF-κB are linked in coregulatory (in macrophages, <xref rid=\"B98\" ref-type=\"bibr\">Lowe et al., 2014</xref>) and antagonistic (HeLa cells, <xref rid=\"B70\" ref-type=\"bibr\">Huang et al., 2007</xref>) manners. The importance of NF-κB in senescence is highlighted by studies where NF-κB suppression allows oncogene-induced IMR-90 fibroblasts to bypass senescence (<xref rid=\"B23\" ref-type=\"bibr\">Chien et al., 2011</xref>) and reduces senescence in osteoarthritic cartilage (<xref rid=\"B181\" ref-type=\"bibr\">Wu et al., 2015</xref>).</p><p>Indeed, the SASP (e.g., TGFβ) can potentiate senescence in neighbouring cells (<xref rid=\"B1\" ref-type=\"bibr\">Acosta et al., 2013</xref>), but also promote senescent cell clearance by attracting immune cells (<xref rid=\"B81\" ref-type=\"bibr\">Kang et al., 2011</xref>; <xref rid=\"B156\" ref-type=\"bibr\">Tasdemir et al., 2016</xref>). The induction of senescence in, and clearance of, premalignant cells consequently reinforces tumour suppression. Paradoxically, the SASP can also drive pre-cancerous development in proximal tissues as many SASP proteins are potent mitogenic factors (e.g., VEGF, <xref rid=\"B29\" ref-type=\"bibr\">Coppé et al., 2006</xref>, <xref rid=\"B28\" ref-type=\"bibr\">2010</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Collado et al., 2007</xref>). The plasticity of the SASP across different microenvironments, cell types and stimuli (<xref rid=\"B18\" ref-type=\"bibr\">Campisi, 2013</xref>; <xref rid=\"B100\" ref-type=\"bibr\">Lupa et al., 2015</xref>; <xref rid=\"B101\" ref-type=\"bibr\">Maciel-Baron et al., 2016</xref>; <xref rid=\"B154\" ref-type=\"bibr\">Sun et al., 2018</xref>) further complicates our understanding of its role within tissues. However, it is clear that senescence, and the SASP, remain important regulators of normal physiology and pathology. Tissue consequences of senescent cells and their SASP are summarised in <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Characteristics of senescent cells and their tissue consequences. Senescent cells feature morphological changes <bold>(1)</bold>, chromatin modifications <bold>(2)</bold>, loss of nuclear envelope lamin B1 <bold>(3)</bold>, increased p16 and p21 <bold>(4)</bold>, senescence-associated beta galactosidase activity <bold>(5)</bold> and production of a senescence-associated secretory phenotype (SASP, <bold>6</bold>). SASP components (red) alter cellular processes (e.g., inflammation and angiogenesis) within the microenvironment. A chronic SASP causes negative outcomes, while a short-lived, transient SASP is beneficial.</p></caption><graphic xlink:href=\"fcell-08-00773-g001\"/></fig></sec><sec id=\"S4\"><title>Roles for Senescence During Development and Regeneration</title><p>Many crucial biological processes require cells to undergo cell cycle arrest and differentiation to terminal states. For example, developmental lineage-specification requires cells to differentiate in a temporospatial manner in order to properly form tissues (<xref rid=\"B35\" ref-type=\"bibr\">Da Silva-Álvarez et al., 2019</xref>). In the skin, basal keratinocytes first proliferate, and then differentiate, transiting through the epidermis to replenish the non-viable stratum corneum (<xref rid=\"B52\" ref-type=\"bibr\">Fuchs and Byrne, 1994</xref>). In fact, to allow effective keratinocyte differentiation, p21 is activated initially (<xref rid=\"B111\" ref-type=\"bibr\">Missero et al., 1996</xref>), but then suppressed (<xref rid=\"B40\" ref-type=\"bibr\">Di Cunto et al., 1998</xref>). It is therefore unsurprising that tumour suppressor genes also aid development and regeneration through control of quiescence, terminal differentiation, apoptosis and senescence (e.g., <xref rid=\"B41\" ref-type=\"bibr\">Di Giovanni et al., 2006</xref>; <xref rid=\"B173\" ref-type=\"bibr\">Watkins et al., 2013</xref>).</p><p>Three main roles have been put forward for the presence of senescent cells during embryogenesis: (1) to promote the regression of transient structures; (2) to balance cell populations and/or; (3) to act as a signalling hub to regulate tissue morphogenesis (<xref rid=\"B35\" ref-type=\"bibr\">Da Silva-Álvarez et al., 2019</xref>). In embryonic development, temporal induction of senescence is required for tissue patterning in the developing limb bud. Here, p21 induction leads to SASP factor expression (e.g., FGF), stimulating cell proliferation and tissue formation. Resulting senescent (and apoptotic) cells are then effectively cleared by macrophages, prior to tissue remodelling. Indeed, genetic knockdown of p21 to attenuate senescence leads to mild patterning defects in murine limbs (<xref rid=\"B150\" ref-type=\"bibr\">Storer et al., 2013</xref>). In a corroborating study, p21 was shown to contribute to senescence-linked development in a p53-independent manner in human and murine embryos (<xref rid=\"B116\" ref-type=\"bibr\">Muñoz-Espín et al., 2013</xref>). In this case, however, loss of p21 was compensated for by increased apoptosis. Thus, p21-regulated senescence and apoptosis can perform synergistic roles during organismal development.</p><p>Akin to development, lower organisms and anamniotes are able to regenerate their tissues to full form and function, either as juveniles or throughout their lives (<xref rid=\"B16\" ref-type=\"bibr\">Brockes and Kumar, 2005</xref>). In fact, it has recently been shown that senescence may play an important role in these regenerative processes. In salamanders, senescence induction occurs at the intermediate stages of limb regeneration and then diminishes due to effective clearance by macrophages (<xref rid=\"B190\" ref-type=\"bibr\">Yun et al., 2015</xref>). Senescence is similarly invoked during pectoral fin regeneration in zebrafish, and impaired when senescence is blocked with the senolytic compound, ABT-263 (<xref rid=\"B34\" ref-type=\"bibr\">Da Silva-Álvarez et al., 2020</xref>). Given the importance of senescence in regulating tissue formation throughout development and regeneration, it is logical to ask whether senescence could play a role in the reparative responses of higher vertebrates.</p></sec><sec id=\"S5\"><title>Senescence in Normal Tissue Repair</title><p>Tissue repair is necessary for all life. While it seldom leads to full regeneration, the process prevents exsanguination and infection, and aids structural and functional restoration required for survival. Tissue repair is rapid and highly dynamic, comprising multiple cell types and overlapping processes that broadly include haemastasis, inflammation, cell proliferation and dermal remodelling (<xref rid=\"B177\" ref-type=\"bibr\">Wilkinson and Hardman, 2017</xref>). During haemastasis, an insoluble blood clot is formed and endothelial cells from damaged vasculature enter the wound, depositing a temporary fibrin scaffold and releasing factors to attract both circulating immune cells and resident skin cells (<xref rid=\"B164\" ref-type=\"bibr\">Velnar et al., 2009</xref>). Inflammatory cells are rapidly recruited to the site of damage, first dominated by neutrophils and pro-inflammatory macrophages to remove bacteria and necrotic tissue (<xref rid=\"B188\" ref-type=\"bibr\">Young and McNaught, 2011</xref>). Later stage healing is characterised by a switch to anti-inflammatory macrophages, which phagocytose any remaining pro-inflammatory cells, supporting fibroplasia and wound resolution (<xref rid=\"B86\" ref-type=\"bibr\">Korns et al., 2011</xref>). To allow effective repair, keratinocytes undergo partial epithelial-to-mesenchymal transition and begin migrating to close the wound gap, a process known as re-epithelialisation (<xref rid=\"B143\" ref-type=\"bibr\">Shaw and Martin, 2016</xref>). Formation of new vasculature (angiogenesis) is essential to provide sustenance during the highly proliferative stage of healing (<xref rid=\"B12\" ref-type=\"bibr\">Baum and Arpey, 2005</xref>). Finally, the immature matrix laid down during early healing is replaced by stronger scaffold proteins, such as mature collagen fibres produced and remodelled by fibroblasts (<xref rid=\"B94\" ref-type=\"bibr\">Li et al., 2007</xref>). Each stage of wound repair involves extensive cellular communication, orchestrated by cytokines, chemokines, growth factors and components of the extracellular milieu. The plasticity of the response, and the cellular behaviours that occur, are homologous to those observed in cancer (e.g., immune cell infiltration, invasion and epithelial-to-mesenchymal transition, <xref rid=\"B140\" ref-type=\"bibr\">Schäfer and Werner, 2008</xref>). It is therefore not unreasonable to suggest that senescence, and associated mechanisms, could significantly contribute to wound healing.</p><p>Indeed, pertinent roles for senescence in tissue injury have been emerging, largely focusing on the beneficial, transient initiation of senescence during repair. Here, induction of senescence following liver damage (<xref rid=\"B89\" ref-type=\"bibr\">Krizhanovsky et al., 2008</xref>) and cutaneous injury (<xref rid=\"B75\" ref-type=\"bibr\">Jun and Lau, 2010</xref>) was shown to prevent excessive fibrosis that would otherwise cause tissue dysfunction. <xref rid=\"B89\" ref-type=\"bibr\">Krizhanovsky et al. (2008)</xref> confirmed that reduced fibrosis was the result of senescence-linked fibrolytic enzyme production, and immune-regulated clearance of injury-expanded cell populations that would otherwise contribute to excessive matrix deposition. Likewise, senescence decreased fibrosis in a model of cardiac injury, where genetic ablation of p53 and p16 accelerated fibrosis (<xref rid=\"B109\" ref-type=\"bibr\">Meyer et al., 2016</xref>). Ectopic expression of Ccn1, which increased cardiac senescence, also limited fibrosis in this model. Interestingly, <xref rid=\"B75\" ref-type=\"bibr\">Jun and Lau (2010)</xref>, the first authors to observe transient senescence during skin repair, revealed that Ccn1 causes fibroblast senescence via an oxidative-stress dependent mechanism. Upregulation of Ccn1 was vitally important to prevent excessive fibrosis. More recently, the same authors demonstrated that topical application of another Ccn family member, Ccn2, similarly actuates senescence and reduces fibrosis in cutaneous murine wounds (<xref rid=\"B76\" ref-type=\"bibr\">Jun and Lau, 2017</xref>).</p><p>By contrast, when <xref rid=\"B37\" ref-type=\"bibr\">Demaria et al. (2014)</xref> ablated p16- and p21-expressing cells in mice they observed impaired extracellular matrix deposition and a decreased rate of wound closure. Intriguingly, by day 15 post-injury, these senescent-deficient wounds were excessively fibrotic. Similar to previous research (<xref rid=\"B75\" ref-type=\"bibr\">Jun and Lau, 2010</xref>), transient senescence appeared limited to fibroblast-like cells, which produced a PDGFA-enriched SASP to stimulate appropriate skin repair (<xref rid=\"B37\" ref-type=\"bibr\">Demaria et al., 2014</xref>). Studies continue to explore the importance of transient senescence during acute wound healing, with <xref rid=\"B67\" ref-type=\"bibr\">Hiebert et al. (2018)</xref> recently reporting that overexpression of nrf2 promotes fibroblast senescence, which is accompanied by accelerated wound re-epithelialisation and extracellular matrix deposition. Although at present limited to murine models, these key investigations provide clear evidence that temporal induction of senescence is necessary for effective skin repair. Yet, many questions remain unanswered. For instance, does transient wound-induced senescence arise through intrinsic cell factors or environmental influences? And how are these senescent cells so effectively cleared once they are no longer required?</p></sec><sec id=\"S6\"><title>Senescence in Aged and Diabetic Wound Healing</title><p>The above studies provide substantial insight into the importance of senescence for the healing of experimental wounds. What they do not address is the potential differential influences of acute vs. chronic senescence to tissue repair, nor how senescence could be involved in pathological healing. These are important considerations for the clinical setting, where effective healing can mean the difference between life or death (<xref rid=\"B58\" ref-type=\"bibr\">Han and Ceilley, 2017</xref>). Chronic, non-healing wounds are a huge socioeconomic burden, reducing quality of life and costing billions each year to treat (<xref rid=\"B54\" ref-type=\"bibr\">Guest et al., 2015</xref>). Considered a “silent epidemic” (<xref rid=\"B95\" ref-type=\"bibr\">Lindholm and Searle, 2016</xref>), chronic wounds display diverse aetiology, with incomplete molecular and cellular understanding (<xref rid=\"B51\" ref-type=\"bibr\">Frykberg and Banks, 2015</xref>). Inadequate current treatments mean it is fundamentally important to further understand why chronic wounds fail to heal, and ultimately develop more effective therapies.</p><p>It has long been appreciated that chronic wound pathology is almost entirely restricted to those who are elderly and/or diabetic. This is fascinating, as the biological processes of ageing and diabetes are themselves notably linked to senescence (<xref rid=\"B178\" ref-type=\"bibr\">Wilkinson and Hardman, 2020</xref>). Senescence is both a characteristic feature of <xref rid=\"B10\" ref-type=\"bibr\">Baker et al. (2013)</xref>, <xref rid=\"B183\" ref-type=\"bibr\">Xu et al. (2015)</xref>, and <xref rid=\"B63\" ref-type=\"bibr\">Helman et al. (2016)</xref> and contributor to <xref rid=\"B9\" ref-type=\"bibr\">Baker et al. (2008</xref>, <xref rid=\"B11\" ref-type=\"bibr\">2011)</xref> widespread tissue ageing. Epigenetic modifications are one feature of ageing that is linked to senescence. Genomic instability and DNA methylation changes correlate with chronological ageing in mice (<xref rid=\"B151\" ref-type=\"bibr\">Stubbs et al., 2017</xref>) and humans (<xref rid=\"B68\" ref-type=\"bibr\">Horvath, 2013</xref>). Interestingly, the repressive mark, H3K27me3, showed altered DNA coverage on aged vs. young stem cells (<xref rid=\"B96\" ref-type=\"bibr\">Liu et al., 2013</xref>; <xref rid=\"B153\" ref-type=\"bibr\">Sun et al., 2014</xref>), which may contribute to their reduced renewal capacity.</p><p>Another attribute of normal metabolic ageing that is experimentally linked to senescence is cumulative oxidative damage. For example, human diploid fibroblasts (<xref rid=\"B45\" ref-type=\"bibr\">Duan et al., 2005</xref>) and endothelial cells (<xref rid=\"B135\" ref-type=\"bibr\">Ruan et al., 2014</xref>) undergo senescence in the presence of heightened reactive oxygen species (ROS), while replicative lifespan can be extended in cell culture by lowering oxygen tension (<xref rid=\"B120\" ref-type=\"bibr\">Parrinello et al., 2003</xref>). More notably, exposure to ultraviolet radiation simulates photoageing by increasing ROS production in skin (<xref rid=\"B65\" ref-type=\"bibr\">Herrling et al., 2006</xref>), while ROS upregulates p16 in skin cells (<xref rid=\"B73\" ref-type=\"bibr\">Jenkins et al., 2011</xref>). Skin ageing is also characterised by cell accumulation of p16 (<xref rid=\"B168\" ref-type=\"bibr\">Waaijer et al., 2012</xref>) and senescence-associated beta galactosidase (<xref rid=\"B43\" ref-type=\"bibr\">Dimri et al., 1995</xref>; <xref rid=\"B128\" ref-type=\"bibr\">Ressler et al., 2006</xref>). This association is causally corroborated by <xref rid=\"B182\" ref-type=\"bibr\">Xu et al. (2018)</xref>, who demonstrated that transplantation of senescent cells to young mice accelerated ageing, while <xref rid=\"B11\" ref-type=\"bibr\">Baker et al. (2011)</xref> revealed that eradication of p16-positive cells alleviated features of premature ageing in a murine progeroid model.</p><p>The link between diabetes and senescence is less well-established, but is an area of intense current research. As previously mentioned, senescent cells cause widespread disruption to normal tissue architecture by virtue of their SASP (<xref rid=\"B28\" ref-type=\"bibr\">Coppé et al., 2010</xref>). Major SASP constituents influence senescence by targeting immunological pathways, such as NF-κB (<xref rid=\"B138\" ref-type=\"bibr\">Salminen et al., 2011</xref>). This leads to matrix proteolysis and increased inflammation, primary features of aged and diabetic wounds (<xref rid=\"B103\" ref-type=\"bibr\">Makrantonaki et al., 2017</xref>; <xref rid=\"B180\" ref-type=\"bibr\">Wilkinson et al., 2019c</xref>). Indeed, growing evidence suggests that a heightened intrinsic immune response, or “sterile” inflammation, contributes to age- and diabetes-related pathology (reviewed in <xref rid=\"B125\" ref-type=\"bibr\">Prattichizzo et al., 2016</xref>). Characteristic features of diabetes that drive immune cell accumulation, and therefore potentiate senescence, include obesity and hyperglycaemia (<xref rid=\"B186\" ref-type=\"bibr\">Yokoi et al., 2006</xref>; <xref rid=\"B110\" ref-type=\"bibr\">Minamino et al., 2009</xref>; <xref rid=\"B102\" ref-type=\"bibr\">Maeda et al., 2015</xref>; <xref rid=\"B141\" ref-type=\"bibr\">Schafer et al., 2016</xref>). These processes most likely promote senescence via increasing advanced glycation end-products and causing widespread oxidative damage (<xref rid=\"B32\" ref-type=\"bibr\">Coughlan et al., 2011</xref>; <xref rid=\"B48\" ref-type=\"bibr\">Fang et al., 2016</xref>).</p><p>Turning specifically to the skin, it is clear that in diabetic and aged tissue, accumulation of senescent cells extends to both uninjured skin and wounds (<xref rid=\"B128\" ref-type=\"bibr\">Ressler et al., 2006</xref>; <xref rid=\"B168\" ref-type=\"bibr\">Waaijer et al., 2012</xref>; <xref rid=\"B176\" ref-type=\"bibr\">Wilkinson et al., 2019a</xref>). Previous authors have demonstrated that chronic venous leg ulcers harbour senescent fibroblasts (<xref rid=\"B107\" ref-type=\"bibr\">Mendez et al., 1998</xref>; <xref rid=\"B162\" ref-type=\"bibr\">Vande Berg et al., 1998</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Agren et al., 1999</xref>; <xref rid=\"B169\" ref-type=\"bibr\">Wall et al., 2008</xref>). The presence of senescent fibroblasts in chronic wounds may even exacerbate pathology, where it was shown that ulcers containing over 15% senescent cells were hard to heal (<xref rid=\"B149\" ref-type=\"bibr\">Stanley and Osler, 2001</xref>). We recently reported a novel mechanistic link between senescence and healing in diabetic wounds (<xref rid=\"B176\" ref-type=\"bibr\">Wilkinson et al., 2019a</xref>). Here, intrinsically senescent macrophages were observed to promote impaired wound healing in a non-aged, murine model of diabetic pathological repair.</p><p>Indeed, many SASP factors attract monocytes and macrophages (e.g., MCP-1; <xref rid=\"B80\" ref-type=\"bibr\">Kamei et al., 2006</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Coppé et al., 2008</xref>; <xref rid=\"B126\" ref-type=\"bibr\">Prattichizzo et al., 2018</xref>), often with a pro-inflammatory phenotype (<xref rid=\"B114\" ref-type=\"bibr\">Mosser and Edwards, 2008</xref>; <xref rid=\"B99\" ref-type=\"bibr\">Lujambio et al., 2013</xref>). Excessive immune cell recruitment and inappropriate retention is a hallmark of chronic wound pathology. This may be even be exacerbated by other local factors, such as iron, which induces a pro-inflammatory phenotype in macrophages and leads to fibroblast senescence in chronic venous leg ulcers (<xref rid=\"B145\" ref-type=\"bibr\">Sindrilaru et al., 2011</xref>). Thus, macrophages are likely a nexus for uncontrolled local inflammation in both diabetic pathogenesis and senescence, ultimately delivering poor wound healing. Moreover, the impaired function of macrophages (and other immune cell types) in aged (<xref rid=\"B155\" ref-type=\"bibr\">Swift et al., 2001</xref>) and diabetic (<xref rid=\"B179\" ref-type=\"bibr\">Wilkinson et al., 2019b</xref>) wounds likely contributes to prolonged, rather than transient, senescence due to ineffective clearance mechanisms.</p><p>Senescence in the wound environment is probably not limited to fibroblasts and macrophages, as other wound cells, including keratinocytes (<xref rid=\"B146\" ref-type=\"bibr\">Smirnov et al., 2016</xref>) and endothelial cells (<xref rid=\"B135\" ref-type=\"bibr\">Ruan et al., 2014</xref>), are capable of undergoing senescence in response to environment cues. Senescent keratinocytes are certainly observed in aged skin (<xref rid=\"B163\" ref-type=\"bibr\">Velarde et al., 2012</xref>) and are suggested to influence the reduced regenerative capacity of aged epidermis (<xref rid=\"B194\" ref-type=\"bibr\">Zouboulis et al., 2008</xref>). Chronic wounds also harbour pathogenic microorganisms (<xref rid=\"B79\" ref-type=\"bibr\">Kalan et al., 2019</xref>) that may contribute to senescence by stimulating ROS production in keratinocytes and exacerbating inflammation (<xref rid=\"B53\" ref-type=\"bibr\">Grange et al., 2009</xref>). Indeed, this may occur via specific bacterial virulence factors, as pyocyanin from <italic>Pseudomonas aeruginosa</italic> can induce senescence in fibroblasts (<xref rid=\"B115\" ref-type=\"bibr\">Muller et al., 2009</xref>).</p><p>It is clear that the chronic ulcer milieu, which is rich in pro-inflammatory factors, indirectly causes senescence via exacerbating inflammation. However, as wound fluid from venous leg ulcers directly induces senescence in neonatal fibroblasts (<xref rid=\"B106\" ref-type=\"bibr\">Mendez et al., 1999</xref>), it is likely that the local microenvironment also stimulates cellular senescence. Intrinsically senescent wounds cells, such as fibroblasts, are similarly capable of potentiating senescence across neighbouring cell types in a paracrine manner, via their SASP (<xref rid=\"B1\" ref-type=\"bibr\">Acosta et al., 2013</xref>; <xref rid=\"B176\" ref-type=\"bibr\">Wilkinson et al., 2019a</xref>). Moreover, in local environments where the SASP is insufficient to directly induce cellular senescence, it may still promote pathological cellular phenotypes, such as epidermal hyperproliferation (<xref rid=\"B5\" ref-type=\"bibr\">Albanesi et al., 2018</xref>) and excessive dermal proteolysis (via MMPs; <xref rid=\"B17\" ref-type=\"bibr\">Caley et al., 2015</xref>).</p><p>To add a further level of complexity, evidence for the disparities between transient and chronic senescence is beginning to emerge, with clear implications for wound healing. For instance, stemness and reprogramming in keratinocytes is promoted by a transient SASP, yet inhibited when the SASP becomes chronic (<xref rid=\"B130\" ref-type=\"bibr\">Ritschka et al., 2017</xref>). Transient senescence also encourages matrix deposition following tissue injury (<xref rid=\"B37\" ref-type=\"bibr\">Demaria et al., 2014</xref>), but prevents excessive fibrosis (<xref rid=\"B75\" ref-type=\"bibr\">Jun and Lau, 2010</xref>), while chronic senescence is linked fibrotic disease (<xref rid=\"B184\" ref-type=\"bibr\">Yanai et al., 2015</xref>). Taken together, published and emerging studies are certainly challenging the dogma that senescence is primarily limited to age-related dysfunction and cancer. Indeed, evolving understanding of the concept of transient vs. chronic senescence is likely to deliver important new insight into the processes that occur during acute and pathological repair. Current understanding of senescence contribution to normal and pathological wound healing is summarised in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Roles for senescence in acute vs. chronic wound repair. Late-stage wound healing is characterised by extracellular matrix (ECM) deposition, full wound closure and blood vessel perfusion <bold>(left)</bold>. Senescent fibroblasts appear during late-stage healing and contribute to ECM deposition by producing a pro-healing senescence-associated secretory phenotype (SASP, blue stars) containing PDGFA. Senescent fibroblasts prevent excessive fibrosis via CCN1 and CCN2. The senescent cells are then effectively cleared by anti-inflammatory macrophages (AI MΦs) and the skin is restored. Chronic healing wounds feature wound edge hyperproliferation and excessive inflammation <bold>(right)</bold>. Here senescent fibroblasts and MΦs (blue) exacerbate inflammation via a pro-inflammatory (PI) SASP of cytokines and proteases. The SASP may also exert a paracrine effect, causing senescence in other wound cell types. High bacterial load stimulates further inflammation and oxidative stress, which can cause hyperproliferation or senescence in keratinocytes. Senescent cells are not effectively cleared by the dysfunctional chronic wound immune cells, thus tissue damage persists and the wound fails to heal.</p></caption><graphic xlink:href=\"fcell-08-00773-g002\"/></fig></sec><sec id=\"S7\"><title>Cellular Senescence as a Therapeutic Target in Pathological Wounds</title><p>The widespread causative biological effects of cellular senescence in tissue ageing pathology make the therapeutic modulation of senescence an attractive target for a plethora of age-related diseases. Genetic studies positively support this idea, with inducible knockdown of p16 alleviating hallmark features of ageing in progeroid murine models (<xref rid=\"B11\" ref-type=\"bibr\">Baker et al., 2011</xref>; <xref rid=\"B139\" ref-type=\"bibr\">Sato et al., 2015</xref>). In fact, the well-documented effects of caloric restriction, which both extends mammalian lifespan (<xref rid=\"B148\" ref-type=\"bibr\">Sohal and Weindruch, 1996</xref>) and delays the onset of age-related disease (<xref rid=\"B174\" ref-type=\"bibr\">Weindruch and Walford, 1982</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Colman et al., 2014</xref>), may be a physical manifestation of tissue senescence modulation. Caloric restriction has been shown to reduce cardiac senescence (<xref rid=\"B144\" ref-type=\"bibr\">Shinmura et al., 2011</xref>), and senescence in hepatocytes and intestinal crypt cells <italic>in vivo</italic> (<xref rid=\"B171\" ref-type=\"bibr\">Wang et al., 2010</xref>). At the epigenetic level, caloric restriction protects against age-related changes in DNA methylation (<xref rid=\"B55\" ref-type=\"bibr\">Hahn et al., 2017</xref>). Caloric restriction also decreases senescence partly by upregulating the epigenetically linked sirtuin pathway, promoting anti-apoptosis and anti-inflammatory mechanisms (<xref rid=\"B15\" ref-type=\"bibr\">Bonda et al., 2011</xref>). Subsequent effects include slowing metabolic processes that contribute to cellular ageing (e.g., oxidative stress, <xref rid=\"B185\" ref-type=\"bibr\">Yang et al., 2016</xref>), increasing antioxidant production (<xref rid=\"B108\" ref-type=\"bibr\">Meydani et al., 2011</xref>), and increasing autophagy to remove damaged and unimportant intracellular components (reviewed in <xref rid=\"B33\" ref-type=\"bibr\">Cuervo, 2008</xref>). Moreover, sirtuins may play important roles in preventing age-related decline in skin repair, as SIRT1 deficiency exacerbates healing pathology in diabetic wounds (<xref rid=\"B158\" ref-type=\"bibr\">Thandavarayan et al., 2015</xref>).</p><p>Although caloric restriction (without malnutrition) provides a multitude of health benefits, it retains poor feasibility as a clinical intervention, requiring high compliance and patient discipline. Many lifestyle choices, such as obesity, are actually strongly associated with social status (<xref rid=\"B44\" ref-type=\"bibr\">Drewnowski and Specter, 2004</xref>). Similarly, those suffering from uncontrolled type II diabetes and severe chronic wounds are often from socially deprived backgrounds (<xref rid=\"B6\" ref-type=\"bibr\">Anderson et al., 2018</xref>), a difficult population in which to manage compliance. For all of these reasons, a considerably more attractive proposition is the use of senescence-targeted drugs, otherwise known as senolytics. These drugs affect unique features of senescent cells, such as resistance to apoptosis (<xref rid=\"B138\" ref-type=\"bibr\">Salminen et al., 2011</xref>). Senescent cells upregulate prosurvival pathways, particularly BCL-2 (<xref rid=\"B119\" ref-type=\"bibr\">Ovadya and Krizhanovsky, 2018</xref>). This opens up drug repurposing opportunities around the numerous BCL-2 inhibitors that were developed for the treatment of cancer (<xref rid=\"B131\" ref-type=\"bibr\">Roberts et al., 2016</xref>; <xref rid=\"B113\" ref-type=\"bibr\">Montero and Letai, 2018</xref>). Results have been promising. Targeting BCL-2 <italic>in vivo</italic> induces apoptosis and thus eliminates senescent cells in the lung following irradiation (<xref rid=\"B187\" ref-type=\"bibr\">Yosef et al., 2016</xref>) and throughout the body following irradiation or natural ageing (<xref rid=\"B20\" ref-type=\"bibr\">Chang et al., 2016</xref>). <xref rid=\"B20\" ref-type=\"bibr\">Chang et al. (2016)</xref> further established that senescent human and murine fibroblasts, and human renal epithelial cells, are more susceptible to BCL-2 inhibitor (ABT-263) than non-senescent cells, proposing potent and specific effects. Unfortunately, traditional BCL-2 inhibitors possess activity against other BCL class proteins, such as BCL-XL, raising questions around off-target effects in the clinic, including thrombocytopenia and neutropenia. As a result, more specific BCL-2 inhibitors with lower toxicity are being tested (<xref rid=\"B85\" ref-type=\"bibr\">King et al., 2017</xref>). It has even been suggested that low-dose, combinatorial use of senolytics may be an effective and less harmful alternative (<xref rid=\"B119\" ref-type=\"bibr\">Ovadya and Krizhanovsky, 2018</xref>).</p><p>Other senolytics that have demonstrated experimental efficacy include the tyrosine kinase inhibitor, Dasatinib, used to treat leukaemia (<xref rid=\"B82\" ref-type=\"bibr\">Keskin et al., 2016</xref>), and the flavonoid p53 activator, Quercetin (<xref rid=\"B83\" ref-type=\"bibr\">Khan et al., 2016</xref>). Combinatorial treatment with Dasatinib and Quercetin extends lifespan, alleviates frailty (<xref rid=\"B182\" ref-type=\"bibr\">Xu et al., 2018</xref>), and improves vasomotor function (<xref rid=\"B134\" ref-type=\"bibr\">Roos et al., 2016</xref>) in aged mice. Dasatinib and Quercetin have also shown promise in a phase I trial in diabetic kidney disease patients, where reduced senescent cells and circulating SASP factors were observed following administration (<xref rid=\"B66\" ref-type=\"bibr\">Hickson et al., 2019</xref>). Alternative flavonoids are now being tested for their potential senolytic effects, such as Fisetin, which is able to eliminate senescent cells and, crucially, restore tissue function in aged mice (<xref rid=\"B189\" ref-type=\"bibr\">Yousefzadeh et al., 2018</xref>).</p><p>The importance of transient senescence for effective healing should not be underestimated. As noted previously, temporary induction of senescence aids rapid tissue reformation (<xref rid=\"B37\" ref-type=\"bibr\">Demaria et al., 2014</xref>; <xref rid=\"B67\" ref-type=\"bibr\">Hiebert et al., 2018</xref>). During a normal damage response, these senescent cells are effectively cleared by natural killer cells (<xref rid=\"B89\" ref-type=\"bibr\">Krizhanovsky et al., 2008</xref>) and macrophages (<xref rid=\"B190\" ref-type=\"bibr\">Yun et al., 2015</xref>). Nevertheless, in chronic situations, senescent cells persist, likely due to elevated immunosenescence and resulting impaired immunological functions (<xref rid=\"B56\" ref-type=\"bibr\">Hall et al., 2016</xref>). It follows that treatments to boost immune system function, for instance by aiding senescent cell recognition, could be beneficial in the context of transient senescence and tissue repair. Generally, senescent cells express stimulatory ligands that bind to NK2GD receptors on natural killer cells, thus initiating a killing response (<xref rid=\"B137\" ref-type=\"bibr\">Sagiv et al., 2016</xref>). However, senescent fibroblasts in aged skin have recently been shown to express HLA-E, which bypasses recognition and clearance by natural killer and T cells (<xref rid=\"B123\" ref-type=\"bibr\">Pereira et al., 2019</xref>). Here, approaches developed in the cancer field may also be useful, for example engineering T cells to express receptors that target specific cellular (tumour) proteins (reviewed in <xref rid=\"B77\" ref-type=\"bibr\">June et al., 2018</xref>). Studies to identify and validate new senescent cell receptors will be essential to the development and clinical application of such immune-regulated approaches.</p><p>Indeed, the emergence of global profiling methodologies, such as single-cell RNA sequencing, could provide the basis to understanding senescence-linked changes in ageing and pathology by identifying unique cell-based transcriptomic signatures within tissues. <xref rid=\"B84\" ref-type=\"bibr\">Kimmel et al. (2019)</xref> used this approach to compare cell frequency, heterogeneity and age-related transcriptomic changes between aged and young murine tissues. Similarly, <xref rid=\"B7\" ref-type=\"bibr\">Angelidis et al. (2019)</xref> combined transcriptomics and proteomics to not only identify the epigenetic and transcriptional consequences of ageing in the lung, but also determine their functional implications. Future harnessing of these technologies could therefore facilitate the identification and targeting of key senescence-linked receptors and biomarkers in a tissue and pathology-specific manner.</p><p>Alternative strategies to diminish or limit senescence and alleviate pathology instead target the SASP or specific senescence-linked receptors directly (summarised in <xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). Certainly, the SASP is transcriptionally regulated by NF-κB and others (<xref rid=\"B138\" ref-type=\"bibr\">Salminen et al., 2011</xref>), and contributes heavily to tissue deterioration, both driving widespread destruction and reinforcing senescence (<xref rid=\"B133\" ref-type=\"bibr\">Rodier et al., 2009</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Acosta et al., 2013</xref>). SASP inhibitors affect key transcriptional mediators, blocking signalling and preventing SASP production (<xref rid=\"B112\" ref-type=\"bibr\">Moiseeva et al., 2013</xref>). Interestingly, Metformin, a widely used anti-diabetic drug, is an effective SASP inhibitor (reviewed in <xref rid=\"B127\" ref-type=\"bibr\">Rena et al., 2017</xref>) able to directly accelerate healing in diabetic mice (<xref rid=\"B59\" ref-type=\"bibr\">Han et al., 2017</xref>). Rapamycin, another SASP inhibitor, was the first drug revealed to extend lifespan in mice (<xref rid=\"B60\" ref-type=\"bibr\">Harrison et al., 2009</xref>), and enhance the replicative lifespan of human keratinocytes (<xref rid=\"B69\" ref-type=\"bibr\">Horvath et al., 2019</xref>) and skin fibroblasts <italic>in vitro</italic> (<xref rid=\"B147\" ref-type=\"bibr\">Sodagam et al., 2017</xref>). Although these studies suggest potential skin-related benefits of SASP inhibitors, removal of the SASP could be deleterious, impairing the healing response and preventing senescent cell clearance (<xref rid=\"B165\" ref-type=\"bibr\">von Kobbe, 2019</xref>). Consequently, it may be more advantageous to target particular SASP components known to impact tissue function, either with antibodies (e.g., IL-1α, <xref rid=\"B118\" ref-type=\"bibr\">Orjalo et al., 2009</xref>), or specific inhibitors (e.g., against CXCR2, <xref rid=\"B176\" ref-type=\"bibr\">Wilkinson et al., 2019a</xref>).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Therapeutic targeting of senescence for chronic healing wounds. Senescent cells accumulate in chronic healing wounds, contributing to inflammation and poor healing. Senescence can be targeted by: <bold>(A)</bold> inhibiting pro-survival pathways with BCL inhibitors and broad spectrum drugs (e.g., quercetin) to cause apoptosis; <bold>(B)</bold> engineering chimeric antigen receptor (CAR) T cells to target senescent cell receptors, or modulating expression of natural killer (NK) cell receptors NKG2A and NKG2D to increase clearance; <bold>(C)</bold> using Metformin or other SASP inhibitors to reduce NF-κB-mediated inflammation and bystander senescence and; <bold>(D)</bold> inhibiting receptors known to potentiate wound senescence (e.g., CXCR2). Red arrows/left panels = negative outcomes. Green arrows/right panels = positive outcomes. MΦ= macrophage. Senescent cells = blue.</p></caption><graphic xlink:href=\"fcell-08-00773-g003\"/></fig><p>We remain a long way from implementing senescence-targeted treatments for pathological wound healing, yet it is reassuring to see that current senolytic drugs display efficacy across a wide range of tissues and pathologies. In a number of studies, systemic senolytic treatments have been shown to have clear effects in peripheral target tissues across a range of treatment regimens. For example, a single dose of BCL inhibitor (<xref rid=\"B187\" ref-type=\"bibr\">Yosef et al., 2016</xref>), and dosing over consecutive days (<xref rid=\"B20\" ref-type=\"bibr\">Chang et al., 2016</xref>), was able to reverse irradiation-induced senescence in different tissues. In the work by <xref rid=\"B182\" ref-type=\"bibr\">Xu et al. (2018)</xref>, aged mice showed improved physical performance following biweekly oral treatments of Dasatinib and Quercetin for 4 months, yet reduced SASP was observed in human <italic>ex vivo</italic> cultured adipose tissue within 48 h of treatment. Moreover, a single 3 day oral treatment of Dasatinib and Quercetin was able to reduce senescence in the adipose tissue of diabetic patients in a phase I trial (<xref rid=\"B66\" ref-type=\"bibr\">Hickson et al., 2019</xref>). These studies therefore suggest that senolytic treatments not only have rapid effects in target peripheral tissues, but can overcome established tissue senescence.</p><p>Experimental studies do show beneficial effects of modulating senescence in the skin. For example, elimination of senescent cells from the epidermis restored proliferative capacity in hair follicle stem cells (<xref rid=\"B187\" ref-type=\"bibr\">Yosef et al., 2016</xref>), known to participate in wound healing (<xref rid=\"B74\" ref-type=\"bibr\">Joost et al., 2018</xref>). Further, blockade of the potential senescence receptor, CXCR2 (<xref rid=\"B2\" ref-type=\"bibr\">Acosta et al., 2008</xref>), directly accelerated healing in human <italic>ex vivo</italic> skin wounds and diabetic murine wounds <italic>in vivo</italic> (<xref rid=\"B176\" ref-type=\"bibr\">Wilkinson et al., 2019a</xref>). Here, a CXCR2 antagonist was administered to wounds topically (<italic>ex vivo</italic>) and subcutaneously (<italic>in vivo)</italic>, suggesting direct delivery to the wound site as a viable administration route. Indeed, elevated CXCR2 has previously been observed in diabetic wounds (<xref rid=\"B175\" ref-type=\"bibr\">Wetzler et al., 2000</xref>), and more recently in T cells from human diabetic patients (<xref rid=\"B92\" ref-type=\"bibr\">Lau et al., 2019</xref>). We note with interest that pharmacological inhibition of CXCR1/2 additionally prevents inflammation-mediated damage to pancreatic islets, thus prohibiting streptozocin-induced diabetes in mice (<xref rid=\"B25\" ref-type=\"bibr\">Citro et al., 2015</xref>). Therefore, CXCR2 appears a common factor in both the ontology and local pathology of diabetes. Senolytics should certainly be considered for the treatment of human chronic wounds characterised by high levels of senescence (<xref rid=\"B149\" ref-type=\"bibr\">Stanley and Osler, 2001</xref>). However, given that knockdown of CXCR2 (<xref rid=\"B39\" ref-type=\"bibr\">Devalaraja et al., 2000</xref>) and ablation of senescent cells (<xref rid=\"B37\" ref-type=\"bibr\">Demaria et al., 2014</xref>) actually delays acute wound healing, future senescence-targeted therapies should be reserved for the treatment of chronic conditions.</p></sec><sec id=\"S8\"><title>Conclusion</title><p>Despite seemingly contradictory roles in many cancers, the detrimental contribution of cumulative senescence to ageing and age-related disease is now well-established. By contrast, the short-lived, transient senescence observed to benefit tissue development, regeneration and repair, remains significantly less well-characterised. In wound repair, a paradigm is emerging where local transient senescence predominately constrains fibrosis, while chronic senescence drives diabetic wound pathology. Indeed, experimentally blocking the senescence-linked receptor, CXCR2, <italic>in vivo</italic> reverses pathology and accelerates diabetic healing. These observations now pave the way to explore the beneficial effects of senescence-targeted therapies for the treatment of chronic wounds.</p></sec><sec id=\"S9\"><title>Author Contributions</title><p>HW wrote the manuscript and prepared the figures. MH provided critical appraisal. Both authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by the Medical Research Council (United Kingdom) Ph.D. studentship (MR/M016307/1).</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Acosta</surname><given-names>J. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Med (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Med (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Med.</journal-id><journal-title-group><journal-title>Frontiers in Medicine</journal-title></journal-title-group><issn pub-type=\"epub\">2296-858X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850892</article-id><article-id pub-id-type=\"pmc\">PMC7431695</article-id><article-id pub-id-type=\"doi\">10.3389/fmed.2020.00376</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Medicine</subject><subj-group><subject>Mini Review</subject></subj-group></subj-group></article-categories><title-group><article-title>MicroRNAs in Synovial Pathology Associated With Osteoarthritis</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Tavallaee</surname><given-names>Ghazaleh</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1007784/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Rockel</surname><given-names>Jason S.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1034417/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Lively</surname><given-names>Starlee</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/152915/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Kapoor</surname><given-names>Mohit</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/956294/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Arthritis Program, Krembil Research Institute, University Health Network</institution>, <addr-line>Toronto, ON</addr-line>, <country>Canada</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Laboratory Medicine and Pathobiology, University of Toronto</institution>, <addr-line>Toronto, ON</addr-line>, <country>Canada</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Surgery, University of Toronto</institution>, <addr-line>Toronto, ON</addr-line>, <country>Canada</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Francisco Airton Castro Rocha, Federal University of Ceará, Brazil</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Mandy J.Peffers, University of Liverpool, United Kingdom; Rita A. Moura, Universidade de Lisboa, Portugal</p></fn><corresp id=\"c001\">*Correspondence: Mohit Kapoor <email>mohit.kapoor@uhnresearch.ca</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Rheumatology, a section of the journal Frontiers in Medicine</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>7</volume><elocation-id>376</elocation-id><history><date date-type=\"received\"><day>17</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>18</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Tavallaee, Rockel, Lively and Kapoor.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Tavallaee, Rockel, Lively and Kapoor</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Osteoarthritis (OA) is the most common type of arthritis, a disease that affects the entire joint. The relative involvement of each tissue, and their interactions, add to the complexity of OA, hampering our understanding of the underlying molecular mechanisms, and the generation of a disease modifying therapy. The synovium is essential in maintaining joint homeostasis, and pathologies associated with the synovium contribute to joint destruction, pain and stiffness in OA. MicroRNAs (miRNAs) are post-transcriptional regulators dysregulated in OA tissues including the synovium. MiRNAs are important contributors to OA synovial changes that have the potential to improve our understanding of OA and to act as novel therapeutic targets. The purpose of this review is to summarize and integrate current published literature investigating the roles that miRNAs play in OA-related synovial pathologies including inflammation, matrix deposition and cell proliferation.</p></abstract><kwd-group><kwd>osteoarthritis</kwd><kwd>synovium</kwd><kwd>microRNA</kwd><kwd>inflammation</kwd><kwd>fibrosis</kwd></kwd-group><counts><fig-count count=\"1\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"72\"/><page-count count=\"8\"/><word-count count=\"6536\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Osteoarthritis (OA) is the most common chronic debilitating disease imposing a significant socioeconomic burden and affecting the quality of life of millions of people worldwide (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). OA affects the whole joint and involves progressive articular cartilage degradation, subchondral bone remodeling, ectopic bone formation, ligament degeneration, menisci degradation, and synovial inflammation and hypertrophy (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). Many OA studies largely focus on cartilage health as it facilitates joint movement and is highly susceptible to OA; however other tissues, notably the synovium, are now recognized to be involved in OA pathology (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B4\" ref-type=\"bibr\">4</xref>). OA alters the homeostatic functions of cells residing in the synovium, but we are only starting to elucidate the underlying gene expression and regulatory mechanisms responsible, and how these changes contribute to disease progression. Gene expression profiles of the synovium are also altered during OA, which is accomplished by multiple regulatory mechanisms. At the post-transcriptional level, gene transcripts are regulated by a class of small non-coding RNAs called microRNAs (miRNAs). A single miRNA can target a large number of transcripts contributing to tissue specific gene expression (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). The complex network of miRNAs that regulate the pathophysiology of cartilage degeneration during OA has been previously reviewed (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>); however, very little is known regarding the role of miRNAs in regulating synovial gene expression during OA. In this review, we summarize the contributions of the synovium to OA pathology and how focusing on the role of miRNAs in regulating the activity of fibroblast-like synoviocytes (FLS) warrants further study to further elucidate mechanisms contributing to OA pathologies.</p></sec><sec id=\"s2\"><title>Cellular Interactions in the OA Synovium</title><p>The synovium is a loose connective tissue that encapsulates the joint and aids in maintaining joint homeostasis through the functions of its resident cells: synovial macrophages and the more abundant FLS [reviewed in (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B4\" ref-type=\"bibr\">4</xref>)]. FLS are mesenchyme-derived cells that share characteristics with other fibroblasts, such as the expression of collagens IV and V, vimentin and CD90, but also show unique expression that differentiates them from other resident fibroblasts, notably cadherin-11 expression by FLS in the synovial lining (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). In healthy synovium, FLS are the major source of extracellular matrix (ECM) and synovial fluid, while resident macrophages remove metabolites and products of matrix degradation (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). As OA progresses, the synovium undergoes hyperplasia, sublining fibrosis, increased vascularization, and increased cell proliferation, migration and invasion (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>).</p><p>In the context of OA, FLS are the major contributors to the observed excessive synovial ECM deposition and fibrosis (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). While they are involved in the production of proinflammatory and profibrotic mediators, resident synovial macrophages also respond and contribute to OA progression and inflammatory responses (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). Accumulation of macrophages in the synovium is a defining characteristic of synovitis, notably adjacent to areas of cartilage degradation (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Macrophages are highly plastic cells; and although a broad spectrum of activated states exists, macrophages are generally classified as pro-inflammatory (M1) and inflammatory resolving (M2) (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). In healthy conditions, macrophages are thought to be in an M2-like phenotype that maintains tissue homeostasis and repair (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Inflammatory mediators, such as interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α), as well as catabolic enzymes, such as matrix metalloproteases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs), are produced by synoviocytes and secreted into synovial fluid in quantifiable levels (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). These changes contribute to the excessive ECM deposition and increased synovial thickness detected in OA patients and animal models, and impact joint integrity.</p><p>In the synovial fluid of patients with knee OA, the balance of M1 and M2 macrophage markers is skewed toward a pro-inflammatory state, and the degree of the shift is positively associated with the OA severity (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>). In the OA synovium, the majority of macrophages possess pro-inflammatory profiles, driving responses that promote synovitis and osteophyte formation (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B15\" ref-type=\"bibr\">15</xref>). In addition to modulating local inflammatory responses, activated macrophages secrete various MMPs and ADAMTSs, which remodel the synovial matrix, and enhance fibrosis-promoting activities of FLS (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). The master driver of fibrosis is transforming growth factor-beta 1 (TGF-β1) as it stimulates FLS expression of other profibrotic mediators, including α-smooth muscle actin (α-SMA), vascular endothelial growth factor (VEGF), procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), tissue inhibitors of metalloproteinases-1 (TIMP-1), and collagen type I, as well as OA FLS proliferation and migration (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). FLS in turn influence macrophage activity (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). Thus, interactions of FLS with macrophages can also contribute to the pathological changes in the synovium during OA and is an important consideration for future studies.</p></sec><sec id=\"s3\"><title>miRNA Biogenesis and Function</title><p>MicroRNAs (miRNAs) are single stranded endogenous small non-coding RNA molecules of 21–24-nucleotide (nt) length that are transcribed by RNA polymerase II. MiRNAs are expressed in polyadelynated and capped nascent transcripts ~ 200 nt (pri-miRNA) with hairpin structures. Pri-miRNAs are recognized by DiGeorge syndrome critical region gene 8 (Dgcr8, an RNA binding protein) and cleaved into ~70 nt stem loop precursors (pre-miRNA) in the nucleus by Drosha, a nuclease of the RNase III family, and transported to cytoplasm by Exportin 5. Pre-miRNAs are processed into miRNA duplexes in the cytoplasm by the enzyme Dicer. One strand (mature miRNA) asymmetrically assembles into the Argonaute (AGO) protein of the RNA-induced silencing complex (RISC) and the other one is destroyed. Mature miRNAs then bind mRNAs of target genes in a sequence-specific manner via “seed” sequences, 2–8 nucleotides from the 5′ end of miRNAs, usually resulting in cleavage of target mRNAs or translational repression [reviewed in detail in (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B20\" ref-type=\"bibr\">20</xref>)].</p></sec><sec id=\"s4\"><title>miRNAs in OA Synovial Pathology</title><p>OA studies to date mostly focus on the role of miRNAs in regulating cartilage maintenance and degradation. However, miRNAs also regulate other aspects of OA, including synovial pathology. This is an understudied area and consequently, much less is known. For the purpose of this review, we searched PubMed using “Osteoarthritis + synovium + miRNA” and “Osteoarthritis + synovitis + miRNA” for studies published until March 2020. A total of 83 articles were identified. Thirty-five articles focused exclusively on articular cartilage or tissues other than synovium or on OA symptoms, rather than synovial pathologies, leaving 48 articles relevant to this review.</p><p>Considering FLS as essential participants in joint homeostasis and contributors to OA synovial pathology, it is not surprising that OA FLS show differential miRNA profiles. Recently, deep sequencing identified 245 differentially expressed genes in OA FLS and bioinformatics analyses highlighted “ECM organization and altered cellular movement” as one of the most enriched OA FLS functions connected to the differentially expressed genes and miRNA network (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). OA FLS also exhibit an independent miRNA signature from rheumatoid arthritis (RA) FLS, negatively correlating to the expression levels of their putative target genes (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). Elevated levels of miR-625 and miR-124 in OA FLS are associated with decreased expression of their target genes, while miR-155b and miR-203 are expressed at lower levels concomitant with higher expression of their target genes (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). In addition to <italic>in vitro</italic> studies, animal models aid in the understanding of differentially expressed miRNAs in OA synovium. Kung et al. found 394 miRNAs transiently expressed at 1 vs. 6 weeks in the synovium of the destabilization of the medial meniscus (DMM) mouse model of knee OA (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). Thus, several miRNAs modulated in the synovium potentially contribute to joint destruction, synovial inflammation, and fibrosis (summarized in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> and <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). However, the individual and combined contributions of these miRNAs to synovial pathology warrant further investigation to comprehensively understand their role and signaling mechanisms in OA.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Role of some miRNAs in the synovial pathology during OA.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>MiRNA</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Species/model system</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Role in OA</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>References</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-181c</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human <break/> OA FLS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Suppresses expression of MMP13, IL-6, and IL-8 and targets OPN to reduce FLS proliferation.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B24\" ref-type=\"bibr\">24</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-770</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human <break/> OA FLS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Suppresses proliferation of OA FLS.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B25\" ref-type=\"bibr\">25</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-26a-5p</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human <break/> OA FLS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Targets COX2 to reduce Bcl-2, IL-6, TNF-α, and IL-8 expression.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B26\" ref-type=\"bibr\">26</xref>)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rat instability model of OA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Alleviates synovial inflammation.</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-146a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human <break/> OA FLS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dampens IL-1β signaling.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B27\" ref-type=\"bibr\">27</xref>–<xref rid=\"B29\" ref-type=\"bibr\">29</xref>)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mouse <break/> Knockout</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exhibits synovial hyperplasia.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B30\" ref-type=\"bibr\">30</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-122</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human <break/> OA FLS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reduces IL-1α levels.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B31\" ref-type=\"bibr\">31</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-381a-3p</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human <break/> OA FLS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Targets IκBα to enhance NF-κB activity.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B32\" ref-type=\"bibr\">32</xref>)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rat <break/> MIA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Upregulates in the synovium of MIA rats.</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-34a <break/> miR-146a <break/> miR-181a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human <break/> OA FLS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Promote inflammatory mechanisms and oxidative stress.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B33\" ref-type=\"bibr\">33</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-29a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human <break/> OA FLS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Targets VEGF and suppresses ECM production.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B34\" ref-type=\"bibr\">34</xref>)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mouse <break/> CIOA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protects the synovium from hyperplasia and macrophage infiltration.</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-338-3p</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human <break/> OA FLS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Targets TRAP-1 to regulate TGF-β responsive genes.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B35\" ref-type=\"bibr\">35</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-125</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human <break/> HUVEC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Enhances glycolysis and angiogenesis.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B36\" ref-type=\"bibr\">36</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-128</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mouse <break/> ACLT</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Promotes synovial membrane thickness and fibroblast activation.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B37\" ref-type=\"bibr\">37</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">miR-101</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rats <break/> MIA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MiR-101 inhibition reduces cytokine expression in the MIA rats synovium.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B38\" ref-type=\"bibr\">38</xref>)</td></tr></tbody></table><table-wrap-foot><p><italic>ACLT, anterior cruciate ligament transection; CIOA, collagenase-induced osteoarthritis; FLS, fibroblast-like synoviocytes; HUVEC, human umbilical vein endothelial cells; OA, osteoarthritis; MIA, monosodium iodoacetate; ECM, extracellular matrix; MMP13, matrix metalloprotease 13; IL-6, interleukin-6; IL-8, interleukin-8; OPN, osteopontin; COX2, cyclooxygenase-2; Bcl-2, B-cell lymphoma-2; TNF-α, tumor necrosis factor alpha; IL-1β, interleukin-1 beta; IL-1α, interleukin-1 alpha; IκBα, inhibitor of nuclear factor kappa B alpha; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; VEGF, vascular endothelial growth factor; TRAP-1, TNF receptor-associated protein-1; TGF-β, transforming growth factor-beta</italic>.</p></table-wrap-foot></table-wrap><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Schematic showing some miRNAs involved in human OA synovial pathology. MiR-381a-3p, miR-34a, miR-146a, and miR-181a promote inflammatory mechanisms (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B33\" ref-type=\"bibr\">33</xref>). MiR-26a-5p, miR-146a, miR-122, and miR-181c suppress the expression of inflammatory cytokines (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>, <xref rid=\"B26\" ref-type=\"bibr\">26</xref>, <xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B31\" ref-type=\"bibr\">31</xref>). MiR-181c and miR-770 suppress fibroblast-like synoviocyte proliferation (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>, <xref rid=\"B25\" ref-type=\"bibr\">25</xref>). MiR-29a and miR-338-3p exhibit anti-fibrotic effects (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>, <xref rid=\"B35\" ref-type=\"bibr\">35</xref>).</p></caption><graphic xlink:href=\"fmed-07-00376-g0001\"/></fig></sec><sec id=\"s5\"><title>miRNAs and Synovial Inflammation in OA</title><p>MiRNAs play key roles in OA-related synovial inflammation. The expression levels of inflammatory-related miRNAs measured in the synovium from OA patients and animal models show unique signatures when compared to normal controls. When comparing inflamed areas with normal areas of synovium from OA patients, 31 miRNAs are identified in an OA-specific regulatory network comprised of 97 interactions of 38 transcription factors and 35 genes (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>). Many miRNAs are upregulated during OA that exacerbate inflammatory responses in the synovium. MiR-381a-3p is upregulated in the synovium of both OA patients and in the monosodium iodoacetate (MIA)-injected rat model of OA pain; and miR-381a-3p enhances nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity in cultured human OA FLS by targeting inhibitor of nuclear factor kappa B alpha (IκBα) (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). Inhibition of miR-101 in MIA-injected rats reduces cytokine expression in the synovium (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>). Furthermore, blocking miR-128 reduces both synovial membrane thickness and fibroblast activation protein (FAP)-positive FLS accumulation in the mouse anterior cruciate ligament transection (ACLT) model of OA (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>). Thus, fine-tuning synovial inflammation through miRNA modulation is a promising avenue of research for future OA therapeutic targets.</p><p>Some miRNAs have been shown to exhibit anti-inflammatory effects in the synovium during OA. For instance, administration of human bone mesenchymal stem cell-derived exosomes overexpressing miR-26a-5p to cultured OA FLS targets cyclooxygenase-2 (COX2), reducing B-cell lymphoma 2 (Bcl-2), IL-6, TNF-α and IL-8 expression, and increasing Bcl-2-associated X protein (Bax) expression and caspase cleavage, alleviating synovial inflammation in a rat joint instability model of OA (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). MiR-146a is highly expressed in the synovium during OA and when knocked-down in mouse models, NOTCH1 and IL-6 expression are increased in the synovium concomitant with synovial hyperplasia (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). When overexpressed in OA FLS, miR-146a decreases the expression of inflammatory mediators, including IL-1-induced TNF receptor associated factor 6 (TRAF 6), IL-1 receptor-associated kinase 1 (IRAK 1), COX2, IL-8, MMP13, and ADAMTS5 expression (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). Denbinobin, a naturally occurring 1,4-phenanthrenequinone, promotes histone acetyltransferase activity, resulting in increased miR-146a expression and inhibition of nuclear factor (NF)-κB activity, dampening IL-1β-elicited expression of cell adhesion molecules and monocyte adhesion to OA FLS (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). Intriguingly, histone deacetylase inhibitors also promote miR-146a expression in IL-1β-treated OA FLS by facilitating NF-κB binding to miR-146a promoter, which reduces downstream responses including IL-6 secretion (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). Thus, the acetylation pattern of miR-146a is an important aspect to its expression and function in OA FLS. MiR-122 is another miRNA with anti-inflammatory potential as its overexpression in OA FLS reduces IL-1α levels (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). Taken together, miRNAs have the potential to regulate inflammation positively or negatively in the OA synovium; but timing, source, and context of their expression in relation to OA-related inflammatory responses needs to be better understood.</p><p>RA FLS have been shown to mount greater inflammatory responses compared to OA FLS, expressing higher levels of certain inflammation-inducing miRNAs. For instance, miR-146a, miR-155, and miR-223 are expressed at higher levels in synovial tissues and RNA extracted from paraffin embedded RA synovial sections relative to OA samples (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>, <xref rid=\"B41\" ref-type=\"bibr\">41</xref>). OA tissue is routinely used as control comparisons in these instances. As a result, much more is known about the role of miRNA in RA FLS and synovial tissue. In RA FLS, miR-155 suppresses MMP1 and MMP3 expression (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>). Inhibition of miR-155 in synovial fluid-derived macrophages reduces TNF-α production <italic>in vitro</italic> (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>). Mir-221-3p is also expressed at higher levels in RA synovial tissue and fluid, and inhibits the anti-inflammatory arm of macrophages by suppressing the JAK3/STAT3 axis and increasing the expression of inflammatory mediators such as IL-6 and IL-8 (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>). Similarly, miR-145-5p and miR-143-3p are expressed at higher levels in RA synovium and FLS compared to OA (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>). MiR-145-5p targets osteoprotegerin, aggravating bone erosion in collagen-induced arthritis, and also regulates semaphorin 3A (SEMA3A) to modulate the phenotype of RA FLS (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>, <xref rid=\"B46\" ref-type=\"bibr\">46</xref>). MiR-143-3p targets insulin-like growth factor1 receptor (IGF1R) and insulin-like growth factor binding protein 5 (IGFBP5) expression, regulating the Ras/p38 MAPK signaling pathway, contributing to FLS proliferation and apoptosis (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>, <xref rid=\"B47\" ref-type=\"bibr\">47</xref>). Additionally, miR-203 promotes NF-κB activation and secretion of MMP1 and IL-6, thereby accelerating RA FLS activation (<xref rid=\"B48\" ref-type=\"bibr\">48</xref>). Overall, miRNAs clearly modulate the inflammatory profile of synovial macrophages and FLS in RA.</p><p>However, it is now appreciated that OA FLS exhibit an independent miRNA signature from RA FLS (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). Intriguingly, several miRNAs that negatively regulate inflammation or FLS proliferation are expressed at higher levels in OA synovium and FLS compared to RA, including miR-34a-3p, miR-124a, miR-30a, miR-10a, miR-140-3p, and miR-140-5p (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>–<xref rid=\"B53\" ref-type=\"bibr\">53</xref>). MiR-34a-3p expression is decreased in RA FLS, leading to increased inflammation and proliferation (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>). Downregulation of miR-34a passenger strand (miR-34a<sup>*</sup>) in RA FLS, due to methylation of its promoter, promotes apoptosis resistance (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>). MiR-124a also suppresses proliferation and inflammation by directly targeting cyclin-dependent kinase 2 (CDK-2) and monocyte chemoattractant protein-1 (MCP-1) in RA FLS (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>). Furthermore, decreased levels of miR-30a in RA synovium correlate with reduced apoptosis and enhanced autophagy (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>), while lower expression of miR-10a is thought to promote excessive secretion of inflammatory cytokines via NF-κB regulation (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>). OA is considered a low-grade inflammatory disease compared to RA or other types of inflammatory arthritis (<xref rid=\"B55\" ref-type=\"bibr\">55</xref>); thus, it is not surprising that many miRNAs are differentially expressed in RA compared to OA synovial cells. However, it does not preclude the possibility that these miRNAs also contribute to synovial inflammation and OA progression. Detailed comparisons of the differential miRNA profiles detected in RA and OA synovial cells coupled with mechanistic studies could offer a jumping point for future investigations into their contributions to OA pathogenesis.</p></sec><sec id=\"s6\"><title>miRNAs and Synovial Fibrosis</title><p>In general, a limited number of studies have investigated the role of miRNAs in processes associated with OA synovial fibrosis. For instance, miR-29a targets VEGF and its inhibition in OA FLS promotes the expression of ECM genes (collagen III, TGF-β1, PLOD2, TIMP1, ADAM12, MMP9, MMP13, and ADAMTS5) (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). Conversely, miR-29a overexpression decreases VEGF and ECM gene expression levels. In a mouse model of collagenase-induced OA (CIOA), intra-articular administration of a miR-29a precursor protects the synovium from hyperplasia and macrophage infiltration (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). Thus, miR-29a, which is decreased in OA synovium, appears to reduce profibrotic activities in the healthy synovium by tightly regulating angiogenesis and ECM production. MiR-338-3p is another ECM-regulating miRNA decreased in OA synovium and synovial effusions compared to synovial tissues from patients with joint trauma. MiR-338-3p counteracts TGF-β1-induced expression of vimentin, type I collagen and TIMP1 in FLS by directly targeting TNF receptor-associated protein 1 (TRAP-1) and regulating Smad 2/3 signaling pathways (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). Overall, these miRNAs exhibit anti-fibrotic regulatory effects; however, there are likely more miRNAs with similar activities that remain to be identified in addition to miRNAs with profibrotic effects that exacerbate synovitis associated with OA.</p><p>Profibrotic mediators have also been shown to regulate miRNA expression, contributing to OA synovial pathology. For instance, TGF-β1 enhances the expression of anti-inflammatory factor hemeoxygenase 1 (HO-1) by reducing the expression of miRNA-519b in human OA FLS (<xref rid=\"B56\" ref-type=\"bibr\">56</xref>). TGF-β1 also inhibits miR-92a to promote the expression of forkhead box class O 3 (FOXO3) in OA FLS, lowering mRNA and protein levels of TNF-α, IL-1β, VEGF, and C-C Motif Chemokine Ligand 2 (CCL2) (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>). Another profibrotic growth factor, connective tissue growth factor (CTGF), increases miR-210 expression in OA FLS by activation of PI3K, AKT, ERK, and NF-κB/ELK1 pathways, contributing to VEGF-dependent angiogenesis (<xref rid=\"B58\" ref-type=\"bibr\">58</xref>). It is noteworthy that profibrotic mediators such as TGF-β1 and CTGF modulate select miRNAs to regulate certain aspects of synovitis including inflammation and angiogenesis. This effect can be counteracted by other miRNAs. MiR-125a is expressed at higher levels in OA synovium compared to psoriatic arthritis and modulates glycolysis in human umbilical vein endothelial cells (HUVEC) to inhibit angiogenesis (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). MiRNAs are dysregulated in the synovium during OA, but the way in which they regulate inflammation, angiogenesis or ECM modulation, and how they interact to maintain the joint homeostasis, remains poorly understood and requires extensive investigation in near future.</p></sec><sec id=\"s7\"><title>Mechanisms Regulating miRNAs in OA Synovium</title><p>Adipocyte-derived molecules (adipokines) are elevated in the joint during OA and play an important role in cartilage and bone turnover (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>). In addition, adipokines alter miRNA expression levels, modulating synovial inflammatory responses. Visfatin and resistin upregulate miR-34a, miR-146a and miR-181a in OA FLS, which when inhibited, decreases proinflammatory responses and oxidative stress (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>). Adipokines can also inhibit miRNAs to enhance inflammatory responses. For instance, visfatin inhibits miR-199a-5p expression in OA FLS through ERK, p38, and JNK signaling pathways, which promotes IL-6 and TNF-α production (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>). Similarly, resistin suppresses miR-33a and miR-33b in OA FLS resulting in increased MCP-1 transcription, facilitating the migration of monocytes (<xref rid=\"B61\" ref-type=\"bibr\">61</xref>). Thus, select miRNAs are regulated by adipokines influencing OA-related inflammatory responses.</p><p>Just as miRNAs regulate mRNAs, miRNAs are also regulated through interaction with RNA partners, specifically long non-coding (lnc) RNAs and circular (circ) RNAs (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>, <xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B62\" ref-type=\"bibr\">62</xref>). Both act as sponges, binding directly to miRNAs and regulating their free concentration. Evidence suggests that these regulatory RNAs have the capacity to fine-tune miRNA activity in OA FLS. For example, lncRNA nuclear enriched abundant transcript 1 (NEAT1) binds miR-181c, inhibiting osteopontin (OPN) expression and regulating OA FLS proliferation (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). Similarly, the lncRNA prostate cancer gene expression marker 1 (PCGEM1) binds miR-770, promoting OA FLS proliferation and survival (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). In fact, 122 circRNAs are differentially expressed in the OA synovium, with over 1,000 miRNAs and 28,000 circRNA-miRNA interaction pairs. Intriguingly, 641 miRNAs are predicted to interact with six circRNAs (<xref rid=\"B62\" ref-type=\"bibr\">62</xref>). These findings indicate that many miRNAs can be modulated by a handful of circRNAs, adding complexity to the network that regulates synoviocyte expression profiles. CircRNAs and lncRNAs represent an opportunity to modulate several miRNAs simultaneously, and thus hold great therapeutic potential to modulate OA synovial pathology.</p></sec><sec id=\"s8\"><title>Future Directions</title><p>An important aspect overlooked in many OA studies using animal models is that OA is an age-related disease and experiments are routinely conducted in young animals. As with other organ systems, cellular activity in joint tissues is altered with age, including abnormal ECM, cytokine and reactive oxygen species (ROS) production, which likely contribute to OA pathology differently than post-traumatic or metabolic-induced OA (<xref rid=\"B63\" ref-type=\"bibr\">63</xref>, <xref rid=\"B64\" ref-type=\"bibr\">64</xref>). Little is known regarding how aging alters synovial homeostasis and function over time, and how that might contribute to OA progression. Expression of many miRNAs change with age in various tissues, altering processes like cell senescence (<xref rid=\"B65\" ref-type=\"bibr\">65</xref>). MiR-126-3p, which is important for cell attachment to the ECM, is downregulated in aged OA chondrocytes relative to their younger counterparts (<xref rid=\"B66\" ref-type=\"bibr\">66</xref>). Improving our understanding of how miRNAs are differentially expressed with age, and how this alters joint homeostasis and OA progression will be essential for future translational success.</p><p>Integrated analyses examining miRNA and transcript profiles in parallel will help elucidate dysregulated miRNA and RNA interactions occurring in OA. In OA, Chen et al. performed RNA sequencing alongside small RNA sequencing in OA FLS compared to those derived from healthy tissue (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). Putative targets of dysregulated miRNAs were predicted by bioinformatic approaches, including 14 genes (11 upregulated and 3 downregulated) that require further biological validations (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). Another study attempted to identify differential mRNA and miRNA expression in the DMM mouse OA model using microarray and RT-qPCR, but found no evidence of differential expression of miRNAs and RNAs levels between sham and DMM-induced OA mice at 1 or 6 weeks after surgery (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). However, the time after surgery examined, the small sample size used and variability observed within the groups, might be masking some relevant changes, and further investigation is warranted.</p><p>In addition to holding therapeutic potential, miRNAs in the synovial fluid or synovium-derived extracellular vesicles (EVs) might also act as biomarkers (<xref rid=\"B67\" ref-type=\"bibr\">67</xref>, <xref rid=\"B68\" ref-type=\"bibr\">68</xref>). Increased levels of miR-23a-3p, miR-24-3p, miR-186-5p, miR-29c-3p, miR-34a-5p, and miR-27b-3p are found in the synovial fluid of OA patients with late-stage compared to early-stage radiographic knee OA (<xref rid=\"B69\" ref-type=\"bibr\">69</xref>). Some of these miRNAs are highly expressed in the OA synovium. MiR-210 is increased in the synovial fluid of both early- and late-stage radiographic knee OA patients compared to healthy donors and positively correlates with VEGF levels (<xref rid=\"B70\" ref-type=\"bibr\">70</xref>). Other synovial fluid miRNAs suggested as OA biomarkers include miR-29b-3p and miR-140, which show positive and negative correlations with radiographic knee OA severity, respectively (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>, <xref rid=\"B72\" ref-type=\"bibr\">72</xref>). As we continue to unearth the biomarker potential of some of these miRNAs, understanding the release mechanism as well as the exact cellular source of secreted miRNAs in the joint will advance our understanding of miRNA contributions to OA pathology. Profiling miRNA content of cells and tissues using next generation sequencing not only helps to identify the source of miRNAs, but also has the added advantage of identifying novel miRNAs, expanding the rapidly-growing human miRNA repository and promoting investigations into new regulatory mechanisms and therapeutic targets. Sequencing datasets are routinely deposited on-line, and this open format is not only idea-generating but can also be used to substantiate novel findings. MiRNAs are currently being explored as potential therapeutic targets to counteract cartilage degeneration and synovitis in OA. For example, inhibition of miR-101 and miR-128 has been shown to rescue cartilage degeneration and synovitis in MIA and ACLT animal models of OA, respectively (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>). Extensive research is underway to identify the best mode of delivery of miRNA-based therapies (mimics or inhibitors) in preclinical models of OA.</p></sec><sec sec-type=\"conclusions\" id=\"s9\"><title>Conclusions</title><p>Taken together, miRNAs contribute to synovial homeostasis, inflammation, fibrosis, angiogenesis, cell survival and cell apoptosis, contributing to OA synovial pathology. MiRNAs have been a focus of OA research since their discovery and they are attracting more attention due to their biomarker and therapeutic potential. However, research on the role of miRNAs in OA-related synovial pathology is only in its infancy. Most research on synovitis is performed in samples from RA patients or animal models where OA tissues are often used as a control reference. This has hampered our understanding of the mechanisms modulated by miRNAs in OA synovitis. Additional studies are needed to comprehensively understand the role miRNAs play in OA-related synovial pathology and to identify novel disease modifying targets for therapeutic development.</p></sec><sec id=\"s10\"><title>Author Contributions</title><p>GT and SL performed the relevant literature searches. GT, JR, and SL wrote the manuscript. MK critically reviewed the manuscript and provided important intellectual and funding contributions. All authors have critically read and approved the manuscript.</p></sec><sec id=\"s11\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work is supported by grants to MK by The Natural Sciences and Engineering Research Council of Canada (NSERC; Grant #RGPIN-2017-06360). MK is the recipient of a Tier 1 CRC from the Canada Research Chairs Program. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Psychol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Psychol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Psychol.</journal-id><journal-title-group><journal-title>Frontiers in Psychology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-1078</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849009</article-id><article-id pub-id-type=\"pmc\">PMC7431696</article-id><article-id pub-id-type=\"doi\">10.3389/fpsyg.2020.01649</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Psychology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Atypical Frequency Sweep Processing in Chinese Children With Reading Difficulties: Evidence From Magnetoencephalography</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Natalie Yu-Hsien</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/346145/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Chiang</surname><given-names>Chun-Han</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/779449/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Hsiao-Lan Sharon</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/212338/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Tsao</surname><given-names>Yu</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/680790/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Audiology and Speech-Language Pathology, Asia University</institution>, <addr-line>Taichung</addr-line>, <country>Taiwan</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Special Education, National Pingtung University</institution>, <addr-line>Pingtung</addr-line>, <country>Taiwan</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Special Education, National Taiwan Normal University</institution>, <addr-line>Taipei</addr-line>, <country>Taiwan</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Research Center for Information Technology Innovation, Academia Sinica</institution>, <addr-line>Taipei</addr-line>, <country>Taiwan</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Duo Liu, The Education University of Hong Kong, Hong Kong</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Timo Ruusuvirta, University of Turku, Finland; Chunming Lu, Beijing Normal University, China</p></fn><corresp id=\"c001\">*Correspondence: Hsiao-Lan Sharon Wang, <email>hlw36@ntnu.edu.tw</email></corresp><corresp id=\"c002\">Yu Tsao, <email>yu.tsao@citi.sinica.edu.tw</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Educational Psychology, a section of the journal Frontiers in Psychology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1649</elocation-id><history><date date-type=\"received\"><day>29</day><month>11</month><year>2019</year></date><date date-type=\"accepted\"><day>17</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Wang, Chiang, Wang and Tsao.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Wang, Chiang, Wang and Tsao</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Chinese lexical tones determine word meaning and are crucial in reading development. Reduced tone awareness is widely reported in children with reading difficulties (RD). Lexical-tone processing requires sensitivity to frequency-modulated sound changes. The present study investigates whether reduced tone awareness in children with RD is reflected in basic auditory processing and the level at which the breakdown occurs. Magnetoencephalographic techniques and an oddball paradigm were used to elicit auditory-related neural responses. Five frequency sweep conditions were established to mirror the frequency fluctuation in Chinese lexical tones, including one standard (level) sweep and four deviant sweeps (fast-up, fast-down, slow-up, and slow-down). A total of 14 Chinese-speaking children aged 9–12 years with RD and 13 age-matched typically developing children were recruited. The participants completed a magnetoencephalographic data acquisition session, during which they watched a silent cartoon and the auditory stimuli were presented in a pseudorandomized order. The results revealed that the significant between-group difference was caused by differences in the level of auditory sensory processing, reflected by the P1m component elicited by the slow-up frequency sweep. This finding indicated that auditory sensory processing was affected by both the duration and the direction of a frequency sweep. Sensitivity to changes in duration and frequency is crucial for the processing of suprasegmental features. Therefore, this sensory deficit might be associated with difficulties discriminating two tones with an upward frequency contour in Chinese.</p></abstract><kwd-group><kwd>reading difficulties</kwd><kwd>basic auditory processing</kwd><kwd>frequency sweep</kwd><kwd>magnetoencephalography</kwd><kwd>P1m</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Ministry of Science and Technology, Taiwan<named-content content-type=\"fundref-id\">10.13039/501100004663</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"3\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"45\"/><page-count count=\"9\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Reading development is associated with phonological awareness—the ability to identify and manipulate the components of speech sound (<xref rid=\"B22\" ref-type=\"bibr\">National Reading Panel, 2000</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Kirby et al., 2003</xref>). Reduced phonological awareness has been widely observed among children with reading difficulties (RD) across language systems (<xref rid=\"B45\" ref-type=\"bibr\">Ziegler and Goswami, 2005</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Goswami et al., 2011</xref>). Therefore, phonological processing deficits are considered the root cause of RD. A possible explanation for this deficit is that difficulties in basic auditory processing lead to inaccurate speech sound identification and mismatched representations, which constrain phonological processing (<xref rid=\"B34\" ref-type=\"bibr\">Tallal and Gaab, 2006</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Pasquini et al., 2007</xref>). Basic auditory processing difficulties are likely to affect various domains of speech sound processing, such as detecting the fluctuations of sound intensity over time (amplitude-modulated speech signal; AM), the fluctuations of sound frequency over time (frequency-modulated speech signal; FM), and changes in phoneme duration. FM sound processing has received particular attention in research on basic auditory processing deficits in Chinese-speaking children with RD because of the close association between Chinese lexical tones and FM sound (<xref rid=\"B21\" ref-type=\"bibr\">Meng et al., 2005</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Wang et al., 2019</xref>). Despite that behavioral evidence has reported reduced performance on the FM sound processing (<xref rid=\"B39\" ref-type=\"bibr\">Wang et al., 2019</xref>), it remains unclear how the deficit is reflected in neural response. This study aims to identify the neural processes associated with the atypical auditory processing of FM signals in Chinese-speaking children with RD.</p><sec id=\"S1.SS1\"><title>Lexical Awareness, Frequency Modulation, and Reading Ability</title><p>Chinese has four lexical-tone variations. All Chinese characters are monosyllabic, and the meaning of each character is defined by the lexical tone accompanying the syllable. The lexical tone is a suprasegmental feature that carries both prosodic and semantic information. For instance, associating the syllable/<italic>ba</italic>/with tone 1 signifies <italic>eight</italic> [八]; however, the meaning changes into <italic>pull</italic> [拔], <italic>target</italic> [爸], and <italic>father</italic> [爸] when the same syllable is associated with tones 2, 3, and 4, respectively. As isolated syllables, the patterns of the four tones are categorized by their fundamental frequency (F0) and pitch contour into high-level, high-rising, low-falling-rising, and high-falling (<xref rid=\"B43\" ref-type=\"bibr\">Xu et al., 2002</xref>). Because of the function of lexical tone, being aware of and familiar with the variation of lexical tones is necessary for Chinese character reading and learning new vocabulary (<xref rid=\"B36\" ref-type=\"bibr\">Tong et al., 2015</xref>).</p><p>Sensitivity to lexical tones is developmental and unique to tonal language speakers. Chinese-speaking children can accurately perceive variations in lexical tones at the age of approximately 1 year (<xref rid=\"B37\" ref-type=\"bibr\">Tsao, 2008</xref>, <xref rid=\"B38\" ref-type=\"bibr\">2017</xref>). Mounting evidence demonstrates a correlation between lexical-tone awareness and reading-related abilities (<xref rid=\"B19\" ref-type=\"bibr\">McBride-Chang et al., 2008</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Shu et al., 2008</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Li and Ho, 2011</xref>). For example, Chinese character recognition (<xref rid=\"B40\" ref-type=\"bibr\">Wang et al., 2017</xref>), and reading comprehension (<xref rid=\"B3\" ref-type=\"bibr\">Choi et al., 2017</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Liu and Tsao, 2017</xref>) are strongly correlated with performance on lexical-tone awareness tasks among children. A recent study investigating 90 children with RD and a matched number of their typically developing peers revealed a later developmental ceiling of lexical-tone awareness in children with RD (<xref rid=\"B40\" ref-type=\"bibr\">Wang et al., 2017</xref>). In a study by <xref rid=\"B40\" ref-type=\"bibr\">Wang et al. (2017)</xref>, primary school children with and without RD from three different age groups (second-, fourth-, and sixth-grader students) were recruited to investigate lexical-tone awareness, verbal short-term memory, rapid automatic naming, and phonological awareness, which involves phoneme discrimination. Children with RD exhibited lower performance than their typically developing peers in all tasks. Moreover, in the fourth grade, the maturation of lexical-tone awareness was observed in typically developing children but not in children with RD. Therefore, lexical-tone awareness was considered a sensitive indicator of RD throughout primary school years.</p></sec><sec id=\"S1.SS2\"><title>Basic Auditory Processing in RD</title><p>Evidence from alphabetic language speakers has indicated that children and adults with RD have difficulty detecting longer time-scale patterns of prosody-related sound features, such as duration, rhythm, AM, FM, and amplitude envelope rise time (<xref rid=\"B2\" ref-type=\"bibr\">Baldeweg et al., 1999</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Goswami et al., 2002</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Richardson et al., 2004</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Pasquini et al., 2007</xref>). For instance, <xref rid=\"B29\" ref-type=\"bibr\">Richardson et al. (2004)</xref> assessed the performance of children with and without RD in discriminating linear rise and fall time envelopes, detecting rise time onset, duration discrimination, intensity detection, and rapid pitch discrimination. These tasks required amplitude envelope rise time processing, which was reported to account for the significant variance in phonological processing performances. <xref rid=\"B26\" ref-type=\"bibr\">Pasquini et al. (2007)</xref> reported similar findings in adults with RD; reading attainment was predicted by their performance on rise time perception and temporal order judgment. Strategies to compensate for these hearing deficits were not observed, as academic high achievers with reading problems performed worse than their peers on AM and FM detection tasks (<xref rid=\"B41\" ref-type=\"bibr\">Witton et al., 2002</xref>).</p><p>Basic auditory processing deficits are a probable contributor to reduced lexical-tone awareness because phonological deficits in RD are a cross-linguistic phenomenon. However, few studies have investigated basic auditory processing in the Chinese-speaking population with RD. Based on the hypothesis that lexical-tone processing involves sensitivity to spectrotemporal acoustic cues, <xref rid=\"B39\" ref-type=\"bibr\">Wang et al. (2019)</xref> investigated the relationship between basic auditory processing, lexical-tone awareness, and Chinese word recognition. The two auditory processing tasks required the participants to discriminate two sound sequences with frequency variations and identify the direction of frequency sweeps, which mimic the frequency changes in Chinese lexical tones. Their findings revealed that the ability to identify frequency sweep direction correlated strongly with both lexical-tone awareness and Chinese character recognition. Moreover, frequency sweep direction identification accounted for 11% and 19% of the variance in Chinese character recognition and lexical-tone awareness, respectively, indicating that basic auditory processing contributes to linguistic processing in Chinese-speaking children with RD.</p></sec><sec id=\"S1.SS3\"><title>Neural Auditory and Processing Responses to Lexical Tone</title><p>Few event-related potentials (ERP) are associated with the sound discrimination used to identify the sensitivity of phonological awareness and the corresponding basic auditory processing. Mismatch negativity (MMN) and late discriminative negativity (LDN) are the most widely used components for measuring pre-attentive detection of an odd stimulus in a sound sequence. MMN peaks at approximately 150–250 ms from change onset, whereas LDN peaks at 300–600 ms. The two components are typically investigated using an oddball paradigm, in which one standard sound occurs regularly, and several deviant sounds with one or more different features appear irregularly in a sound sequence. ERP studies have reported that children with RD demonstrated atypical MMN or LDN patterns in perceiving both speech and basic auditory stimuli (<xref rid=\"B1\" ref-type=\"bibr\">Alonso-Búa et al., 2006</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Huttunen et al., 2007</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Neuhoff et al., 2012</xref>). <xref rid=\"B1\" ref-type=\"bibr\">Alonso-Búa et al. (2006)</xref> reported longer MMN latency in children with RD during the processing of speech stimuli (syllables). Atypical LDN, lower amplitude, and reduced latency were observed during the processing of both speech and non-speech (complex tones) stimuli. However, <xref rid=\"B9\" ref-type=\"bibr\">Hämäläinen et al. (2013)</xref> reported that kindergarten children with a high risk of RD exhibited stronger N250 responses, resembling MMN, to both speech stimuli (pseudowords), and non-speech stimuli (sounds with formant and frequency mimicking real phonemes) compared with a control group. Evidence regarding MMN and LDN is inconsistent across studies, which may be because of the task designs and the profiles of the populations involved. However, this discrepancy indicates that atypical auditory and speech processing occurs at the neural level.</p><p>P300 (P3) is another ERP component that is reportedly associated with RD. P3 is a late positive component that occurs at approximately 300 ms after the stimulus onset. It is linked to the process of context updating, working memory, and decision-making. Similar to MMN and LDN, P3 is usually elicited by using an oddball paradigm, which reflects higher-cognitive functions that may influence the speed of auditory processing (<xref rid=\"B27\" ref-type=\"bibr\">Polich, 1987</xref>). Studies have indicated that P3 may be an effective indicator of central auditory system integrity and auditory attention functioning (<xref rid=\"B20\" ref-type=\"bibr\">Mendonça et al., 2013</xref>). <xref rid=\"B17\" ref-type=\"bibr\">Maciejewska et al. (2013)</xref> reported prolonged P3 latencies in children with RD during a non-speech auditory processing task. <xref rid=\"B17\" ref-type=\"bibr\">Maciejewska et al. (2013)</xref> highlighted that P3 latency was modulated by age in typically developing children. Older children should exhibit a shorter latency; however, the age effect was absent in children with RD. This finding suggests that maturation of non-speech auditory processing may be delayed, which contributes neurological evidence to the behavioral-based account presented by <xref rid=\"B40\" ref-type=\"bibr\">Wang et al. (2017)</xref>. Furthermore, <xref rid=\"B25\" ref-type=\"bibr\">Papagiannopoulou and Lagopoulos (2017)</xref> reported a reduced P3 amplitude and localization response in children with RD, suggesting a delayed processing speed, and a diminished processing capacity.</p><p>The phonological deficit hypothesis for RD has been proven to be universal, and processing of FM sound and lexical-tone awareness are closely associated; therefore, Chinese-speaking children with and without RD may exhibit neural-level differences in basic auditory processing. Little research has been conducted to verify this hypothesis. <xref rid=\"B21\" ref-type=\"bibr\">Meng et al. (2005)</xref> clarified neural-level differences that may reflect the difficulties in lexical-tone processing. They reported a reduced performance in tone frequency and temporal discrimination tests in children with RD. Furthermore, atypical ERPs were elicited by a steady pure tone discrimination task. Although the results were not statistically significant, children with RD demonstrated lower MMN than their typically developing peers. However, steady pure tone stimuli do not optimally reflect the dynamic nature of Chinese lexical-tone variations, because only tone 1 is flat and the other tones are composed of rising or falling frequency changes. Furthermore, the MMN component alone does not necessarily reflect the level of auditory processing breakdown. Therefore, further research is needed to explore auditory processing difficulties among Chinese-speaking children with RD.</p></sec><sec id=\"S1.SS4\"><title>Aims of the Study</title><p>Few behavioral studies have investigated the association between lexical-tone processing and basic auditory processing. Therefore, this study focused on verifying whether the difference in basic auditory processing between children with and without RD occurred at the neural level. A magnetoencephalography (MEG) technique was used, because this approach provides a spatial resolution more favorable than that of electroencephalography (EEG). A frequency sweep was employed as the auditory stimuli; this created a non-linguistic condition that resembled the characteristics of Chinese lexical tones.</p></sec></sec><sec id=\"S2\"><title>Methodology</title><sec id=\"S2.SS1\"><title>Participants</title><p>In Taiwan, the majority of children with RD are diagnosed after the third grade; a potential diagnosis is made for those who demonstrate relatively severe reading impairments at an early stage of primary school education. Third- and fourth-grader students with normal hearing were recruited for this study, including 13 children with RD and 14 of their typically developing peers (controls). The children with RD (mean age = 114.8 months; <italic>SD</italic> = 11.82; 11 boys and 2 girls) were identified from the database of the Special Education Division, Department of Education of Taipei City Government. The main inclusion criteria for the children with RD were significant Chinese character decoding failure and a demonstrated delay > 1 year compared with the controls. The controls were age-matched (mean age = 112.57; <italic>SD</italic> = 7.2; 10 boys and 4 girls) children from local primary schools who volunteered for the study. The sex ratio did not differ between groups [<italic>X</italic><sup>2</sup>(1) = 0.68, <italic>p</italic> = 0.65]. According to reports from parents and schoolteachers, the controls had no learning difficulties and no neurological or psychiatric disorders. Before the experiment, participants and their parents were debriefed, and informed consent was obtained. The study was approved by the Research Ethics Office of National Taiwan University (Application No. 201310EM016).</p><p>All children were assessed using the Graded Chinese Character Recognition Test (<xref rid=\"B10\" ref-type=\"bibr\">Huang, 2001</xref>) and the Abbreviated Wechsler Intelligence Scale for children, to evaluate their character recognition performance and ensure a normal intelligence quotient (IQ). As demonstrated in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, both groups had a full-scale IQ of >80, and children with RD exhibited results 1.5 standard deviations lower than the controls in Chinese character recognition.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Average scores of participants’ age, IQ, and Chinese character recognition.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>CA (<italic>N</italic> = 14)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>RD (<italic>N</italic> = 15)</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>t</italic></bold></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Age (months)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">109.79 (8.54)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">114.20 (11.93)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.138</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IQ (full scale)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110.39 (10.56)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92.59 (11.89)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.249**</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chinese character recognition</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">85.71 (26.17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">49.53 (23.60)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.914*</td></tr></tbody></table><table-wrap-foot><attrib><italic>Standard deviations are presented in the bracket. *<italic>p</italic> < 0.01; **<italic>p</italic> < 0.001.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S2.SS2\"><title>Stimuli and Procedure for the MEG Experiment</title><p>Four sound sequences for FM sweep were used as auditory stimuli. Each sound sequence was composed of the repetition of a standard and four FM sweep deviant variations (fast-up, fast-down, slow-up, and slow-down). The standard FM sweep was created with a combination of 180- and 600-Hz stimuli; the duration of the sound was 240 ms. The directions of the deviant FM sweeps were either upward or downward, and the conditions of the speed of frequency change were either 80 and 160 ms. The fast-up FM sweep involved a frequency increase from 180 to 270 Hz over 80 ms, whereas in the fast-down FM sweep the frequency decreased from 270 to 180 Hz. The slow-up and slow-down FM sweeps involved a frequency increase from 600 to 900 Hz and a frequency drop from 900 to 600 Hz, respectively, over 160 ms.</p><p>The order in which the FM sweeps were presented in the sound sequences was pseudorandomized. Each sound sequence contained 360 deviant FM sweeps (90 per deviant condition) and 840 standard sounds. A total of 20 standard sounds were placed at the beginning of a sound sequence, and the other standard sounds were inserted between the deviant FM sweeps to ensure that the spacing was appropriate but unpredictable. The overall duration of a sound sequence was approximately 5 min. The visual stimulus was a silent cartoon divided into four sections, equal in length to the sound sequence.</p><p>The MEG experiment adopted an oddball paradigm with a block design. During MEG data acquisition, the children were presented with a silent cartoon along with the FM sweep sequence. The children were told that no response was needed. The experiment contained four blocks, and the overall time of data acquisition was 25–30 min.</p></sec><sec id=\"S2.SS3\"><title>MEG Data Acquisition</title><p>Data acquisition was performed using a whole-head 306 sensor MEG device (Elekta Neuromag, Helsinki, Finland) with 102 arrays, containing one magnetometer and two orthogonal, planar gradiometers. To monitor the head location in relation to the MEG sensors, four Head Position Indicator coils with a 293–321-Hz sinusoidal current were attached to the scalp. The sampling rate was 1000 Hz, and a band-pass filter of 1–40 Hz was employed. Eye movements during the acquisition were recorded with two electrodes attached above and to the side of the left eye.</p></sec><sec id=\"S2.SS4\"><title>MEG Data Analysis</title><p>The data were preprocessed using single subspace separation (<xref rid=\"B35\" ref-type=\"bibr\">Taulu et al., 2005</xref>) in the MaxFilter program (Elekta Neuromag) to reduce the strength of external noise. Head movement compensation (200 ms) was also performed using the MaxFilter program to transform the head origin to the same position for each participant.</p><p>The data were exported into BESA Research 6.0 (BESA GmbH, Gräfelfing, Germany) for averaging and dipole-source localization. A segmentation process was used to extract the time sequences between -200 and 800 ms for preprocessing with a 1-Hz high-pass filter and a 40-Hz low-pass filter. BESA Statistics was used to perform permutation tests—a non-parametric statistic method for channel and time-point clustering—to examine between-group and within-subject differences. <italic>P</italic>-values were calculated to assess statistical significance. The permutation testing of 1000 permutations was significant at alpha = 0.05, and the channel neighbor distance was 4 cm (for permutation testing, see <xref rid=\"B18\" ref-type=\"bibr\">Maris and Oostenveld, 2007</xref>). The equivalent current dipole models and CLARA source localization model in BESA Research 6.0 were used to investigate the brain regions responsible for the group differences.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Effects of FM Sweeps on Event-Related Fields</title><p>The results of the permutation tests revealed that the four deviant FM sweep conditions elicited Event-Related Fields (ERFs) that were significantly different from the standard condition. However, the effects were reflected differently in the control and RD groups. In the control group, the fast-up sweep elicited deviant waveforms at five different time points, including a positive peak at 117 ms (<italic>p</italic> < 0.05) and negative peaks at 192 (<italic>p</italic> < 0.001), 212 (<italic>p</italic> < 0.0001), 358 (<italic>p</italic> < 0.05), and 478 ms (<italic>p</italic> < 0.01), as illustrated in <xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>. The positive peak, observed in the right anterior and left posterior temporal areas, corresponded to the P1m component. The negative change in the left middle temporal and right anterior temporal area waveforms resembled the MMNm component (192 and 212 ms) and the LDNm component (478 ms). The fast-down sweep elicited a negative change in the waveform, corresponding to the MMNm component at 162 (<italic>p</italic> < 0.0001) and 179 ms (<italic>p</italic> < 0.0001) and corresponding to the LDNm component at 377 (<italic>p</italic> < 0.05) and 391 ms (<italic>p</italic> < 0.01) in the bilateral temporal lobes, as illustrated in <xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>. Furthermore, the P3m component was observed at 278 ms (<italic>p</italic> < 0.01). The slow-up sweep, illustrated in <xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref>, induced negative waveforms associated with the MMNm component at 164 (<italic>p</italic> < 0.01) and 175 ms (<italic>p</italic> < 0.01) and associated with the LDNm component at 383 ms (<italic>p</italic> < 0.01), in the right temporal area. A positive peak that corresponded to the P3m component was identified at 277 ms (<italic>p</italic> < 0.0001). For the slow-down sweep condition, illustrated <xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>, only the P3m component at 342 ms (<italic>p</italic> < 0.01) was observed in the left posterior temporal lobe.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>ERFs elicited in the control participants by <bold>(A)</bold> the fast-up sweep, <bold>(B)</bold> the fast-down sweep, <bold>(C)</bold> the slow-up sweep, and <bold>(D)</bold> the slow-down sweep.</p></caption><graphic xlink:href=\"fpsyg-11-01649-g001\"/></fig><p>In the RD group, the fast-up sweep, illustrated in <xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>, triggered negative waveform changes that corresponded to the MMNm component at 283 ms (<italic>p</italic> < 0.01) and the LDNm component at 363 ms (<italic>p</italic> < 0.0001) in the bilateral temporal regions. The P3m component, at 303 ms (<italic>p</italic> < 0.01), was only observed in the left temporal area. The fast-down sweep elicited the MMNm component at 192 (<italic>p</italic> < 0.001) and 196 ms (<italic>p</italic> < 0.01) in the bilateral temporal lobes, as illustrated in <xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>. Negative changes in the waveforms were also observed at 354 (<italic>p</italic> < 0.001) and 370 ms (<italic>p</italic> < 0.01), which were associated with the LDNm component, in the right posterior frontal area and the left temporal area. A positive peak was observed at 301 ms (<italic>p</italic> < 0.01) in the right temporal area. Similarly, the slow-up sweep, illustrated in <xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>, triggered negative waveform changes associated with the MMNm component at 177 ms (<italic>p</italic> < 0.0001) and the LDNm component at 490 ms (<italic>p</italic> < 0.01) in the bilateral temporal lobes. A positive waveform peak at 278 ms, associated with the P3m component, was also observed in this area. For the slow-down sweep, the MMNm component at 184 ms (<italic>p</italic> < 0.0001), and LDNm component at 460 ms (<italic>p</italic> < 0.0001) were observed in the left and the right temporal lobes, respectively, as illustrated in <xref ref-type=\"fig\" rid=\"F2\">Figure 2D</xref>.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>ERFs elicited in the children with RD by <bold>(A)</bold> the fast-up sweep, <bold>(B)</bold> the fast-down sweep, <bold>(C)</bold> the slow-up sweep, and <bold>(D)</bold> the slow-down sweep.</p></caption><graphic xlink:href=\"fpsyg-11-01649-g002\"/></fig></sec><sec id=\"S3.SS2\"><title>Group Effect on ERFs</title><p>A permutation test demonstrated between-group differences in the waveform changes. Significant between-group differences were reported in three of the four FM sweep conditions. First, a significantly different cluster in the central frontal field map was identified in the fast-up sweep condition. As illustrated in <xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>, the difference occurred at 30–95 ms (<italic>p</italic> = 0.041) with a peak time at 52 ms. The equivalent current dipole models and CLARA source localization model, with 49.59% residual variance, further revealed that the ERF source was in the left inferior frontal gyrus.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Between-group differences in ERF observed for three different time points and sources: ERF with peak time at <bold>(A)</bold> 52 ms in the left inferior frontal gyrus, <bold>(B)</bold> 121 ms in the left frontal region, and <bold>(C)</bold> 550 ms in the left middle frontal gyrus.</p></caption><graphic xlink:href=\"fpsyg-11-01649-g003\"/></fig><p>Second, a between-group difference was observed in the slow-up sweep condition between 105 and 140 ms (<italic>p</italic> = 0.035), as displayed in <xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>. The distribution of the upward field peaked at 121 ms in the left frontal area. However, source localization models (residual variance = 19.33%) further revealed differences in the left inferior frontal gyrus (blue dipole) and the postcentral gyrus in the right parietal lobe (red dipole). Third, a between-group difference in the slow-down condition was revealed. A downward current was observed during 535–570 ms with a peak at 550 ms (<italic>p</italic> = 0.034) in the left occipital area, as displayed in <xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>. The source localization results suggested that the significant clusters were close to the left middle frontal gyrus (blue dipole) and the cingulate gyrus in the right hemisphere (red dipole). The residual variance was 27.63%.</p></sec></sec><sec id=\"S4\"><title>Discussion</title><p>This study explored the differences in basic auditory processing between Chinese-speaking children with and without RD. The results demonstrated that the oddball paradigm elicited three auditory components (i.e., MMNm, LDNm, and P3m) commonly associated with detecting sound changes in both groups. However, the effects of sound changes were inconsistent across the frequency sweep conditions and groups. For the MMNm component, the control group exhibited a larger amplitude than the children with RD in the fast-up, fast-down, and slow-up conditions; the MMNm component was not observed in the slow-down frequency sweep condition in the control group, and the RD group demonstrated a relatively low amplitude. The patterns of the LDNm component mirrored the MMNm in the larger amplitude observed in the control group in all frequency sweep conditions except the slow-down condition, which exhibited no effect in the control group and a low amplitude effect in the RD group. Although differences in neural response patterns may be observed, permutation tests failed to reveal group effects on these pre-attentive auditory components. However, the P3 component was less stable and was only observed in the fast-down and slow-up frequency sweep conditions. Although the delayed latency was observed for the fast-down frequency sweep in the children with RD, the evidence is inconclusive regarding deficits in auditory attention or central auditory system functioning. However, other cognitive deficits, including attention disorders, were exclusion criteria when recruiting the children with RD and thus the neural responses of attention-related components were not significantly different from those in typically developing children.</p><p>The findings of this study indicate that basic auditory processing deficits in children with RD may not be as strongly associated with pre-attentive auditory processing as originally expected. Although the MMN component identified in children with RD differed from that in the control group in terms of the amplitude and latency of ERP, the differences were not significant in phonological or basic auditory passive listening tasks. This accorded with findings reported by <xref rid=\"B21\" ref-type=\"bibr\">Meng et al. (2005)</xref>. These combined findings suggest that the MMN component may not be an efficient indicator of basic auditory processing deficits in Chinese-speaking children with RD. The main difference between these two studies was the design of basic auditory processing stimuli. <xref rid=\"B21\" ref-type=\"bibr\">Meng et al. (2005)</xref> used steady pure tones of different frequencies, whereas the present study employed frequency sweeps that reflected the frequency fluctuation within Chinese lexical tones. Nonetheless, the findings were consistent, suggesting the pre-attentive auditory processing is not the major contributor to the basic auditory processing deficits observed in Chinese-speaking children with RD. Analysis of the LDNm component, which also reflects pre-attentive auditory processing, did not reveal significant differences between the groups, supporting the argument that the basic auditory processing deficits may not involve the pre-attentive level.</p><p>However, between-group differences were observed over three different time frames, namely, 30–95, 105–140, and 535–570 ms. Only the positive peaks at 52 and 121 ms were considered as auditory evoked fields. The findings were interpreted carefully because the two early peaks may reflect auditory sensory processing in the P1m component, which matures with age (<xref rid=\"B28\" ref-type=\"bibr\">Ponton et al., 2000</xref>). P1m deflection in newborns peaks at approximately 250 ms, but, as children age, it becomes smaller, and its latency decreases (<xref rid=\"B28\" ref-type=\"bibr\">Ponton et al., 2000</xref>). According to <xref rid=\"B24\" ref-type=\"bibr\">Paetau et al. (1995)</xref>, the auditory evoked field of P1m peaks at approximately 50 ms in adults, whereas the peak occurs at approximately 100 ms in children aged 3 months to 12 years. Therefore, we considered that the peak observed at 121 ms resembled the P1m component in the children involved in this study. The P1m component has been associated with early auditory sensory processing in the central auditory system. This study demonstrated that the children with RD demonstrated weaker P1m response compared with their typically developing peers, suggesting potential auditory sensory processing deficits.</p><p>In the existing literature, the P1(m) component is often investigated along with the N2(m) component, which is a negative waveform that occurs around 200 ms, to reflect auditory sensory processing in the perception of basic auditory stimuli which employed mainly duration and frequency (<xref rid=\"B33\" ref-type=\"bibr\">Swink and Stuart, 2012</xref>). Sensitivity to sound duration and frequency are closely associated with the processing of suprasegmental features, such as stress, tone, and intonation. <xref rid=\"B4\" ref-type=\"bibr\">Chung and Bidelman (2016)</xref> suggested that healthy individuals with different levels of language proficiency (native and non-native adult speakers of English) demonstrated distinct waveform patterns during the processing of pseudowords with a legal English stress. This finding suggested that auditory sensory processing could be an indicator of phonological awareness at the suprasegmental level.</p><p>Studies on alphabetic-language-speaking children with RD or with a high risk of RD have demonstrated an altered P1(m) component in various basic auditory processing tasks (<xref rid=\"B16\" ref-type=\"bibr\">Lovio et al., 2010</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Khan et al., 2011</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Stefanics et al., 2011</xref>). For instance, <xref rid=\"B16\" ref-type=\"bibr\">Lovio et al. (2010)</xref> reported in their ERP study that children with a risk of RD demonstrated altered neural response to syllable duration. The appearance of a syllable in an oddball paradigm elicited different P1 responses in children at risk of RD and the control group. The children at risk of RD demonstrated overall smaller P1 amplitudes than the controls in the frontocentral regions. The amplitude envelop rise time is a critical acoustic property underlying syllable rate (<xref rid=\"B5\" ref-type=\"bibr\">Drullman, 2006</xref>), and thus, sensitivity to the rise time may reflect the ability to process speech (<xref rid=\"B6\" ref-type=\"bibr\">Goswami, 2011</xref>). <xref rid=\"B32\" ref-type=\"bibr\">Stefanics et al. (2011)</xref> determined that reduced sensitivity to rise time in children with RD was associated with atypical P1 responses in the frontocentral regions; furthermore, the effect of stimuli conditions (speed of rise time) engendered variations in the responses of children with RD, whereas this phenomenon was not observed in the control group. The children with RD had smaller P1 responses to slower rise time stimuli than for faster rise time stimuli. <xref rid=\"B32\" ref-type=\"bibr\">Stefanics et al. (2011)</xref> hypothesized that difficulties in processing were associated with an extended rise time. These findings partially accorded with the results of the present study.</p><p>In this study, the atypical P1m responses in the children with RD were also observed in the frontocentral regions, including the left inferior frontal gyrus and the postcentral gyrus in the right parietal lobe. However, the atypical responses in children with RD were unexpected, because the slow-up frequency sweep exhibited a between-group difference in the P1m component. Furthermore, the children with RD demonstrated a larger amplitude than the controls in the processing of slow-up frequency. These findings contribute cross-linguistic evidence that children with RD exhibit auditory sensory processing deficits. Concordant with the aforementioned findings among English-speaking children with RD (<xref rid=\"B32\" ref-type=\"bibr\">Stefanics et al., 2011</xref>), the atypical P1m responses in Chinese-speaking children were elicited by stimuli with a slower rise time (the slow-up frequency sweep). This finding indicates that auditory sensory processing deficits were particularly evident for relatively extended auditory stimuli. However, the lack of group differences in P1m response to processing the slow-down frequency sweep suggested that the frequency sweep direction and the overall duration of a stimulus affect the basic auditory processing at the sensory level. A behavioral study by <xref rid=\"B39\" ref-type=\"bibr\">Wang et al. (2019)</xref> adopted upward and downward frequency sweeps with relatively short durations, seven variations between 5 and 80 ms. Between-group differences in discrimination of frequency sweep directions were reported; however, the effects of overall duration and direction on discrimination accuracy were not discussed. Therefore, we cannot conclude whether the manipulation of speed and frequency sweep direction in the study by Wang et al. and the current study have shared patterns. Moreover, in this study, the group differences in neural responses were elicited with stimuli of 160 ms rather than 80 ms, leading to the question of whether frequency sweeps with longer durations reflect children’s ability to discriminate frequency sweep direction more accurately. Further research combining behavioral and neurological evaluation is needed to clarify this aspect.</p><p>The findings contribute to the relevant literature on basic auditory processing in Chinese-speaking children and demonstrate that the reduced sensitivity to non-speech sounds has neurological origins. Furthermore, difficulty in distinguishing tone 2 from tone 3 is a commonly reported characteristic of Chinese-speaking children with RD, which may also be related to the phenomena observed in this study. Tone 3 resembles tone 2 in its rising contour; therefore, distinguishing the tones requires detection of the timing of turning point in tone 3 (<xref rid=\"B30\" ref-type=\"bibr\">Shen and Lin, 1991</xref>). Moreover, the overall durations of tone 2 (417.7 ms), and tone 3 (484 ms) are relatively longer than those of tones 1 (416.2 ms) and 4 (307.8 ms) in monosyllabic conditions (<xref rid=\"B44\" ref-type=\"bibr\">Yang et al., 2017</xref>). This discrimination is more challenging, even for typically developing children. <xref rid=\"B42\" ref-type=\"bibr\">Wong et al. (2005)</xref> reported that typically developing preschoolers perceived tone 1, tone 2, and tone 4 more accurately than tone 3, with accuracies of 90, 87, and 89%, respectively; the accuracy for identifying tone 3 was only 70%. Tone 3 was most frequently misidentified as tone 2. The group difference in neural responses was caused by the frequency sweep condition that resembled the relative duration and the sweep direction of the two easily confused tones. However, the present experimental design does not allow us to verify whether the P1m responses were directly associated with children’s accuracy in discriminating tones 2 and 3. Future research is needed to provide a deeper understanding of neural responses and children’s lexical-tone awareness and, specifically, whether the P1m component is particularly sensitive to the difficulty in tone 2 and tone 3 discrimination.</p><p>In conclusion, this study investigated whether Chinese-speaking children with RD demonstrated basic auditory processing and at what level the auditory processing deficits occurred. The findings of this study provide evidence that accords with studies that have reported basic auditory processing deficits in children with RD. Moreover, the major contribution of this study is the evidence that basic auditory processing deficits may result from disrupted auditory sensory processing rather than pre-attentive auditory processing, which has been a focus of investigation in numerous studies. Furthermore, the atypical auditory sensory processing was particularly evident when the frequency sweep direction was upward and the overall duration was longer. Auditory sensory processing difficulties may be tuned to the characteristics of auditory stimuli in children with RD. The interaction between stimuli duration and frequency sweep direction requires further research, verifying the effect on auditory sensory processing and clarifying the association with Chinese lexical-tone processing.</p></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>The datasets generated for this study are available on request to the corresponding author.</p></sec><sec id=\"S6\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by Research Ethics Office of National Taiwan University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>NW was responsible for data analysis and writing up the manuscript. C-HC was responsible for data collection and analysis. NW was the PI of this research project and supports the writing up of the manuscript. YT was the co-PI of this research and responsible for creating the auditory stimuli used for the experiment. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by the Ministry of Science and Technology, Taiwan (MOST 103-2420-H-003-008-MY3, 109-2634-F-008, and 109-2221-E-001-016). C-HC was funded by Graduate Students Study Abroad Program of MOST, Taiwan (104-2917-I-003-002).</p></fn></fn-group><ack><p>We thank all of the participants and their parents for taking part in this study, the Imaging Center for Integrated Body, Mind and Culture Research, National Taiwan University for technical and facility supports, and the Department of Psychology, University of Jväskylä for hosting C-HC as a visiting student.</p></ack><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alonso-Búa</surname><given-names>B.</given-names></name><name><surname>Díaz</surname><given-names>F.</given-names></name><name><surname>Ferraces</surname><given-names>M. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Psychol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Psychol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Psychol.</journal-id><journal-title-group><journal-title>Frontiers in Psychology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-1078</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849105</article-id><article-id pub-id-type=\"pmc\">PMC7431697</article-id><article-id pub-id-type=\"doi\">10.3389/fpsyg.2020.01884</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Psychology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Social Cognition in Children With Non-specific Intellectual Disabilities: An Exploratory Study</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Jacobs</surname><given-names>Emilie</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/964104/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Simon</surname><given-names>Poline</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/965014/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Nader-Grosbois</surname><given-names>Nathalie</given-names></name><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/31037/overview\"/></contrib></contrib-group><aff><institution>UCLouvain, Psychological Sciences Research Institute</institution>, <addr-line>Louvain-la-Neuve</addr-line>, <country>Belgium</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Ilaria Grazzani, University of Milano-Bicocca, Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Elisabetta Conte, University of Milano-Bicocca, Italy; Christina Breil, Julius Maximilian University of Würzburg, Germany</p></fn><corresp id=\"c001\">*Correspondence: Nathalie Nader-Grosbois, <email>nathalie.nader@uclouvain.be</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Developmental Psychology, a section of the journal Frontiers in Psychology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1884</elocation-id><history><date date-type=\"received\"><day>27</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>08</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Jacobs, Simon and Nader-Grosbois.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Jacobs, Simon and Nader-Grosbois</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Social cognitive abilities – notably, Theory of Mind (ToM) and social information processing (SIP) – are key skills for the development of social competence and adjustment. By understanding affective and cognitive mental states and processing social information correctly, children will be able to enact prosocial behaviors, to interact with peers and adults adaptively, and to be socially included. As social adjustment and inclusion are major issues for children with intellectual disabilities (IDs), the present study aimed to explore their social cognitive profile by combining cluster analysis of both ToM and SIP competence, and to investigate the structure of relations between these skills in children with IDs. Seventy-eight elementary school children with non-specific IDs were recruited. They had a chronological age ranging from 4 years and 8 months to 12 years and 6 months and presented a preschool developmental age. Performance-based measures were administered to assess ToM and SIP abilities. Questionnaires were completed by the children’s parents to evaluate the children’s social competence and adjustment and their risk of developing externalizing or internalizing behaviors. Exploratory analysis highlighted strengths and weaknesses in the social cognitive profiles of these children with IDs. It also emphasized that the understanding of affective and cognitive mental states was used differently when facing appropriate vs. inappropriate social behaviors. The present study leads to a better understanding of the socio-emotional profile of children with IDs and offers some suggestions on how to implement effective interventions.</p></abstract><kwd-group><kwd>social cognition</kwd><kwd>theory of mind</kwd><kwd>social information processing</kwd><kwd>intellectual disability</kwd><kwd>social behavior</kwd></kwd-group><counts><fig-count count=\"3\"/><table-count count=\"7\"/><equation-count count=\"0\"/><ref-count count=\"97\"/><page-count count=\"19\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>In typically developing children, theoretical conceptions and developmental studies emphasize that the preschool period, ranging from 3 to 6 years, corresponds to a “critical” period of development of emotional and social abilities (<xref rid=\"B87\" ref-type=\"bibr\">Vygotsky, 1978</xref>; <xref rid=\"B65\" ref-type=\"bibr\">Perner, 1991</xref>; <xref rid=\"B89\" ref-type=\"bibr\">Wellman, 1991</xref>; <xref rid=\"B90\" ref-type=\"bibr\">Wellman et al., 2001</xref>; <xref rid=\"B91\" ref-type=\"bibr\">Wellman and Liu, 2004</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Astington and Baird, 2005</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Astington and Edward, 2010</xref>). The diversity of social interactions with peers and adults in different contexts increases, and social environments require more and more respect for social conventions or rules. Children have to develop new abilities to understand emotional and social situations and to interact successfully with others, in order to be perceived as socially adjusted and to create harmonious social relationships. However, children with intellectual disabilities (IDs) find it difficult to acquire these emotional and social abilities. In the definition of intellectual disability, limitations in both cognitive and adaptive functioning, including social skills (<xref rid=\"B71\" ref-type=\"bibr\">Schalock et al., 2010</xref>; <xref rid=\"B5\" ref-type=\"bibr\">American Psychiatric Association, 2013</xref>), are recognized as diagnostic criteria. To navigate the world, children with IDs have to develop skills in social cognition to be able to interact in a socially appropriate and adaptive way (<xref rid=\"B95\" ref-type=\"bibr\">Yeates et al., 2007</xref>). However, a majority of children with IDs face difficulties in social cognition and perform at a lower level in comparison with typically developing children with the same chronological or developmental age (<xref rid=\"B53\" ref-type=\"bibr\">Leffert and Siperstein, 2002</xref>). Depending on whether subjects are matched for developmental or chronological age, studies have shown that there is either a deficit or a delay in social cognitive development in children with IDs, in comparison with typically developing children. These comparisons determine whether impairments are related to a child’s specific disorder or to developmental difficulties (<xref rid=\"B76\" ref-type=\"bibr\">Skwerer, 2017</xref>). Social cognition in children with IDs has been explored according to either a developmental approach through the concept of Theory of Mind (ToM) or a functional view based on the social information processing (SIP) model. By contrast with previous studies investigating specific aspects of either ToM or SIP, our study examined social cognitive profiles according to both approaches.</p><sec id=\"S1.SS1\"><title>ToM in Children With IDs</title><p>ToM is described as the ability to understand one’s own and other people’s mental states, and to infer other people’s mental states in order to predict social behavior and to behave in a socially adapted way (<xref rid=\"B28\" ref-type=\"bibr\">Denham et al., 2003</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Barisnikov et al., 2002</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Deneault and Ricard, 2013</xref>). From a Vygotskyan perspective, ToM development is related to language acquisition and social interactions in the family, social and cultural environment (<xref rid=\"B68\" ref-type=\"bibr\">Ricard et al., 1999</xref>). Children with IDs face difficulties in both these areas but also in developing early prerequisites of ToM abilities (such as imitation, pretend play or joint attention; <xref rid=\"B16\" ref-type=\"bibr\">Charman et al., 2000</xref>; <xref rid=\"B82\" ref-type=\"bibr\">Tourrette et al., 2000</xref>; <xref rid=\"B60\" ref-type=\"bibr\">Meltzoff, 2002</xref>; <xref rid=\"B67\" ref-type=\"bibr\">Rakoczy, 2008</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Barthélémy and Tartas, 2009</xref>) and empathy (<xref rid=\"B74\" ref-type=\"bibr\">Sigman et al., 1992</xref>). The distinction between affective and cognitive ToM is relevant here, as the nature of the mental states being considered makes a difference to whether a delay or a deficit is reported (<xref rid=\"B27\" ref-type=\"bibr\">Deneault and Ricard, 2013</xref>). Affective mental states include desires and emotions, while cognitive ones include beliefs, pretense, visual perception, intentions, false beliefs, knowledge, thinking, and attention (<xref rid=\"B32\" ref-type=\"bibr\">Flavell, 1999</xref>). In terms of affective ToM, children with IDs present a delay in their understanding of causes and consequences of emotions (<xref rid=\"B34\" ref-type=\"bibr\">Garitte, 2003</xref>; <xref rid=\"B80\" ref-type=\"bibr\">Thirion-Marissiaux and Nader-Grosbois, 2008b</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Fiasse and Nader-Grosbois, 2012</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Baurain and Nader-Grosbois, 2013</xref>), when compared with typically developing children matched for developmental age. In terms of cognitive ToM, results of past studies have suggested the existence either of a delay (<xref rid=\"B35\" ref-type=\"bibr\">Giaouri et al., 2010</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Fiasse and Nader-Grosbois, 2012</xref>) or of a deficit (<xref rid=\"B18\" ref-type=\"bibr\">Charman and Campbell, 2002</xref>; <xref rid=\"B79\" ref-type=\"bibr\">Thirion-Marissiaux and Nader-Grosbois, 2008a</xref>), depending on the tasks (e.g., unexpected content task, appearance-reality task, change of location task), thus revealing an intertask variability. More particularly, perspective-taking competence seems deficient in children with borderline IDs, who struggle with distancing themselves from their own perspective to understand what others know (<xref rid=\"B8\" ref-type=\"bibr\">Baglio et al., 2016</xref>). Finally, <xref rid=\"B56\" ref-type=\"bibr\">Légaré et al. (2019)</xref> observed that mothers also perceived difficulties in both affective and cognitive ToM competence in their children with IDs, compared with parents of typically developing children.</p><p>ToM abilities lead to better social interaction and adjustment (<xref rid=\"B17\" ref-type=\"bibr\">Charman and Campbell, 1996</xref>, <xref rid=\"B18\" ref-type=\"bibr\">2002</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Barisnikov et al., 2002</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Abbeduto and Murphy, 2004</xref>; <xref rid=\"B50\" ref-type=\"bibr\">Jervis and Baker, 2004</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Deneault and Ricard, 2013</xref>). More particularly, some specific and variable links have been emphasized between, on the one hand, affective and cognitive ToM abilities in children with IDs, and on the other hand, their social or prosocial behavior during interactions with peers and adults as perceived by teachers (<xref rid=\"B30\" ref-type=\"bibr\">Fiasse and Nader-Grosbois, 2012</xref>; <xref rid=\"B81\" ref-type=\"bibr\">Thirion-Marissiaux and Nader-Grosbois, 2008c</xref>) or observed in dyadic play (<xref rid=\"B15\" ref-type=\"bibr\">Baurain and Nader-Grosbois, 2013</xref>).</p></sec><sec id=\"S1.SS2\"><title>SIP in Children With IDs</title><p>The SIP model was conceived to explain the cognitive processes that contribute to the understanding and resolving of social situations in typically developing children and children presenting externalized behavior disorders (<xref rid=\"B23\" ref-type=\"bibr\">Crick and Dodge, 1994</xref>). Individuals treat social information according to specific steps – (1) the encoding of emotional and social cues, (2) the interpretation of these cues, (3) goal clarification, (4) the generation of possible responses, and (5) response selection – displayed in social situations (<xref rid=\"B23\" ref-type=\"bibr\">Crick and Dodge, 1994</xref>). Problems in one or more steps will lead to a risk of maladjusted social behavior. More recently, this SIP model has been applied in studies of children with IDs (<xref rid=\"B83\" ref-type=\"bibr\">van Nieuwenhuijzen et al., 2006</xref>). This functional approach has improved our understanding of impairments in social problem-solving skills in children with IDs (<xref rid=\"B15\" ref-type=\"bibr\">Baurain and Nader-Grosbois, 2013</xref>; <xref rid=\"B92\" ref-type=\"bibr\">Wieland et al., 2014</xref>), by identifying the SIP steps in which the deficiencies lie. Some studies have shown that children with IDs display difficulties in encoding (step 1) and interpreting social and emotional cues (step 2) (<xref rid=\"B84\" ref-type=\"bibr\">van Nieuwenhuijzen et al., 2004</xref>; <xref rid=\"B85\" ref-type=\"bibr\">van Nieuwenhuijzen et al., 2009</xref>). These difficulties are particularly observed in socially ambiguous or provoking situations, in which these children are more likely to have faulty detection of information and to misidentify cues indicating unintentional actions (<xref rid=\"B49\" ref-type=\"bibr\">Jahoda et al., 2006</xref>; <xref rid=\"B86\" ref-type=\"bibr\">van Nieuwenhuijzen and Vriens, 2012</xref>). In situations where negative cues occur, hostile attribution bias is more likely to occur in children with IDs (<xref rid=\"B55\" ref-type=\"bibr\">Leffert et al., 2010</xref>), whereas once it is clear that there are no hostile intentions, children display no difficulty (<xref rid=\"B23\" ref-type=\"bibr\">Crick and Dodge, 1994</xref>; <xref rid=\"B54\" ref-type=\"bibr\">Leffert et al., 2000</xref>). Problems in encoding and interpretation result in deficits in steps 4 and 5 (<xref rid=\"B52\" ref-type=\"bibr\">Leffert and Siperstein, 1996</xref>; <xref rid=\"B85\" ref-type=\"bibr\">van Nieuwenhuijzen et al., 2009</xref>; <xref rid=\"B86\" ref-type=\"bibr\">van Nieuwenhuijzen and Vriens, 2012</xref>), such as positive evaluation of maladjusted behavior and production of aggressive reactions. Difficulties in social competence and adjustment, such as behavioral problems (<xref rid=\"B52\" ref-type=\"bibr\">Leffert and Siperstein, 1996</xref>; <xref rid=\"B84\" ref-type=\"bibr\">van Nieuwenhuijzen et al., 2004</xref>, <xref rid=\"B85\" ref-type=\"bibr\">2009</xref>; <xref rid=\"B86\" ref-type=\"bibr\">van Nieuwenhuijzen and Vriens, 2012</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Baurain and Nader-Grosbois, 2013</xref>), could therefore be explained by the specific SIP profile of these children.</p></sec><sec id=\"S1.SS3\"><title>Objectives of the Present Study</title><p>No previous study has examined the social cognition of children with IDs by combining analysis of both ToM and SIP profiles in order to better understand how their particular profiles contribute to their social adjustment or to the risk of maladjustment in family or school contexts. Studies have reported that these children are more at risk of displaying externalizing behaviors (such as opposition, resistance or aggressiveness; <xref rid=\"B78\" ref-type=\"bibr\">Taylor, 2002</xref>; <xref rid=\"B70\" ref-type=\"bibr\">Rojahn et al., 2012</xref>) and internalizing behaviors (such as withdrawal, isolation or anxiety; <xref rid=\"B61\" ref-type=\"bibr\">Merrell and Holland, 1997</xref>; <xref rid=\"B81\" ref-type=\"bibr\">Thirion-Marissiaux and Nader-Grosbois, 2008c</xref>), or even both kinds of behavioral problems (<xref rid=\"B26\" ref-type=\"bibr\">Dekker et al., 2002</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Baker et al., 2003</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Dekker and Koot, 2003</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Emerson, 2003</xref>; <xref rid=\"B63\" ref-type=\"bibr\">Nader-Grosbois et al., 2013</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Hauser-Cram and Woodman, 2016</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Bailey et al., 2019</xref>). To make it possible to give effective support to children with IDs in developing their social abilities and gaining social inclusion, the strengths and weaknesses in their social cognitive profiles, particularly in affective and cognitive ToM as well as in SIP, need to be explored. The present study aimed firstly to identify clinical homogenous groups of children depending on their affective and cognitive ToM abilities and/or SIP competence. Cluster analyses were applied considering children’s social cognitive skills and between-group comparisons were realized regarding their individuals’ characteristics and social behaviors. We hypothesized that children with IDs belonging to a cluster described by better ToM and SIP abilities should have higher developmental age and better social emotional and behavioral competence, in comparison with a cluster presenting less social cognitive abilities. The second objective was to analyze the structure of relations between skills related to affective and cognitive ToM and SIP processes in positive or negative social situations in children with IDs. We hypothesized that ToM abilities and SIP skills would be related depending on the situation. Social situations required ToM abilities such as perspective taking, comprehension of beliefs or emotions, and SIP skills needed to process social information adequately and to solve social problems.</p></sec></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Participants</title><p>Seventy-eight children (56 boys and 21 girls) with non-specific IDs were recruited in special primary schools from French-speaking areas of Belgium. They had been diagnosed as having mild to moderate IDs (intelligence quotient between 50 and 70), according to AAIDD (<xref rid=\"B4\" ref-type=\"bibr\">American Association on Intellectual and Developmental Disabilitites, 2011</xref>) and DSM-V (the <italic>Diagnostic and Statistical Manual of Mental Disorders</italic>) criteria. The intelligence quotient was not assessed by the experimenter but was checked through a cognitive assessment made previously by a professional. Children had to present an intelligence quotient between 50 and 70 to meet inclusion criteria. As can be seen in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, the children were approximatively 9 years, with a mean chronological age of 109.86 months (<italic>SD</italic> = 21.19), ranging from 56 to 150 months. Their global developmental age was about 5 years, with a mean of 63.97 months (<italic>SD</italic> = 13.65), ranging from 40 and 91 months. Moreover, their estimated verbal developmental age was between 37 and 86 months (mean = 62.88; <italic>SD</italic> = 13.71). Before we started the recruitment, an ethics committee of the faculty of psychology at UCLouvain approved the research procedure, notably by attesting to the respect of the ethical guidelines of the declaration of Helsinki. Recruitment was restricted on the basis of exclusion criteria. Children with Williams’s syndrome or autistic spectrum disorder could not be included. Children also had to be able to form sentences of three to four words and display a global developmental age higher than 36 months. Before connecting with parents, we asked school directors to indicate children who potentially met these criteria. Despite this and due to the application of these strict criteria, six children were excluded. Parents received a consent form from teachers explaining the research goal and procedure. This consent form also offered the possibility to receive, at the end of the procedure, a report of the child’s competence based on the completion of the different measures. The children’s families presented a low socioeconomic status. On a nine-level scale describing range of monthly income from 0–500 to 4,000 and more, the parents reported a low income (mean = 3.14) corresponding to a monthly income (salaries and benefits) of 1,000–1,500 euros, compared to a mean monthly salary of about 1,527 euros in Belgium. In terms of the parents’ levels of education, the mothers had typically completed secondary school (mean = 3.24), while the fathers had typically completed an apprenticeship contract (mean = 4). Our sample therefore revealed a certain homogeneity of cultural and socioeconomic status.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Demographic and individual characteristics: mean scores and standard deviations in Theory of Mind, Social information processing, and social (mal)adjustment measures.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" colspan=\"2\" rowspan=\"1\">Variables</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mean</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Children with non-specific IDs (<italic>n</italic> = 78)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sex (% boys)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">73%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CA (in months)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">109.86 (21.2)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GDA (in months)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">63.97 (13.65)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VDA (in months)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">62.88 (13.71)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Family measures</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Family income</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.14 (1.06)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mothers’ education (max = 7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.24 (2.25)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fathers’ education (max = 7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (1.72)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Explicit ToM measures</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM Task Battery total (max = 15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.14 (2.37)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Affective ToM Task Battery (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.04 (1.11)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cognitive ToM Task Battery (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.58 (1.45)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mixed ToM Task Battery (max = 3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.63 (0.91)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM emotions (max = 12)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.62 (2.33)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM emotions – causes (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.07 (1.46)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM emotions – consequences (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.21 (1.78)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM beliefs (max = 5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.9 (1.32)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Problem-solving task</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Judgment score on appropriate vignettes (max = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.71 (0.49)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Judgment score on inappropriate vignettes (max = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.82 (0.24)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Identification score on appropriate vignettes (max = 1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.78 (0.28)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Identification score on inappropriate vignettes (max = 1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.78 (0.19)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Justification score on appropriate vignettes (max = 7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.01 (1.38)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Justification score on inappropriate vignettes (max = 7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.04 (1.18)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Social (mal)adjustment</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EASE total (max = 98)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">57.9 (17.18)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EASE ToM (max = 52)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.84 (9.34)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EASE Social Skills (max = 46)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.06 (8.36)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Externalizing problems</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">68.69 (17.97)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Internalizing problems</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70.94 (15.54)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Social competence</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">108.31 (27.72)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – General adjustment</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">247.93 (51.44)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Depressive-happy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">34.96 (8.28)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Anxious-secure</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.63 (9.06)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Isolated-integrated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.87 (8.49)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Dependent-autonomous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.99 (8.66)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Angry-tolerant</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.19 (9.45)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Aggressive-controlled</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.02 (7.38)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Egoistic-prosocial</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.04 (8.77)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Resistant-cooperative</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.24 (9.26)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CBCL Externalizing Behaviors</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.36 (9.71)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CBCL Internalizing Behaviors</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.21 (8.54)</td></tr></tbody></table><table-wrap-foot><attrib><italic><italic>IDs, intellectual disabilities; CA, chronological Age; GDA, Global Developmental Age; VDA, Verbal Developmental Age; ToM, Theory of Mind; EASE, Social Adjustment Scale for Children; SCBE, Social Competence and Behavior Evaluation; CBCL, Child Behavior Checklist.</italic></italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S2.SS2\"><title>Measures</title><sec id=\"S2.SS2.SSS1\"><title>Wechsler Preschool and Primary Scales (WPPSI-III; <xref rid=\"B88\" ref-type=\"bibr\">Wechsler, 2004</xref>)</title><p>Four subtests – “information,” “vocabulary,” “block design,” and “matrix reasoning” – of the well-known WPPSI-III were administered. The results indicated the children’s verbal and non-verbal cognitive functioning and global developmental age. This evaluation ensured that children displayed a preschool developmental age, in order to meet the criteria for inclusion.</p></sec><sec id=\"S2.SS2.SSS2\"><title>ToM-Emotions Tasks (<xref rid=\"B80\" ref-type=\"bibr\">Thirion-Marissiaux and Nader-Grosbois, 2008b</xref>)</title><p>ToM-emotions is a computerized instrument (on Eprime) that assesses the comprehension of causes and consequences of emotions (namely, joy, sadness, anger, and fear). There are three tasks: (1) The first is a preliminary task evaluating facial expression recognition for the four emotions; (2) The second task assesses the comprehension of causes of emotions. A script describing a situation of joy, sadness, anger, or fear is presented to the children, who have to predict the protagonist’s emotion depending on the story. Concretely, the children need to identify the emotion and justify their response for each story. Emotion recognition receives a score of 1 and the score for coherent justification is 0.5, with a maximum score of 6 for this task. (3) The third task evaluates the comprehension of consequences of emotions by presenting four scripts in which the protagonist feels joy, anger, sadness and fear respectively. Children have to choose one of the three behavioral responses suggested, according to the protagonist’s emotion. These options illustrate a socially adjusted, maladjusted, or neutral behavior. The choice of the socially adjusted card gets a score of 1, whereas the maladjusted or neutral card receives a 0. The children then have to justify their choice. A coherent justification receives 0.5. The maximum score for this third task is 6. The entire ToM-emotions instrument is thus scored out of 12.</p><p>Validation of the original version was conducted on 80 children with and without IDs and matched for preschool developmental age. The recorded evaluations revealed a high level of inter-judge agreement (between 95 and 98%, with Cohen’s kappa between 0.89 and 0.92; Pearson correlation coefficient between 0.93 and 0.96), based on each item score as well as for each task and emotion. A factor analysis revealed two factors related to the causes and consequences subscales (<xref rid=\"B80\" ref-type=\"bibr\">Thirion-Marissiaux and Nader-Grosbois, 2008b</xref>). Analysis of the computerized measure showed the same factors and a Cronbach’s alpha of 0.57 as well as a very high test-retest stability for the two subscales (between 0.56 and 0.68). For the present study, the Cronbach’s alpha coefficients are 0.34 and 0.27 respectively.</p></sec><sec id=\"S2.SS2.SSS3\"><title>ToM-Beliefs Tasks (<xref rid=\"B79\" ref-type=\"bibr\">Thirion-Marissiaux and Nader-Grosbois, 2008a</xref>)</title><p>The ToM-beliefs tasks instrument evaluates the understanding of beliefs through five popular tasks. (1) The deception skills task (<xref rid=\"B64\" ref-type=\"bibr\">Oswald and Ollendick, 1989</xref>) assesses the ability of the child to deceive an adult by hiding a little object in his or her hands. (2) A change of representation task (<xref rid=\"B33\" ref-type=\"bibr\">Flavell et al., 1981</xref>) asks the child to infer what the adult sees on a specific image. (3) The third task is the appearance-reality task (<xref rid=\"B31\" ref-type=\"bibr\">Flavell, 1986</xref>). The experimenter presents an object with an appearance that differs from its real function (e.g., a pencil that looks like a flower). The child has to distinguish appearance from reality. (4) During the fourth task, the unexpected content task (<xref rid=\"B66\" ref-type=\"bibr\">Perner et al., 1987</xref>), the experimenter shows a Smarties box filled with pencils to the child and asks, “What is inside the box?” After demonstrating the content, experimenter fills the box with the pencils again and asks the child, “What did you think was in the box before it was opened?” and “What will your mother think is in the box if she has not seen inside it?” (5) The last task is the change of location task (<xref rid=\"B94\" ref-type=\"bibr\">Wimmer and Perner, 1983</xref>), corresponding to the well-known “Max and the transfer of chocolate” task. Each task gets a score of 1, with a maximum score of 5.</p><p>This measure was validated with typically developing preschoolers and children with IDs (ages 6–15) with a preschool developmental age. Evaluations were recorded and the following analysis demonstrated a very high inter-judge agreement (between 99 and 100%; Cohen’s kappa between 0.98 and 0.99; Pearson correlation coefficient between 0.99 and 1). A test-retest session revealed no significant difference (<xref rid=\"B79\" ref-type=\"bibr\">Thirion-Marissiaux and Nader-Grosbois, 2008a</xref>). Cronbach’s alpha for the present sample is 0.62.</p></sec><sec id=\"S2.SS2.SSS4\"><title>ToM-Task Battery – French Version (<xref rid=\"B42\" ref-type=\"bibr\">Hutchins et al., 2008a</xref>; <xref rid=\"B62\" ref-type=\"bibr\">Nader-Grosbois and Houssa, 2016</xref>)</title><p>This battery was created to assess affective and cognitive ToM. Mental states are evaluated by means of nine tasks: (1) emotion recognition; (2) perspective taking; (3) inference of desire-based emotion; (4) inference of perception-based belief; (5) inference of perception-based action; (6) false belief; (7) inference of belief- and reality-based emotion and second order emotion; (8) message-desire discrepancy; and (9) second-order false belief. Children are asked control (e.g., What does Brigitte want? Where did Anthony put his book?), prompt (e.g., Where is the book now?), and test (e.g., Where will Anthony search his book?) questions. Only test questions are scored. Each task is scored 1 except for the three following: emotion recognition is scored 4 (1 point for recognition of joy, sadness, fear and anger respectively); perspective taking is scored 2 (since the child has to take the perspective of two protagonists); and inference of belief- and reality-based emotion and second-order emotion are scored 2 (1 point for recognition of emotion and of second-order emotion respectively). The total is scored out of 15. Subscores can be calculated to obtain affective, cognitive, and mixed scores.</p><p>This measure was validated for children with autism-spectrum disorder (ages 4.5–12) and revealed good internal consistency (α = 0.91) and test-retest reliability, considering different time variation between two administrations (<xref rid=\"B44\" ref-type=\"bibr\">Hutchins et al., 2008b</xref>). Another validation of the French version was carried out on typically developing preschoolers. Analysis revealed good internal consistency (α = 0.75) and test-retest reliability (<italic>r</italic> = 0.87) (<xref rid=\"B62\" ref-type=\"bibr\">Nader-Grosbois and Houssa, 2016</xref>). The reliability of this measure for the present sample is also good (α = 0.68).</p></sec><sec id=\"S2.SS2.SSS5\"><title>Problem-Solving Task (RES, <xref rid=\"B12\" ref-type=\"bibr\">Barisnikov et al., 2004</xref>)</title><p>The problem-solving task estimates how children identify and judge a protagonist’s social behavior as appropriate or not. Fourteen images illustrate fictitious social situations involving either appropriate (5) or inappropriate (9) behaviors. For each image, children are asked to judge whether the behavior is appropriate or not (judgment score), to identify target behavior by social cues (identification score), and to justify their judgment (justification score). A score of 1 or 2 points is given for correct identification and judgment respectively. The justification score is determined by the children’s responses to the consequence for the protagonist (descriptive level: 2 points), to social consciousness (intersubjective level: 5 points), or to social rules (conventional level: 7 points). This measure mobilizes SIP skills. It is possible to differentiate scores depending on the social behavior depicted in the images. Five vignettes illustrate appropriate behaviors, and nine others illustrate inappropriate actions. In this study, identification, judgment, and justification scores are analyzed from responses to the vignettes, providing six subscores for either appropriate or inappropriate behaviors. The maximum total score is 140: 28 for judgment, 14 for identification, and 98 for justification.</p><p>The validation revealed an inter-judge agreement of 98% on a sample of children with and without IDs (<xref rid=\"B40\" ref-type=\"bibr\">Hippolyte et al., 2010</xref>). For the present study, Cronbach’s alpha is 0.87.</p></sec><sec id=\"S2.SS2.SSS6\"><title>Social Adjustment Scales for Children (EASE; <xref rid=\"B41\" ref-type=\"bibr\">Hughes et al., 1997</xref>)</title><p>This questionnaire measures adults’ perception of children’s social adjustment. Parents estimate how frequent particular behaviors occur in daily interactions (rarely, relatively frequently or usually). Half of the items measure adaptive social skills (e.g., politeness, discipline or civility), and the other half assess social behaviors related to ToM abilities (e.g., considering others’ emotions, desires or beliefs), providing two subscores: one for social skills (maximum 46) and the other for ToM (maximum 52).</p><p>The two subscales have a good internal consistency, with Cronbach’s alpha coefficients of 0.77 and 0.79 respectively (<xref rid=\"B41\" ref-type=\"bibr\">Hughes et al., 1997</xref>). Similarly, good reliability was obtained for the present sample, with Cronbach’s alpha coefficients of 0.78 and 0.83.</p></sec><sec id=\"S2.SS2.SSS7\"><title>Social Competence and Behavior Evaluation Scale (SCBE; <xref rid=\"B51\" ref-type=\"bibr\">LaFrenière et al., 1992</xref>)</title><p>This questionnaire assesses children’s socio-affective profile through 80 items. Parents evaluate to what extent their children display each behavior, using a 6-point Likert scale, from “never” to “always.” The questionnaire provides a complete profile in eight socio-affective domains: angry-tolerant, anxious-secure, depressive-happy, isolated-integrated, dependent-autonomous, resistant-cooperative, egoistic-prosocial, and aggressive-controlled. Each dimension is evaluated on a continuum emphasizing the child’s weaknesses and strengths. Some dimensions are related to the affective domain (depressive-happy; angry-tolerant; anxious-secure), while others reflect interactions with peers (isolated-integrated; egoistic-prosocial; aggressive-controlled) or with adults (dependent-autonomous, resistant-cooperative). The sum of the scores in certain specific dimensions gives four global scales: externalizing problems, internalizing problems, social competence, and general adjustment. The externalizing scale clusters four of the eight dimensions (angry-tolerant, resistant-cooperative, egoistic-prosocial and aggressive-controlled), while the internalizing scale brings together the other four (anxious-secure, depressive-happy, isolated-integrated, dependent-autonomous). The social competence scale covers 40 positive statements and assesses behaviors related to affective maturity, flexibility, and adequate adjustment during social interactions with peers or adults. The general adjustment scale reflects a global score through all 80 statements of this measure. These scores can be converted into T-scores, allowing the results to be compared with standards varying according to participants’ gender and developmental age (more or less than 4 years), and making it possible to identify difficulties or strengths. For all the scales and dimensions, T-scores lower than 38 or above 68 reflect weaknesses or strengths in comparison to a representative sample.</p><p>For the French version, the eight subscales display Cronbach’s alpha coefficients of between 0.79 and 0.82 and for the present sample between 0.78 and 0.90.</p></sec><sec id=\"S2.SS2.SSS8\"><title>Child Behavior Checklist (CBCL; <xref rid=\"B2\" ref-type=\"bibr\">Achenbach, 1991</xref>)</title><p>This well-known questionnaire of 79 items assesses parents’ perception of children’s behavioral and emotional problems. Parents indicate the frequency of children’s behaviors on a 3-point Likert scale, from “not at all” to “often.” This produces, among other things, two scores for the presence of either internalizing or externalizing behaviors. Four subscales, namely “anxious/depressed,” “emotionally reactive,” “withdrawn,” and “somatic complaints,” determine the internalizing behavior score (clinical cutoff > 17), whereas the “attention problems” and “aggressive behavior” subscales are integrated to provide the externalizing behavior score (clinical cutoff > 24). These scores provide information about the sample’s clinical profile and potential risk of behavioral problems.</p><p>Cronbach’s alpha for the different subscales is between 0.63 and 0.86. For the present study, Cronbach’s alpha was 0.85.</p></sec></sec><sec id=\"S2.SS3\"><title>Procedure</title><p>All the described measures were administered to children and parents during a period of 2 weeks. The researcher used direct measures to evaluate the children’s cognition, ToM and SIP skills. The evaluation took place in a quiet room at the child’s school, during two 45 min sessions. At the same time, the parents received questionnaires about their perceptions of their child’s social adjustment and social and behavioral competence, including internalizing or externalizing behaviors problems. Parents could choose either to fill in the questionnaires at home or to make an appointment to fill them in with the researcher’s help. This help was requested by 80% of the parents. Given their low socioeconomic status and education level, many items were difficult for them to understand. During interviews, they also preferred to discuss the challenging behaviors they faced daily.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Participants’ Characteristics</title><p><xref rid=\"T1\" ref-type=\"table\">Table 1</xref> presents average scores and standard deviations for the sample’s demographic and individual characteristics including chronological and developmental age and scores in ToM, SIP, and social abilities. Details about ages and demographic data are given above in the description of the sample. The ToM and SIP measures give indications of strengths and weaknesses, but not standards. Regarding ToM and SIP tasks, children’s scores were not below average, except for cognitive and mixed ToM Task Battery. In terms of socio-affective profile, T-scores for the SCBE scales results indicated that the children in the present sample displayed neither specific weaknesses nor strengths. T-scores for the four global scales (externalizing problems, internalizing problems, social competence, and general adjustment) were 47 respectively. For specific dimensions, T-scores corresponded in order to 49, 47, 48, 42, 45, 49, 48, 48, and 44. No T-scores for global scales and specific dimensions (ranging from 42 to 49) were lower than the cutoff (38). This demonstrated that these children with IDs had competence corresponding to those of a representative sample matched for developmental age. However, by comparing qualitatively the eight dimensions with each other, it can be observed on the one hand that with adults children were more irritable or frustrated and less autonomous and cooperative. On the other hand, scores revealed that they were perceived as particularly happy and controlled in their interactions with peers. In terms of behavior problems, children were at higher risk of developing internalizing problems, given that their score (mean = 16) was at a borderline level (according to CBCL standards).</p></sec><sec id=\"S3.SS2\"><title>Inter-Correlations Between Individuals’ Characteristics and Skills in Social Cognition</title><p><xref rid=\"T2\" ref-type=\"table\">Table 2</xref> presents intercorrelations between individuals’ characteristics and competence related to ToM and SIP within the present sample. It reveals that global and verbal developmental ages were linked in a statistically significant way with all ToM (<italic>r</italic> between 0.252 and 0.674; <italic>p</italic> < 0.05) and SIP abilities (<italic>r</italic> between 0.348 and 0.683; <italic>p</italic> ≤ 0.001), whereas chronological age was related positively and significantly only to certain aspects, namely the total (<italic>r</italic> = 0.241; <italic>p</italic> < 0.05) and affective (<italic>r</italic> = 0.244, <italic>p</italic> < 0.05) scores of ToM Task Battery, ToM beliefs (<italic>r</italic> = 0.362; <italic>p</italic> ≤ 0.001), the judgment score on appropriate vignettes (<italic>r</italic> = 0.364; <italic>p</italic> < 0.05), the identification score on inappropriate vignettes (<italic>r</italic> = 0.228; <italic>p</italic> < 0.05), and the justification score on both appropriate (<italic>r</italic> = 0.276; <italic>p</italic> < 0.05) and inappropriate (<italic>r</italic> = 0.232; <italic>p</italic> < 0.05) vignettes. ToM and SIP abilities were highly and positively interrelated in a statistically significant way (<italic>r</italic> between 0.228 and 0.595; <italic>p</italic> ≤ 0.001).</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Spearman correlations between individuals’ characteristics, Theory of Mind and Social information processing skills.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"justify\" colspan=\"2\" rowspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Individual</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. CA (in months)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.319**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.323**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.086</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.241*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.244*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.137</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.213</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.118</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.081</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.169</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.362**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.354*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.185</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.360</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.228*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.276*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.232*</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">characteristics</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2. GDA (in months)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.915**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.287</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.570**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.602**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.256*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.384**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.483**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.328**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.481**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.674**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.501**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.403**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.507**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.489**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.579**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.656**</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3. VDA (in months)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.237</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.566**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.554**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.252*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.404**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.580**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.391**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.582**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.673**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.476**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.348**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.493**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.521**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.592**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.683**</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4. Family income</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.137</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.216</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.054</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.021</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.031</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.121</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.052</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.162</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.009</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.147</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.023</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.141</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.095</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.203</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Explicit ToM measures</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5. ToM Task Battery total (max = 15)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.748**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.820**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.498**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.344**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.314**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.245*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.510**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.507**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.255*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.499**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.441**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.495**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.510**</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">6. Affective ToM Task Battery (max = 6)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.412**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.319**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.314**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.369**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.169</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.515**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.397**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.351**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.399**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.418**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.392**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.345**</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7. Cognitive ToM Task Battery (max = 6)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.121</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.197</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.233*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.153</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.224</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.356**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.014</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.314**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.242*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.317**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.272*</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8. Mixed ToM Task Battery (max = 3)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.151</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.068</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.095</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.354**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.377**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.218</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.367**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.182</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.268*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.490**</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">9. ToM emotions (max = 12)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.716**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.850**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.496**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.277*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.259*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.416**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.507**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.471**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.401**</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">10. ToM emotions – causes (max = 6)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.392**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.469**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.213</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.228*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.370**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.426**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.361**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.302**</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">11. ToM emotions – consequences (max = 6)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.473**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.327**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.213</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.400**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.545**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.488**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.412**</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">12. ToM beliefs (max = 5)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.505**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.441**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.563**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.595**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.562**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.530**</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Problem-solving task</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">13. Judgment score on appropriate vignettes (max = 2)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.210</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.798**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.484**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.699**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.503**</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14. Judgment score on inappropriate vignettes (max = 2)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.292**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.542**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.147</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.407**</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">15. Identification score on appropriate vignettes (max = 1)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.737**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.791**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.597**</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">16. Identification score on inappropriate vignettes (max = 1)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.656**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.640**</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">17. Justification score on appropriate vignettes (max = 7)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.669**</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">18. Justification score on inappropriate vignettes (max = 7)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic><italic>CA, Chronological Age; GDA, Global Developmental Age; VDA, Verbal Developmental Age; ToM, Theory of Mind; *p < 0.05; **p ≤ 0.001.</italic></italic></attrib></table-wrap-foot></table-wrap><p><xref rid=\"T3\" ref-type=\"table\">Table 3</xref> presents correlations between individuals’ characteristics and abilities related to ToM and SIP on the one hand and their social adjustment competence and socio-affective profile on the other hand. The results indicate that global and verbal developmental ages are linked positively and significantly with social adjustment (<italic>r</italic> = 0.333 and 0.379 respectively; <italic>p</italic> ≤ 0.001). They are also linked in a statistically significant way with some dimensions of the socio-affective profile. Global developmental age is correlated with anxious-secure (<italic>r</italic> = 0.281; <italic>p</italic> < 0.05) and isolated-integrated (<italic>r</italic> = 0.272; <italic>p</italic> < 0.05) subscales while verbal developmental age is correlated with depressive-happy (<italic>r</italic> = 0.267; <italic>p</italic> < 0.05), anxious-secure (<italic>r</italic> = 0.378; <italic>p</italic> ≤ 0.001) and isolated-integrated (<italic>r</italic> = 0.357; <italic>p</italic> ≤ 0.001) subscales, whereas chronological age is not. Moreover, a higher verbal developmental age is associated in a statistically significant way with a lower risk of developing internalized problems (<italic>r</italic> = 0.273; <italic>p</italic> < 0.05) and with better social competence (<italic>r</italic> = 0.344; <italic>p</italic> ≤ 0.001). Affective ToM is associated positively and significantly with social adjustment (related to social skills; <italic>r</italic> = 0.234; <italic>p</italic> < 0.05) and competence (<italic>r</italic> = 0.375; <italic>p</italic> ≤ 0.001), as well as with some socio-affective dimensions (anxious-secure, <italic>r</italic> = 0.236; <italic>p</italic> < 0.05; isolated-integrated, <italic>r</italic> = 0.232; <italic>p</italic> < 0.05; dependent-autonomous, <italic>r</italic> = 0.266; <italic>p</italic> < 0.05). A good understanding of affective mental states is related in a statistically significant way to a lower risk of developing internalized problems (<italic>r</italic> = 0.242; <italic>p</italic> < 0.05) and externalizing behaviors (<italic>r</italic> = -0.267; <italic>p</italic> < 0.05). Cognitive ToM measured by ToM beliefs, is associated positively and significantly with social adjustment (<italic>r</italic> = 0.274; <italic>p</italic> < 0.05) and competence (<italic>r</italic> = 0.372; <italic>p</italic> ≤ 0.001). It is also correlated positively and significantly with anxious-secure (<italic>r</italic> = 0.256; <italic>p</italic> < 0.05) and dependent-autonomous (<italic>r</italic> = 0.259; <italic>p</italic> < 0.05) subscales of the SCBE measure.</p><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>Spearman correlations between individuals’ characteristics and abilities in Theory of Mind and social information processing and their social adjustment and socio-affective profile.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"justify\" colspan=\"2\" rowspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">EASE<hr/></td><td valign=\"top\" align=\"center\" colspan=\"12\" rowspan=\"1\">SCBE<hr/></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">CBCL<hr/></td></tr><tr><td valign=\"top\" colspan=\"2\" rowspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Total</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ToM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Social Skills</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Ext. prob.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Int. prob.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Social Compet.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">General adjustment</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Depressive-Happy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Anxious-Secure</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Isolated-Integrated.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Dependent-Autonomous.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Angry- Tolerant</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Aggressive-Controlled</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Egoistic-Prosocial</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Resistant-Coop.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">EB</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">IB</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Individual characteristics</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CA (in months)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.141</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.185</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.104</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.087</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.213</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.010</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.058</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.183</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.187</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.162</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.088</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.084</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.073</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.045</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.103</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.066</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.121</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GDA (in months)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.333**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.349**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.332**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.170</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.206</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.281−</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.194</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.210</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.281*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.272*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.221</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.039</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.144</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.060</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.055</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.067</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.212</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VDA (in months)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.379**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.400**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.378**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.086</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.273*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.344**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.275*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.267*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.378**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.357**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.227</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.038</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.052</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.028</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.100</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.043</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.232</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Family income</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.057</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.010</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.098</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.099</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.027</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.087</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.034</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.036</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.228</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.166</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.035</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.106</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.167</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.016</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.077</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.228</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.184</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Explicit ToM measures</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM Task Battery total (max = 15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.222</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.219</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.235*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.014</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.243*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.308**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.266*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.145</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.263*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.277*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.238*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.067</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.065</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.082</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.126</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.223</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.236</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Affective ToM Task Battery (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.222</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.189</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.234*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.082</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.242*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.375**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.321**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.166</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.236*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.232*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.266*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.083</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.152</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.207</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.221</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.267*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.249</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cognitive ToM Task Battery (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.102</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.094</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.117</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.052</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.165</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.159</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.158</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.066</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.143</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.134</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.095</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.127</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.105</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.055</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.220</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.198</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mixed ToM Task Battery (max = 3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.149</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.188</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.125</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.166</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.106</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.139</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.076</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.091</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.111</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.114</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.159</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.064</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.107</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.155</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.038</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.066</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.202</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM emotions (max = 12)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.412**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.439**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.369**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.078</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.215</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.374**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.330**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.201</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.222</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.310**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.191</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.195</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.131</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.278*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.267*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.086</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.046</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM emotions – causes (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.341**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.380**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.280*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.171</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.258*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.451**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.386**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.176</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.261*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.223</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.355**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.270*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.253*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.338**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.304**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.105</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.052</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM emotions – consequences (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.276*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.280*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.271*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.087</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.091</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.305**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.275*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.139</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.100</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.242*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.033</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.214</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.186</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.289*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.254*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.029</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.060</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM beliefs (max = 5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.274*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.281*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.275*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.177</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.372**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.297*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.163</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.256*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.225</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.259*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.113</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.082</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.140</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.164</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.186</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.185</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Problem-solving task</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Judgment score on appropriate vignettes (max = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.191</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.190</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.214</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.240*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.030</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.132</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.026</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.019</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.046</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.013</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.066</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.136</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.020</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.098</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.053</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.149</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.140</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Judgment score on inappropriate vignettes (max = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.342**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.308**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.344**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.102</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.232*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.256*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.197</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.112</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.209</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.180</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.381**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.057</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.028</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.042</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.054</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.071</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.019</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Identification score on appropriate vignettes (max = 1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.145</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.178</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.139</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.087</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.040</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.229*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.159</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.016</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.077</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.046</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.174</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.082</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.077</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.083</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.021</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.091</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.039</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Identification score on inappropriate vignettes (max = 1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.205</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.229*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.192</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.031</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.192</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.416**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.341**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.090</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.187</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.249*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.330**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.253*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.215</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.212</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.110</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.026</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.051</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Justification score on appropriate vignettes (max = 7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.244*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.270*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.241*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.029</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.191</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.344**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.297*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.097</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.250*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.160</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.232*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.197</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.205</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.121</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.143</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.180</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.197</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Justification score on inappropriate vignettes (max = 7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.190</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.241*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.171</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.132</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.188</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.211</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.168</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.038</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.236*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.207</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.339**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.048</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.016</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.083</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.113</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.226</td></tr></tbody></table><table-wrap-foot><attrib><italic><italic>CA, Chronological Age; GDA, Global Developmental Age; VDA, Verbal Developmental Age; ToM, Theory of Mind; EASE, Social Adjustment Scale for Children; SCBE, Social Competence and Behavior Evaluation; CBCL, Child Behavior Checklist; Ext prob., externalized problems; Int. prob., internalized problems; Compet., Competence; Coop., Cooperative; EB, Externalizing behaviors; IB, Internalizing behaviors. *p < 0.05; **p ≤ 0.001.</italic></italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS3\"><title>Hierarchical Cluster Analysis With Reference to ToM and/or SIP Abilities</title><p>We applied a hierarchical agglomerative cluster analysis using Ward’s method and squared Euclidean distance in order to identify groups that presented different patterns in terms of ToM abilities, SIP competence, or both (using scores for ToM-emotions, ToM-beliefs and subscores for ToM task Battery and RES). The clustering allows exploration of the profile without an explanatory model.</p><p>The hierarchical cluster analysis depending on ToM profiles revealed two groups as shown in the dendrogram (see <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). The average distance between these two clusters was 778.155.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Dendrogram with added line indicating suggested stopping location, resulting from the application of hierarchical cluster analysis with Ward’s method and Euclidian distance and depending on Theory of Mind profile of children with intellectual disabilities.</p></caption><graphic xlink:href=\"fpsyg-11-01884-g001\"/></fig><p><xref rid=\"T4\" ref-type=\"table\">Table 4</xref> presents the individual characteristics and ToM competence of two clusters obtained through hierarchical cluster analysis using scores related to ToM task Battery, ToM-emotions and ToM-beliefs. Independent <italic>t</italic>-tests indicated some differences between these two clusters. Children in the second cluster had a higher chronological age [<italic>t</italic>(1) = 2.12; <italic>p</italic> = 0.037; <italic>d</italic> = 0.48] as well as a higher global [<italic>t</italic>(1) = 2.64; <italic>p</italic> = 0.011; <italic>d</italic> = 0.60] and verbal [<italic>t</italic>(1) = 3.13; <italic>p</italic> = 0.003; <italic>d</italic> = 0.71] developmental age, in comparison with children in the first cluster. In terms of ToM abilities, a marginal difference was revealed in the affective score for the ToM Task Battery [<italic>t</italic>(1) = 1.98; <italic>p</italic> = 0.051; <italic>d</italic> = 0.45], while significant differences were obtained in the understanding of consequence of emotions [ToM emotions-consequences; <italic>t</italic>(1) = 3.48; <italic>p</italic> = 0.001; <italic>d</italic> = 0.79] and of cognitive mental states [ToM beliefs; <italic>t</italic>(1) = 2.12; <italic>p</italic> = 0.034; <italic>d</italic> = 0.49]. The second cluster, displaying higher ages, also presented better ToM competence compared with the first cluster. Regarding social adjustment, a unique difference was significant. Children in the second cluster were perceived as more socially adjusted [EASE total; <italic>t</italic>(1) = 2.12; <italic>p</italic> = 0.026; <italic>d</italic> = 0.48], particularly in situations requiring the understanding of social conventions [EASE – social skills; <italic>t</italic>(1) = 2.12; <italic>p</italic> = 0.038; <italic>d</italic> = 0.48]. They also tended to be evaluated as more cooperative [<italic>t</italic>(1) = 1.74; <italic>p</italic> = 0.085; <italic>d</italic> = 0.40] when interacting with adults.</p><table-wrap id=\"T4\" position=\"float\"><label>TABLE 4</label><caption><p>Between-group comparisons of the two clusters obtained through hierarchical cluster analysis according to Theory of Mind abilities.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Cluster 1 (<italic>n</italic> = 37)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Cluster 2 (<italic>n</italic> = 41)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" colspan=\"2\" rowspan=\"1\">Variables</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mean (<italic>SD</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mean (<italic>SD</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>X</italic><sup>2</sup><italic><sup>/</sup>t</italic> (1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>d</italic></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sex (% boys)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">67%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">78%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.08</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CA (in months)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">104.62 (20.71)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">114.59 (20.76)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.12*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.48</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GDA (in months)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">59.83 (13.83)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">67.69 (12.49)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.64*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.60</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VDA (in months)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">58.03 (14.17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">67.26 (11.82)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.13**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.71</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Family income</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.32 (0.98)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.80 (1.15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.49</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Explicit ToM measures</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM Task Battery total (max = 15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.59 (2.31)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.63 (2.33)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.97<sup>†</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.45</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Affective ToM Task Battery (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.78 (1.22)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.27 (0.95)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.98<sup>†</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.45</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cognitive ToM Task Battery (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.39 (1.14)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.73 (1.66)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.99</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mixed ToM Task Battery (max = 3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.53 (0.76)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.71 (1.01)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.82</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM emotions (max = 12)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.78 (2.35)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.38 (2.06)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.17**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.72</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM emotions – causes (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.78 (1.59)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.33 (1.29)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.66</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM emotions – consequences (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.53 (1.73)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.85 (1.59)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.48***</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.79</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM beliefs (max = 5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.57 (1.4)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.21 (1.19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.16*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.49</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Social (mal)adjustment</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EASE total (max = 98)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53.59 (17.04)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">61.54 (15.99)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.12*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.48</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EASE ToM (max = 52)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25.78 (9.04)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.66 (8.79)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.91<sup>†</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.43</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EASE Social Skills (max = 46)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.81 (8.58)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.88 (8.34)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.12*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.48</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Externalizing problems</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">67.81 (17.71)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">69.51 (18.42)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.40</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Internalizing problems</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70.89 (14.71)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70.99 (16.48)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.03</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Social competence</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">103.77 (25.81)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">112.59 (29.11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.38</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – General adjustment</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">242.48 (45.32)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">253.11 (56.75)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.89</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Depressive-happy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">34.85 (8.09)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35.05 (8.57)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.10</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Anxious-secure</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.56 (8.62)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.69 (9.58)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Isolated-integrated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.35 (8.17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35.31 (8.63)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.52</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Dependent-autonomous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.91 (9.54)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.06 (7.86)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Angry-tolerant</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.59 (9.05)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.76 (9.91)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.53</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Aggressive-controlled</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.76 (6.88)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.26 (7.92)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.29</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Egoistic-prosocial</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.10 (7.73)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.92 (9.67)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.89</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Resistant-cooperative</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.34 (8.59)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35.04 (9.62)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.74<sup>†</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.40</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CBCL Externalizing Behaviors</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17.09 (10.52)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14.41 (9.25)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.05</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CBCL Internalizing Behaviors</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17.91 (9.38)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14.22 (8.74)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.48</td><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic><italic>CA, Chronological Age; GDA, Global Developmental Age; VDA, Verbal Developmental Age; ToM, Theory of Mind; EASE, Social Adjustment Scale for Children; SCBE, Social Competence and Behavior Evaluation; CBCL, Child Behavior Checklist; <sup>†</sup>p ≤ 0.059; *p < 0.05; **p ≤ 0.01; ***p ≤ 0.001.</italic></italic></attrib></table-wrap-foot></table-wrap><p>The hierarchical cluster analysis depending on SIP profiles of the present sample revealed two clusters of cases, with an average distance between them of 113.070. The distribution of cases is presented in the dendrogram (see <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Dendrogram with added line indicating suggested stopping location, resulting from application of hierarchical cluster analysis with Ward’s method and Euclidian distance and depending on social information processing profile of children with intellectual disabilities.</p></caption><graphic xlink:href=\"fpsyg-11-01884-g002\"/></fig><p>In <xref rid=\"T5\" ref-type=\"table\">Table 5</xref>, independent <italic>t</italic>-tests demonstrated some differences between the two clusters obtained through hierarchical cluster analysis using RES scores. Compared with children in the first cluster, children in the second had a higher verbal developmental age [<italic>t</italic>(1) = 2.80; <italic>p</italic> = 0.008; <italic>d</italic> = 0.47] and better abilities at identifying [<italic>t</italic>(1) = 2.32; <italic>p</italic> = 0.028; <italic>d</italic> = 0.61] and justifying [<italic>t</italic>(1) = 2.35; <italic>p</italic> = 0.024; <italic>d</italic> = 0.58] socially inappropriate behavior. In terms of social adjustment, children in the second cluster were perceived as more socially competent [<italic>t</italic>(1) = 2.33; <italic>p</italic> = 0.036; <italic>d</italic> = 0.61] in comparison with those in the first cluster. They were perceived as more cooperative [<italic>t</italic>(1) = 2.20; <italic>p</italic> = 0.036; <italic>d</italic> = 0.59] in their interactions with adults and tended to be less aggressive [<italic>t</italic>(1) = 2.04; <italic>p</italic> = 0.052; <italic>d</italic> = 0.57] when interacting with peers.</p><table-wrap id=\"T5\" position=\"float\"><label>TABLE 5</label><caption><p>Between-group comparisons of the two clusters obtained through hierarchical cluster analysis according to Social information processing abilities.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Cluster 1 (<italic>n</italic> = 56)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Cluster 2 (<italic>n</italic> = 22)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" colspan=\"2\" rowspan=\"1\">Variables</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>M</italic> (<italic>SD</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>M</italic> (<italic>SD</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>X</italic><sup>2</sup><italic><sup>/</sup>t</italic> (1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>d</italic></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sex (% boys)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">72%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">73%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CA (in months)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">105.50 (20.13)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">111.57 (21.54)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.17</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GDA (in months)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">59.45 (13.46)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">65.74 (13.42)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.86<sup>†</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.47</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VDA (in months)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">56.32 (12.81)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">65.45 (13.29)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.80**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.47</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Family income</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.23 (1.17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.10 (1.03)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.35</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Problem-solving task</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Judgment score on appropriate vignettes (max = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.67 (0.46)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.72 (0.51)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.36</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Judgment score on inappropriate vignettes (max = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.82 (0.24)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.82 (0.23)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.01</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Identification score on appropriate vignettes (max = 1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.74 (0.25)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.79 (0.29)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.71</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Identification score on inappropriate vignettes (max = 1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.69 (0.23)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.81 (0.16)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.32*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.61</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Justification score on appropriate vignettes (max = 7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.65 (1.42)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.15 (1.36)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.40</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Justification score on inappropriate vignettes (max = 7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.57 (1.09)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.23 (1.18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.35*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.58</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Social (mal)adjustment</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EASE total (max = 98)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53.86 (17.68)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">59.30 (16.45)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.25</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EASE ToM (max = 52)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25.14 (9.64)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.87 (8.69)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.58</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EASE Social Skills (max = 46)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.73 (8.57)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.43 (8.71)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.78</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Externalizing problems</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">63.52 (21.18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70.59 (16.45)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.35</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Internalizing problems</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70.05 (14.55)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">71.27 (16.01)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.31</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Social competence</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">96.15 (27.58)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">112.81 (26.64)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.33*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.61</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – General adjustment</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">229.72 (52.51)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">254.68 (49.84)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.84</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Depressive-happy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.85 (8.89)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35.74 (7.99)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.27</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Anxious-secure</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.05 (8.74)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.22 (9.19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.94</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Isolated-integrated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.72 (8.28)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">34.67 (8.50)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.35</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Dependent-autonomous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.75 (8.57)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.07 (8.77)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.14</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Angry-tolerant</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24.55 (10.61)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.17 (8.89)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.36</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Aggressive-controlled</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.80 (8.86)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.21 (6.45)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.04<sup>†</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.57</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Egoistic-prosocial</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24.77 (8.67)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.87 (8.73)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.36</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Resistant-cooperative</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.22 (9.86)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">34.72 (8.66)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.20*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.59</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CBCL Externalizing Behaviors</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17.35 (11.48)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.30 (9.41)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.65</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CBCL Internalizing Behaviors</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14.88 (7.97)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17.03 (9.75)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.86</td><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic><italic>CA, Chronological Age; GDA, Global Developmental Age; VDA, Verbal Developmental Age; EASE, Social Adjustment Scale for Children; SCBE, Social Competence and Behavior Evaluation; CBCL, Child Behavior Checklist; <sup>†</sup>p ≤ 0.06; *p < 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p = 0.000.</italic></italic></attrib></table-wrap-foot></table-wrap><p>The hierarchical cluster analysis depending on ToM and SIP profiles indicated two clusters. The distribution of cases is presented in the dendrogram (see <xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). The average distance between the two clusters described below is 1036.334.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Dendrogram with added line indicating suggested stopping location, resulting from application of hierarchical cluster analysis with Ward’s method and Euclidian distance and depending on Theory of Mind and social information processing profiles of children with intellectual disabilities.</p></caption><graphic xlink:href=\"fpsyg-11-01884-g003\"/></fig><p><xref rid=\"T6\" ref-type=\"table\">Table 6</xref> presents the individual characteristics and socio-emotional competence of the two clusters obtained through hierarchical cluster analysis in terms of both ToM and SIP abilities. Independent <italic>t</italic>-tests highlighted differences between these two groups. In the first cluster, children displayed a lower developmental [<italic>t</italic>(1) = 4.16; <italic>p</italic> = 0.000; <italic>d</italic> = 0.94] and verbal [<italic>t</italic>(1) = 3.78; <italic>p</italic> = 0.000; <italic>d</italic> = 0.85] age, compared to children in the second cluster. Regarding ToM abilities, children in the second cluster presented more abilities in ToM than those in the first cluster. They displayed a better understanding of affective mental states [<italic>t</italic>(1) = 2.9; <italic>p</italic> = 0.005; <italic>d</italic> = 0.65], such as emotions [<italic>t</italic>(1) = 2.37; <italic>p</italic> = 0.021; <italic>d</italic> = 0.54] and of cognitive mental states [in cognitive ToM task Battery, <italic>t</italic>(1) = 2.49; <italic>p</italic> = 0.033; <italic>d</italic> = 0.58 and in ToM beliefs, <italic>t</italic>(1) = 2.18; <italic>p</italic> = 0.033; <italic>d</italic> = 0.49]. Similarly, compared with children in the first cluster, children in the second cluster identified [<italic>t</italic>(1) = 2.21; <italic>p</italic> = 0.030; <italic>d</italic> = 0.52] and justified [<italic>t</italic>(1) = 2.92; <italic>p</italic> = 0.005; <italic>d</italic> = 0.66] social behaviors in negative situations more easily. In terms of social adjustment, children with a lower developmental age and a lower level of social-cognitive skills also displayed fewer social competence [<italic>t</italic>(1) = -2.67; <italic>p</italic> = 0.009; <italic>d</italic> = 0.62] and adjustment [<italic>t</italic>(1) = -2.06; <italic>p</italic> = 0.043; <italic>d</italic> = 0.46]. These children were perceived as more anxious, especially in social groups, and as more isolated among peers. Children with a higher developmental age seemed to be more autonomous and cooperative with adults, in comparison with the younger cluster. Parents of these children reported more behavioral disorders, and more specifically a higher level of internalizing problems [<italic>t</italic>(1) = -2.62; <italic>p</italic> = 0.011; <italic>d</italic> = 0.61]. The CBCL indicated a mean corresponding to clinical level (>19) for this first cluster.</p><table-wrap id=\"T6\" position=\"float\"><label>TABLE 6</label><caption><p>Between-group comparisons of the two clusters obtained through hierarchical cluster analysis according to Theory of Mind and social information processing abilities.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Cluster 1 (<italic>n</italic> = 36)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Cluster 2 (<italic>n</italic> = 42)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" colspan=\"2\" rowspan=\"1\">Variables</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mean (<italic>SD</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mean (<italic>SD</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>X</italic><sup>2</sup><italic><sup>/</sup>t</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>d</italic></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sex (% boys)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">64%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.87</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CA (in months)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">105.97 (22.61)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">113.19 (19.57)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.49</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GDA (in months)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">57.66 (13.54)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">69.37 (11.34)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.16****</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.94</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VDA (in months)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">57.01 (13.63)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">67.9 (11.77)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−3.78****</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.85</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Family income</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.39 (0.78)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.85 (1.27)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.71</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Explicit ToM measures</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM Task Battery total (max = 15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.19 (2.3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.95 (2.13)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−3.48***</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.79</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Affective ToM Task Battery (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.66 (1.21)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.36 (0.91)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.9***</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.65</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cognitive ToM Task Battery (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.13 (1.26)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.93 (1.5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.49*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.58</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mixed ToM Task Battery (max = 3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.47 (0.76)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.76 (0.99)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.35</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM emotions (max = 12)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.96 (2.46)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.19 (2.07)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.37*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.54</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM emotions – causes (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.9 (1.61)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.21 (1.32)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.94</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM emotions – consequences (max = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.69 (1.69)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.65 (1.75)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.46*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.56</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM beliefs (max = 5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.56 (1.39)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.2 (1.19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.18*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.49</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Problem-solving task</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Judgment score on appropriate vignettes (max = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.69 (0.48)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.71 (0.51)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.17</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Judgment score on inappropriate vignettes (max = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.76 (0.28)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.86 (0.18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.88<sup>†</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.42</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Identification score on appropriate vignettes (max = 1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.77 (0.27)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.79 (0.29)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.21</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Identification score on inappropriate vignettes (max = 1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.72 (0.22)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.82 (0.16)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.21*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.52</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Justification score on appropriate vignettes (max = 7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.69 (1.44)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.28 (1.28)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.88<sup>†</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.43</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Justification score on inappropriate vignettes (max = 7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.64 (1.12)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.39 (1.14)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.92***</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.66</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Social (mal)adjustment</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EASE total (max = 98)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53.61 (17.33)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">61.33 (15.8)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.06*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.46</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EASE ToM (max = 52)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25.67 (9.01)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.67 (8.81)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.98<sup>†</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.45</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EASE Social Skills (max = 46)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.94 (8.86)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.67 (8.18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.93<sup>†</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.44</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Externalizing problems</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">65.94 (16.91)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">71.02 (18.8)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.22</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Internalizing problems</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">66 (15.98)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">75.14 (14.02)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.62*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.61</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Social competence</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.32 (26.79)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">115.94 (26.48)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.67**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.62</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – General adjustment</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">231.25 (48.99)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">262.11 (49.73)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.68**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.62</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Depressive-happy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.17 (8.25)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">36.48 (8.10)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.73</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Anxious-secure</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.18 (8.30)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.71 (9.25)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.22*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.51</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Isolated-integrated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.93 (8.81)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">36.37 (7.43)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.84*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.67</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Dependent-autonomous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.70 (9.92)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.93 (6.97)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.09*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.49</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Angry-tolerant</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25.16 (8.17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.92 (10.21)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.76</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Aggressive-controlled</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.25 (7.3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.67 (7.48)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.83</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Egoistic-prosocial</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25.71 (7.06)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.16 (9.94)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–1.23</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCBE – Resistant-cooperative</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.15 (9.28)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35.86 (8.51)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.74*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CBCL Externalizing Behaviors</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18.14 (10.62)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13.91 (9.09)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.65</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CBCL Internalizing Behaviors</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19.04 (9.35)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13.79 (8.48)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.18*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.59</td></tr></tbody></table><table-wrap-foot><attrib><italic><italic>CA, Chronological Age; GDA, Global Developmental Age; ToM, Theory of Mind; EASE, Social Adjustment Scale for Children; SCBE, Social Competence and Behavior Evaluation; CBCL, Child Behavior Checklist; <sup>†</sup>p ≤ 0.062; *p < 0.05; **p < 0.01; ***p ≤ 0.001; ****p = 0.000.</italic></italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS4\"><title>Exploratory Factor Analysis</title><p>We applied an exploratory factor analysis in principal axis factoring with oblimin rotation on the subscores for ToM and SIP (see <xref rid=\"T7\" ref-type=\"table\">Table 7</xref> for the loadings of each task on the factor and the percentage of explained variance). The first factor included affective ToM and social problem-solving skills related to inappropriate social behaviors. The second integrated cognitive ToM and social problem-solving skills related to appropriate social behaviors. The third one encompassed ToM competence linked to understanding of mixed mental states.</p><table-wrap id=\"T7\" position=\"float\"><label>TABLE 7</label><caption><p>Exploratory factor analysis in principal axis factoring with oblimin rotation in Theory of Mind and social information processing.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Factor</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Loadings on factor</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>SIPin-ToMAffCo</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Judgment score of inappropriate vignettes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.768</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Identification score of inappropriate vignettes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.740</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Justification score of inappropriate vignettes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.354</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Affective ToM Task Battery</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.694</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM-emotions</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.618</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ToM-beliefs</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.690</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Percentage of explained variance</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>46.95%</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Cumulative percentage</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>46.95%</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>SIPa-ToMCo</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Judgment score of appropriate vignettes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.930</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Identification score of appropriate vignettes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.879</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Justification score of appropriate vignettes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.703</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cognitive ToM Task Battery</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.332</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Percentage of explained variance</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>9.05%</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Cumulative percentage</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>56%</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>ToM-mixed</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mixed ToM Task Battery</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.715</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Percentage of explained variance</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>4.86%</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Cumulative percentage</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>60.86%</bold></td></tr></tbody></table><table-wrap-foot><attrib><italic><italic>IP, social information processing; ToM, Theory of Mind.</italic> The bold values corresponded to the percentage of explained variance and the cumulative percentage for each factor.</italic></attrib></table-wrap-foot></table-wrap></sec></sec><sec id=\"S4\"><title>Discussion</title><p>The present study aimed to explore the social cognitive profiles of children with non-specific IDs. To do so, we investigated whether different clusters could be distinguished within one sample according to ToM and/or SIP competence and how these profiles of abilities were related to one another in children with IDs. Results indicated that children with IDs could be distinguished by their social cognitive profiles. Children who displayed better social cognitive abilities had higher chronological and/or global and verbal developmental ages, as well as better social, emotional, and behavioral competence and adjustment. The exploratory factor analysis revealed that ToM abilities and SIP competence are both used during positive or negative social interactions.</p><p>When hierarchical cluster analyses were used with respect to ToM abilities, SIP competence, or both, two groups were always identified, and some differences between them were revealed by comparing means, using independent <italic>t</italic>-tests. The two clusters obtained based on ToM abilities differed by chronological age as well as global and verbal developmental age. Children in the first cluster were younger and displayed lower ToM abilities, particularly in the understanding of emotions, consequences, and cognitive mental states. These results are in line with the literature, which has identified a positive and predictive relationship between developmental age and ToM abilities, notably in typically developing children (<xref rid=\"B36\" ref-type=\"bibr\">Grazzani et al., 2018</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Conte et al., 2019</xref>) and in children with IDs (e.g., <xref rid=\"B18\" ref-type=\"bibr\">Charman and Campbell, 2002</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Baurain and Nader-Grosbois, 2013</xref>; <xref rid=\"B63\" ref-type=\"bibr\">Nader-Grosbois et al., 2013</xref>). Children have to display a certain level of cognitive skills to understand mental states (<xref rid=\"B19\" ref-type=\"bibr\">Cicchetti et al., 1995</xref>). The difference in chronological age highlights the potential impact of social life experiences that become more diversified over time. As they get older, children experience more social interactions with different people, and this gives them opportunities to develop ToM abilities (<xref rid=\"B87\" ref-type=\"bibr\">Vygotsky, 1978</xref>). This conclusion also explains why the older children in this study score more highly on the ToM-beliefs measure, as they had first-hand experience of deception and of perspective taking. Compared with older children, the first cluster presented lower competence in social adjustment, particularly when they had to use and respect social conventions and rules. They also seemed to be slightly less cooperative with adults. Clustering based on SIP competence indicated two groups that differed by verbal developmental age. Studies have shown developmental delay in social interaction abilities (<xref rid=\"B38\" ref-type=\"bibr\">Guralnick, 2006</xref>) and deficits in receptive and expressive language in children with IDs (<xref rid=\"B73\" ref-type=\"bibr\">Sigafoos, 2000</xref>). These delays and deficits limit discussion about critical social situations or the use of language in order to guide the SIP process. Children who displayed a higher verbal developmental age found it easier to identify a social behavior as inappropriate and to justify it by considering the relationship between the protagonists or by referring to social rules. In typically developing children, the relationship between abilities to construct and decide to enact positive social behaviors and expressive (<xref rid=\"B97\" ref-type=\"bibr\">Ziv, 2013</xref>) as well as receptive (<xref rid=\"B21\" ref-type=\"bibr\">Conte et al., 2018</xref>) language has already been revealed. Children with higher SIP skills seemed to be more socially competent and, in particular, less aggressive and more cooperative. These two dimensions are related to the quality of social interactions with peers and with adults respectively. Based on these results, we could speculate that children with higher language and SIP skills could be perceived as more socially competent and therefore interact with others in a more appropriate way, leading to a virtuous circle. In the two clusters obtained on the basis of ToM and SIP skills, children differed from one another in verbal and global developmental age. The older group displayed better competence in affective and cognitive ToM as well as in SIP specifically related to negative situations. With respect to understanding emotions, the difference between the two groups was only in the comprehension of the consequences of emotions. Children in the first cluster displayed lower competence in social cognition and also presented a lower developmental age and lower social adjustment skills. Concretely, these children had a developmental age of 4 years and 9 months, in comparison with the children in the other group, who presented a developmental age of 5 years and 9 months. This observation is in line with previous studies reporting a predictive link between developmental age and ToM (<xref rid=\"B18\" ref-type=\"bibr\">Charman and Campbell, 2002</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Abbeduto and Murphy, 2004</xref>; <xref rid=\"B81\" ref-type=\"bibr\">Thirion-Marissiaux and Nader-Grosbois, 2008c</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Alevriadou and Giaouri, 2011</xref>; <xref rid=\"B63\" ref-type=\"bibr\">Nader-Grosbois et al., 2013</xref>) and a relationship between developmental age and social problem-solving abilities (<xref rid=\"B15\" ref-type=\"bibr\">Baurain and Nader-Grosbois, 2013</xref>; <xref rid=\"B63\" ref-type=\"bibr\">Nader-Grosbois et al., 2013</xref>) in children with IDs. It is also consistent with the empirical observation that typically developing children acquire competence in social cognition as their verbal and non-verbal cognitive capacities increase (<xref rid=\"B91\" ref-type=\"bibr\">Wellman and Liu, 2004</xref>; <xref rid=\"B72\" ref-type=\"bibr\">Schultz et al., 2010</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Grazzani et al., 2018</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Conte et al., 2019</xref>). It highlights the importance of considering children’s developmental age during assessment or intervention rather than their chronological age in order to take account of the proximal zone of development. Compared with others, children with this lower level of competence in social cognition presented more social maladjustment. They were perceived as less socially adjusted in social situations requiring an understanding of mental states or social rules. Children in this first cluster also seemed to be less socially competent in various situations, displaying less positive, appropriate, flexible, and prosocial behaviors. Specifically, these children were perceived as more anxious. In their interactions with peers, they seemed more isolated, while when interacting with adults, they tended to be less autonomous and cooperative. Social maladjustment has been generally associated with a deficit in social cognition, notably in ToM (<xref rid=\"B17\" ref-type=\"bibr\">Charman and Campbell, 1996</xref>; <xref rid=\"B50\" ref-type=\"bibr\">Jervis and Baker, 2004</xref>; <xref rid=\"B63\" ref-type=\"bibr\">Nader-Grosbois et al., 2013</xref>) and SIP (<xref rid=\"B14\" ref-type=\"bibr\">Baurain and Nader-Grosbois, 2012</xref>). Children in the first cluster also tended to present more internalizing problems, even at a clinical level: a number of studies have observed the presence of behavioral problems in children with IDs (<xref rid=\"B26\" ref-type=\"bibr\">Dekker et al., 2002</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Baker et al., 2003</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Dekker and Koot, 2003</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Emerson, 2003</xref>; <xref rid=\"B63\" ref-type=\"bibr\">Nader-Grosbois et al., 2013</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Bailey et al., 2019</xref>), notably internalizing problems (<xref rid=\"B61\" ref-type=\"bibr\">Merrell and Holland, 1997</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Guralnick, 1999</xref>). Some studies have also reported a link between internalizing problems and a deficit in social cognition (<xref rid=\"B84\" ref-type=\"bibr\">van Nieuwenhuijzen et al., 2004</xref>; <xref rid=\"B81\" ref-type=\"bibr\">Thirion-Marissiaux and Nader-Grosbois, 2008c</xref>).</p><p>Hierarchical cluster analyses led to classifying cases into groups that differ from each other but also that include individuals who present common specific characteristics in each group (<xref rid=\"B96\" ref-type=\"bibr\">Yim and Ramdeen, 2015</xref>). With respect to differences, average distances between clusters indicated that when cluster analyses of ToM abilities were run, the two groups were more distant than the two clusters obtained according to SIP skills. These findings indicated that children with IDs differ more according to their ToM abilities than to their SIP skills. While differences between clusters were highlighted above, some similarity could also be underlined. In the three cluster analyses, no difference between the two clusters was obtained for gender, family income, or the presence of externalizing problems. It revealed a homogeneity in these variables in the present sample. When children are clustered in two groups depending on their ToM abilities, differences appeared in social adjustment but not in their socio-affective and behavioral profiles, whereas when children are clustered in two groups depending on their SIP skills, no difference was observed in social adjustment. The goal of the present analyses is not to reveal whether ToM and SIP abilities were related to social competence and adjustment, especially since significant correlations are obtained in preliminary analysis (see <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>) between these variables. Instead, cluster analysis aimed to investigate how diverse observations could be grouped according to different characteristics or variables, as in the present study, social cognitive abilities.</p><p>While ToM and SIP are two distinct and specific concepts, they could have an influence on each other. The present results demonstrated a relationship between the ability to understand mental states and social problem-solving skills in children with IDs. In their model, <xref rid=\"B23\" ref-type=\"bibr\">Crick and Dodge (1994)</xref> underline the particular function of some mental states (namely intentions, emotions and thoughts) in selecting and enacting prosocial behavior. <xref rid=\"B59\" ref-type=\"bibr\">Mazza et al. (2017)</xref> even consider ToM competence as prerequisites for SIP. In their view, children first have to understand their own and other people’s mental states before processing social information in order to behave in a socially appropriate way. Similarly, <xref rid=\"B57\" ref-type=\"bibr\">Lemerise and Arsenio (2000)</xref>, <xref rid=\"B58\" ref-type=\"bibr\">Lemerise et al. (2005)</xref> emphasize the role of emotions in SIP. In our results, affective ToM is related to problem solving during negative situations while cognitive ToM is linked to SIP when an individual faces appropriate social behavior. It seems that when children face hostile intentions, provocation, frustration, or rejection, they make more use of skills related to affective ToM, i.e., the understanding of emotions, whereas in helping or sharing situations they tend to use cognitive ToM. In typically developing children, <xref rid=\"B21\" ref-type=\"bibr\">Conte et al. (2018)</xref> highlight relations on one hand between emotion knowledge and prosocial behavior of helping, and on the other hand, between cognitive ToM and sharing. Given the present result, future research could investigate similar links in children with IDs. Concerning the first factor of the exploratory factor analysis (SIPin-ToMAffCo), it includes items that differentiate children with IDs particularly with different levels of social cognitive skills. In fact, in the present study, affective and cognitive ToM as well as SIP competence in negative situations are competence that clusters children into two different groups. It seemed that children have to consider other people’s perspective or intentions to display prosocial behavior. To go further, it would be interesting to study the effect of ToM on SIP. <xref rid=\"B59\" ref-type=\"bibr\">Mazza et al. (2017)</xref> investigate the role of ToM components on SIP leading to prosocial behaviors in a sample of children with autistic spectrum disorder. In their study, <xref rid=\"B59\" ref-type=\"bibr\">Mazza et al. (2017)</xref> reveal that in children with autistic spectrum disorder, ToM competence does not reduce social problem-solving difficulties, whereas the understanding of emotions and beliefs does help typically developing children to interpret social cues during SIP.</p><p>The present findings revealed an underlying structure between ToM and SIP skills in children with IDs. Results demonstrated that ToM and SIP profiles in children with IDs could be distinguished. Thanks to cluster analyses, differences and similarities were observed. It stresses the importance of considering social cognitive variables separately and together, as well as the weaknesses and the strengths when exploring a child with IDs profile. Moreover, professionals have to pay attention to the relation between ToM abilities and SIP competence in positive or negative social situations during assessment and intervention processes toward children with IDs.</p><sec id=\"S4.SS1\"><title>Future Perspectives</title><p>Some limitations should be taken into account when considering the present results. Yet, they provide insights into future research opportunities. The sample included children with non-specific IDs. Similar studies need to be conducted with children with distinct genetic syndromes (e.g., Down syndrome, Williams’s syndrome) and with a control group of typically developing children. These designs would lead researchers to explore whether the same factors and groupings apply to other samples. This research fit a clinical special education approach. Therefore, we focused on the underlying strengths and weaknesses and chose measures based on developmental age rather than the intelligence quotients of children with IDs. Future research could replicate a study with similar objectives and hierarchical cluster analyses adding children’s intelligence quotients. As for instruments, ToM-emotions tasks presented a low reliability score for the present sample. Even if they provided more information than facial emotion recognition, notably about the understanding of causes and consequences of emotions, related results have to be considered carefully. Nevertheless, the affective subscore of the ToM Task Battery could be used to gather information on the understanding of emotions and desires. Future research could create and validate an assessment device with animated virtual support featuring characters who undergo negative and positive social situations and express various emotions (as used in the Emotion trainer program, conceived by <xref rid=\"B75\" ref-type=\"bibr\">Silver and Oakes, 2001</xref>). This type of device should be validated with children with IDs and typically developing children, in order to evaluate emotions recognition and understanding of causes and consequences of emotions, in a more dynamic way. To collect information on ToM abilities in diverse contexts, parents and/or teachers could fill in a questionnaire such as the Theory of Mind Inventory (<xref rid=\"B43\" ref-type=\"bibr\">Hutchins et al., 2012</xref>), on their perception of children’s ToM abilities. Variability in social cognition profiles could be investigated considering abilities in receptive and expressive language, in executive functions and empathy (<xref rid=\"B40\" ref-type=\"bibr\">Hippolyte et al., 2010</xref>). For instance, several authors have demonstrated that low levels of prosocial behaviors are associated with low empathy-related abilities in preschoolers (<xref rid=\"B77\" ref-type=\"bibr\">Strayer and Roberts, 2004</xref>; <xref rid=\"B93\" ref-type=\"bibr\">Williams et al., 2014</xref>), and that social problem solving is stronger in more empathetic children and adolescents (<xref rid=\"B20\" ref-type=\"bibr\">Coie and Dodge, 1998</xref>; <xref rid=\"B24\" ref-type=\"bibr\">de Wied et al., 2007</xref>). It would therefore be interesting to explore the empathy profile of these children. Given the specific emotion-related socialization behaviors of parents of children with IDs (<xref rid=\"B69\" ref-type=\"bibr\">Rodas et al., 2016</xref>; <xref rid=\"B46\" ref-type=\"bibr\">Jacobs et al., 2019b</xref>; <xref rid=\"B56\" ref-type=\"bibr\">Légaré et al., 2019</xref>), it would be interesting to consider family environment in this kind of study: parental socialization of emotions (e.g., reactions and conversations) has been recently found to affect social adjustment (<xref rid=\"B46\" ref-type=\"bibr\">Jacobs et al., 2019b</xref>), emotion regulation, and ToM abilities (<xref rid=\"B45\" ref-type=\"bibr\">Jacobs et al., 2019a</xref>). To investigate more precisely the causal contribution of ToM and SIP to each other and to the social and emotional competence of children with IDs, longitudinal studies or experimental studies implementing ToM or SIP training need to be conducted. ToM and SIP training studies including a control group and pre- and post-test measures of social cognition, emotion regulation and social adjustment have already highlighted some particular effects (<xref rid=\"B47\" ref-type=\"bibr\">Jacobs and Nader-Grosbois, 2020a</xref>, <xref rid=\"B48\" ref-type=\"bibr\">b</xref>).</p></sec><sec id=\"S4.SS2\"><title>Psychoeducational Implications</title><p>As far as interventions are concerned, the results underscore the importance of considering the developmental age as well as the chronological age of children with IDs. Clinicians need to adapt materials and goals to ToM and SIP profiles (i.e., strengths and weaknesses) and to life experience. Since children display difficulties with both affective and cognitive mental states, it is crucial to assess all nine mental states and to support the understanding of all of them. Similarly, children with IDs tend to present higher difficulties during negative social situations, so SIP intervention should focus on both appropriate and inappropriate social behaviors. The relation between developmental verbal age and better SIP skills also highlights the importance of encouraging children to use self-verbalization. This could be fostered by experimenters in training by using repetitive and successive key questions adapted to SIP. Given the link between ToM and SIP, it could be hypothesized that interventions that aim to promote ToM abilities could impact SIP abilities and vice versa. Finally, both ToM and SIP interventions seem crucial to help children with IDs to be more socially adjusted and to have appropriate social interactions, in order to ultimately assist their integration and social inclusion.</p></sec></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>The datasets presented in this article are not readily available because consent filled by participants ensured that the data will be used only for this research and not shared. Requests to access the datasets should be directed to <email>nathalie.nader@uclouvain.be</email>.</p></sec><sec id=\"S6\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by Ethics committee of the Psychological Sciences Research Institute. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>EJ collected and analyzed the data and wrote the manuscript. PS helped to collect and analyzed the data. NN-G supervised all the study and helped to write the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>We would like to thank the Marguerite-Marie Delacroix Foundation and the Chair Baron Frère in special education for their financial support. We are also thankful to the SMCS for its help in statistical analysis. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Pharmacol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Pharmacol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Pharmacol.</journal-id><journal-title-group><journal-title>Frontiers in Pharmacology</journal-title></journal-title-group><issn pub-type=\"epub\">1663-9812</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32848784</article-id><article-id pub-id-type=\"pmc\">PMC7431698</article-id><article-id pub-id-type=\"doi\">10.3389/fphar.2020.01181</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Pharmacology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Vitexin Possesses Anticonvulsant and Anxiolytic-Like Effects in Murine Animal Models</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>de Oliveira</surname><given-names>Denise Dias</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/1050873\"/></contrib><contrib contrib-type=\"author\"><name><surname>da Silva</surname><given-names>Cassio Prinholato</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/902332\"/></contrib><contrib contrib-type=\"author\"><name><surname>Iglesias</surname><given-names>Bruno Benincasa</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Beleboni</surname><given-names>Renê O.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/659969\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Biotechnology, University of Ribeirão Preto</institution>, <addr-line>Ribeirão Preto</addr-line>, <country>Brazil</country></aff><aff id=\"aff2\"><sup>2</sup><institution>School of Medicine, University of Ribeirão Preto</institution>, <addr-line>Ribeirão Preto</addr-line>, <country>Brazil</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: María Pilar López-Gresa, Universitat Politècnica de València, Spain</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Alexandra Olimpio Siqueira Cunha, University of São Paulo, Brazil; Donatus Wewura Adongo, University of Health and Allied Sciences, Ghana</p></fn><corresp id=\"fn001\">*Correspondence: Renê O. Beleboni, <email xlink:href=\"mailto:rbeleboni@unaerp.br\" xlink:type=\"simple\">rbeleboni@unaerp.br</email>; <email xlink:href=\"mailto:reneusp@yahoo.com\" xlink:type=\"simple\">reneusp@yahoo.com</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p>This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1181</elocation-id><history><date date-type=\"received\"><day>04</day><month>2</month><year>2020</year></date><date date-type=\"accepted\"><day>20</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 de Oliveira, da Silva, Iglesias and Beleboni</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>de Oliveira, da Silva, Iglesias and Beleboni</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Different types of epilepsy and forms of pathological anxiety have been described as significant neurological disorders that may exist as comorbidities. Some of those disorders share the association of affected limbic areas/neuropathological triggers as well as the use of drugs for their clinical management. The aim of this work was to investigate the anticonvulsant and anxiolytic properties of the vitexin (apigenin-8-C-glucoside), since this compound is a flavonoid usually found as one of the major constituents in several medicinal plants claimed as anxiolytics and/or anticonvulsants. This investigation was performed by the use of a series of classical murine animal models of chemically induced-seizures and of anxiety-related tests (open-field, elevated plus-maze, and light-dark box tests). Here, we show that the systemic administration of vitexin (1.25; 2.5 and 5 mg/kg; i.p.) exhibited selective protection against chemically-induced seizures. Vitexin did not block seizures evoked by glutamate receptors agonists (NMDA and kainic acid), and it did not interfere with the latencies for these seizures. Conversely, the same treatments protected the animals in a dose-dependent manner against the seizures evoked by the Gabaergic antagonists picrotoxin and PTZ and rise the latency time for the first seizure on non-protected animals. The higher dose of vitexin protected 100% of animals against the tonic-clonic seizures triggered by GABA antagonists. The results from open-field, elevated plus-maze, and light-dark box tests indicated the anxiolytic properties of vitexin at similar range of doses described for the anticonvulsant action screening. Furthermore, these results pointed that vitexin did not cause sedation or locomotor impairment on animals. The selective action of vitexin against picrotoxin and PTZ may reinforce the hypothesis by which this compound acts mainly by the modulation of GABAergic neurotransmission and/or related pathways. This could be useful to explain the dual activity of vitexin as anticonvulsant and anxiolytic, and highlight the pharmacological interest on this promising flavonoid.</p></abstract><kwd-group><kwd>epilepsy</kwd><kwd>anxiety</kwd><kwd>etnopharmacology</kwd><kwd>flavonoid</kwd><kwd>γ - aminobutyric acid (GABA)</kwd><kwd>glutamate</kwd></kwd-group><counts><fig-count count=\"4\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"41\"/><page-count count=\"9\"/><word-count count=\"4027\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Epilepsy is a progressive chronic disorder characterized by an abnormal and synchronous neuronal firing associated with recurrent and unpredictable seizures (<xref rid=\"B12\" ref-type=\"bibr\">England et al., 2012</xref>). It affects more than 50 million people worldwide. The current antiepileptic drug arsenal fails to evoke a positive response in about 30% of diagnosed cases of epilepsy (<xref rid=\"B37\" ref-type=\"bibr\">World Health Organization, 2019a</xref>). Patients suffering from different types of epilepsy are often affected by psychiatric and behavioral comorbidities such as mood disorders, anxiety, psychoses, motor disorders, cognitive deficits, and social dysfunctions (<xref rid=\"B35\" ref-type=\"bibr\">Salpekar and Mula, 2018</xref>). Besides the broad epidemiological relevance of anxiety disorders at different segments of our modern society, anxiety disorders are usually observed in a large number of epileptic patients, which represents additional negative impacts in their already compromised quality of lives (<xref rid=\"B15\" ref-type=\"bibr\">Gandy et al., 2015</xref>). It is well known that the alleviation of anxiety symptoms may have a positive effect in the progress of treatment of epileptic patients contributing to ameliorate their general health condition (<xref rid=\"B13\" ref-type=\"bibr\">Fisher and Noble, 2017</xref>).</p><p>The pathophysiological mechanism related to the onset and maintenance of different types of epilepsy/seizures, includes the imbalance between excitatory (glutamate) and inhibitory (γ-aminobutyric acid (GABA) synaptic activities that are located at different brain areas (<xref rid=\"B6\" ref-type=\"bibr\">Bialer and White, 2010</xref>). Some brain networks are commonly involved at prevention or appearance of seizures as well as in the regulation of behavior and mood (<xref rid=\"B23\" ref-type=\"bibr\">Kwon and Park, 2014</xref>). Although the huge differences in the molecular and cellular bases involved in onset and development of epilepsy and anxiety, the same imbalance between GABA and Glutamate is also related to anxiety (<xref rid=\"B26\" ref-type=\"bibr\">Masneuf et al., 2014</xref>; <xref rid=\"B16\" ref-type=\"bibr\">Gauthier and Nuss, 2015</xref>). Of course, several intervenient factors can differentially contribute to the neuropathology underlying both disorders, particularly in terms of magnitude, recruited structures, and brain areas (<xref rid=\"B25\" ref-type=\"bibr\">Martin et al., 2009</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Wu et al., 2018</xref>). In any case, the screening for new antiepileptic/anxiolytic drugs that are able to modulate the inhibitory and/or excitatory neurotransmission pathways may be worth for the treatment of epilepsy and/or anxiety (<xref rid=\"B28\" ref-type=\"bibr\">Moto et al., 2018</xref>).</p><p>Vitexin (apigenin-8-C-glucoside) has received great attention by presenting a wide range of pharmacological effects. Vitexin has been found, in some cases as the major constituent, at different medicinal plants potentially useful to treat anxiety and/or epilepsy (<xref rid=\"B29\" ref-type=\"bibr\">Nassiri-Asl et al., 2007</xref>; <xref rid=\"B21\" ref-type=\"bibr\">He et al., 2016</xref>). More specifically, recent studies have shown the protective effects of vitexin against some neurological and psychiatric diseases (<xref rid=\"B2\" ref-type=\"bibr\">Abbasi et al., 2013</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Can et al., 2013</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Guimarães et al., 2015</xref>). In spite of the interesting pharmacological properties of vitexin; only a few and preliminary efforts have been conducted to prove its potential anxiolytic and anticonvulsant activities. Therefore, the main aim of this work is to investigate these potential effects using a complementary set of experiments at a well-established animal model arrangement.</p></sec><sec sec-type=\"materials|methods\" id=\"s2\"><title>Materials and Methods</title><sec id=\"s2_1\"><title>Vitexin and Other Drugs</title><p>Vitexin (apigenin-8-C-glucoside) was purchased from Cayman Chemical Company, USA (CAS Number 3681-93-4, purity degree ≥ 98%). The proconvulsant drugs N-Methyl-D-aspartate (NMDA) (CAS Number: 6384-92-5; ≥ 98%); kainic acid (CAS Number: 58002-62-3; ≥ 98%); Pentylenetetrazol (PTZ) (CAS Number: 54-95-5) and picrotoxin (CAS Number 204-716-6) were purchased from Sigma Aldrich, St. Louis, MO. Diazepam (DZP) (injectable solution in 0.9% saline) and dimethyl sulfoxyde (DMSO) were acquired from União Química (Brazil) and Synth (Brazil), respectively.</p></sec><sec id=\"s2_2\"><title>Animals and Experimental Conditions</title><p>All experiments were carried out using male adult Wistar rats within 4 to 5 weeks of age weighing about 150 to 180 g. Rats were housed five per cage in a controlled condition of humidity and temperature under 12 h light/dark schedule at 6 am/6 pm. They were allowed to acclimatize to our host facilities for at least 3 days prior to any experimental manipulation. Chow and water were provided <italic>ad libitum</italic>. The procedures were in accordance with the University of Ribeirão Preto Ethic Committee (approval number 11/2015) and with the Guide for the Care and Use of Laboratory Animals (National Research Council, US).</p><p>The animals were divided in control and experimental groups (<italic>n</italic>=06) in all sets of experiments. All the experiments were carried out in an acoustic isolated room between 1:00 to 5:00 p.m. All apparatus or arenas used in experiments were cleaned with 70% ethanol after observation of each animal in all of the procedures. Rats were habituated to the testing room for 30-min before experimental procedures. All experiments were recorded by a digital video camera and copies of files kept in our laboratory as official documents for public consultation.</p></sec></sec><sec id=\"s3\"><title>Assessment of Anxiolytic-Like Effects of Vitexin</title><p>The anxiolytic activity of vitexin was evaluated by elevated plus-maze, light-dark box, and open-field tests. The behavior of animals on each experimental apparatus for anxiolytic effects was observed 30 min later after control or experimental treatments. For all experiments, Diazepam 2 mg/kg (dissolved in 0.9% saline) (i.p.) served as positive control while 1% DMSO in 0.9% saline (i.p.) and 0.9% saline solution (i.p.) served as controls. Experimental groups were composed of animals treated with different i.p. doses of vitexin (0.75, 1.25 and 2.5 mg/kg). Doses of vitexin were selected based on pilot scale tests while the dose of DZP were based on previous scientific reports (<xref rid=\"B22\" ref-type=\"bibr\">Kazdoba et al., 2015</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Zemdegs et al., 2018</xref>)</p><sec id=\"s3_1\"><title>Elevated Plus-Maze Test</title><p>The elevated plus maze (EPM) was conducted in accordance with the method validated by <xref rid=\"B32\" ref-type=\"bibr\">Pellow et al. (1985)</xref>.The maze comprised of two wood open arms (50 × 10 cm) surrounded by a short (0.5 cm) acrylic edge to avoid falls and two wood enclosed arms (50 × 10 × 40 cm) arranged such that the two open arms were opposite to each other. The arms were connected by a central platform (10 × 10 cm) and the maze was kept 50 cm above the floor. Each rat was placed in the middle compartment (head facing an open arm) and allowed to freely explore the apparatus for 5 min. It was measured (i) the time spent(s) in the open and closed arms of the EPM, (ii) the percentage of time spent in open arms, (iii) protected and unprotected stretch attend postures, and (iv) protected and unprotected head dipping events. Stretch-attend was defined when the animal stretches forward and retracts without moving its feet. Head dipping was defined as the exploratory movement in which the rodent head protruding over the edge toward the floor on the open arms. Behaviors were defined unprotected when they were exhibited in the open arm region of the EPM and were protected if it occurs in closed arms or central platform of the maze (<xref rid=\"B36\" ref-type=\"bibr\">Walf and Frye, 2007</xref>).</p></sec><sec id=\"s3_2\"><title>Open Field Test</title><p>The open field test was performed according to the method previously described (<xref rid=\"B14\" ref-type=\"bibr\">Funchal and Dani, 2014</xref>).The open-field used was a circular arena measuring 60 cm in diameter with 50-cm high circular acrylic wall (OP0199, Insight). The apparatus floor was divided into 12 squares (4 squares corresponding a central zone and 8 squares to the peripheral zones).Every animal was placed in the central region of the open field, and the number of lines crossed (all four limbs), rearing, grooming, and the times spent(s) in the central zone of the apparatus were recorded for 5 minutes. Also, for an indirect measurement of locomotor performance of animals, the number of crossings was checked in time blocks along the experiment comparing control and experimental groups, since the animals usually explore more of the apparatus in the first blocks of time and less at the end of the task, as they habituate.</p></sec><sec id=\"s3_3\"><title>Light-Dark Box Test</title><p>The light-dark box consist of a <italic>box</italic> (46 x 27 x 30 cm) divided into a dark and illuminated compartment (EP 158, Insight, fluorescent lamp- 20 W). The chambers were connected by a small opening (7.5 × 7.5 cm) in the middle of the wall separating the two chambers. Rats were placed in the middle of the light chamber and then released to explore for 5 min (<xref rid=\"B20\" ref-type=\"bibr\">Hascoet and Bourin, 1998</xref>).The time that rats spent in the light/dark compartments and the number of transitions between them were recorded. Transition was defined as the placement of all four paws in the entry chamber.</p></sec></sec><sec id=\"s4\"><title>Assessment of Anticonvulsant Activity of Vitexin</title><p>The anticonvulsant effect of vitexin was evaluated against chemically-induced seizures by <italic>intraperitoneal (i.p.) injection</italic> of proconvulsant agents: NMDA, 150 mg/kg; kainic acid, 30 mg/kg; picrotoxin, 6 mg/kg; and PTZ 90, mg/kg. NMDA, kainic acid, picrotoxin, and PTZ were dissolved in 0.9% saline while vitexin dissolved in 0.9% saline containing dimethyl sulfoxyde – DMSO 1%. The animals control and experimental groups were divided according to the assessment of anxiolytic-like effects of vitexin, except by the range of vitexin i.p. doses, which were slightly up changed for anticonvulsant screening (1.25, 2.5, and 5 mg/kg). Doses of vitexin were selected based on pilot scale tests while the dose of DZP were selected based on previous scientific reports, as considered above for anxiolytic-related tests. Thirty minutes after the administration of each proconvulsant drug, each animal was maintained individually in a transparent <italic>acrylic arena</italic> (60 cm × 40 cm). The rats were observed for 30, 40, 40, and 120 min respectively for kainic acid, NMDA, picrotoxin, and PTZ assays. It was evaluated the latency time to the first generalized tonic-clonic seizure and the incidence/number of animals exhibiting generalized tonic-clonic seizures (<xref rid=\"B14\" ref-type=\"bibr\">Funchal and Dani, 2014</xref>). Animal behavior seizure rating was classified/certified according to the Racine’s scale, modified by Pinel and Rovner (<xref rid=\"B33\" ref-type=\"bibr\">Pinel and Rovner, 1978</xref>). Generalized tonic-clonic seizures were considered as those rating score 7 or more in accordance to this scale and analyses of our video-recorded files.</p><sec id=\"s4_1\"><title>Statistical Analysis</title><p>Except by the Open-Field test which was performed one and two-way ANOVA test, other experiments statistical analyses were performed using One-way ANOVA, followed by the Tukey post-hoc test (GraphPad Prism; version 7.0, Graphpad Software, USA). Data were expressed as the means ± standard error of mean (SEM) (<italic>n</italic>=06/group). A level of p< 0.05 was accepted as statistically significant.</p></sec></sec><sec sec-type=\"results\" id=\"s5\"><title>Results</title><sec id=\"s5_1\"><title>Anxiolytic-Like Effects of Vitexin</title><sec id=\"s5_1_1\"><title>Elevated Plus-Maze</title><p>The amount of time (sec) spent in the open and closed arms and percentage of time spent in open arms in the elevated plus maze are shown in <xref ref-type=\"fig\" rid=\"f1\"><bold>Figure 1</bold></xref>. The <italic>post hoc</italic> test showed that the time spent on the open arms was longer (<italic>p</italic> < 0.05) in the diazepam treated group (positive control). Also, the data shows that all doses of vitexin increased significantly the time spent in the open arms compared to the saline control (F<sub>(5,60)</sub> = 103.9; p<0.05). Pretreatment with vitexin produced a significant increase in unprotected head dipping (F<sub>(5,30)</sub> = 74.25; p<0.05) and unprotected stretch-attend postures (F<sub>(5,30)</sub> = 27.99; p<0.05).</p><fig id=\"f1\" position=\"float\"><label>Figure 1</label><caption><p>Elevated Plus Maze. <bold>(A)</bold> Total time spent on the open and closed arms, <bold>(B)</bold> percentage of time spent in opens arms, <bold>(C)</bold> protected and unprotected stretch attend postures and <bold>(D)</bold> protected and unprotected head dipping between the groups treated with different doses of vitexin, diazepam (DZP), and vehicle (VEH), compared with control group (Ctrl - Saline Control). Significantly different from saline control group: <sup>∗</sup><italic>p</italic> < 0.05. One-way ANOVA; Tukey <italic>post hoc</italic> test.</p></caption><graphic xlink:href=\"fphar-11-01181-g001\"/></fig></sec><sec id=\"s5_1_2\"><title>Open Field Test</title><p>The number of line crossings during open field test showed that vitexin increased locomotion on apparatus (F<sub>(5,26)</sub> = 45.33; p<0.05) (<xref ref-type=\"fig\" rid=\"f2\"><bold>Figure 2A</bold></xref>). Vitexin significantly improved locomotion mainly in the first 3 minutes of experimentation when compared with the control group (F <sub>(20, 140)</sub> = 26,23 p<0.05) (<xref ref-type=\"fig\" rid=\"f2\"><bold>Figure 2B</bold></xref>). In the open field test, animals treated with vitexin (all doses) spent more time on the center of arena when compared with those from the control group (F<sub>(5,30)</sub> = 76.82; p<0.05) (<xref ref-type=\"fig\" rid=\"f2\"><bold>Figure 2C</bold></xref>). Vertical activity behaviors (rearing and grooming) are shown in <xref ref-type=\"fig\" rid=\"f2\"><bold>Figure 2D</bold></xref>. The analysis of total number of the rearing showed significantly higher for vitexin treatments than that in the control group (saline) (F<sub>(5,27)</sub> = 67.49; p<0.05). The <italic>post hoc</italic> test revealed that vitexin 0.75 and 1.25 mg/kg reduced grooming behaviors (compared with the control group) in a way similar to the diazepam (F<sub>(5,27)</sub> = 40.02; p<0.05) (<xref ref-type=\"fig\" rid=\"f2\"><bold>Figure 2D</bold></xref>).</p><fig id=\"f2\" position=\"float\"><label>Figure 2</label><caption><p>Open Field Test. <bold>(A)</bold> number of crossings made in the open field, <bold>(B)</bold> Crossings in function of the time, <bold>(C)</bold> time spent in the center and <bold>(D)</bold> rearing and grooming behavior of the open field by different doses of vitexin, diazepam (DZP), and vehicle (VEH), compared with control group (Ctrl—saline control). Significantly different from saline control: <sup>∗</sup><italic>p</italic> < 0.05. For data in <bold>(A, C</bold> and <bold>D)</bold> was performed one-way ANOVA and in <bold>(B)</bold> was performed two-way ANOVA. For all data were performed Tukey <italic>post hoc</italic> test.</p></caption><graphic xlink:href=\"fphar-11-01181-g002\"/></fig></sec><sec id=\"s5_1_3\"><title>Light-Dark Test</title><p>Vitexin at the dose of 0.75, 1.25, and 2.5 mg/kg and diazepam (2 mg/kg) induced a significant increase in the time spent by rats on the illuminated side of the apparatus compared with the saline control group (F<sub>(5;50)</sub> = 26.87; p<0.05) (<xref ref-type=\"fig\" rid=\"f3\"><bold>Figure 3A</bold></xref>). Analysis of the light/dark transitions revealed significant greater number of transitions for animals treated with vitexin than in the control group (F<sub>(5,28)</sub> = 21.33; p<0.05) (<xref ref-type=\"fig\" rid=\"f3\"><bold>Figure 3B</bold></xref>).</p><fig id=\"f3\" position=\"float\"><label>Figure 3</label><caption><p>Light dark box test. <bold>(A)</bold> times spent(s) on light/dark area and <bold>(B)</bold> light/dark transitions by different doses of vitexin, diazepam (DZP) and vehicle (VEH) compared with control group (Ctrl—saline control). Significantly different from saline control: <sup>∗</sup><italic>p</italic> < 0.05. One-way ANOVA; Tukey <italic>post hoc</italic> test.</p></caption><graphic xlink:href=\"fphar-11-01181-g003\"/></fig></sec></sec><sec id=\"s5_2\"><title>Anticonvulsant Screening</title><p>Acute administration of vitexin prior to kainic acid and NMDA injections did not protect animals against seizures. However, clonic seizures induced by i.p. administrations of PTZ and picrotoxin were inhibited by all doses of vitexin (<xref ref-type=\"fig\" rid=\"f4\"><bold>Figures 4A, B</bold></xref>). Vitexin significantly protected animals against seizures and showed 16.66, 50 and 100% of protection against picrotoxin-induced seizures (<xref ref-type=\"fig\" rid=\"f4\"><bold>Figure 4C</bold></xref>) and 16.66%, 33.33%, and 100% against PTZ-induced seizures (<xref ref-type=\"fig\" rid=\"f4\"><bold>Figure 4D</bold></xref>), respectively, on the doses of 1.25; 2.5, and 5 mg/kg. Therefore, the anticonvulsant activity of vitexin exhibited a dose-dependent profile on PTZ and picrotoxin assays. In addition, the latency to to the first seizure induced by PTZ and picrotoxin was significantly higher for groups treated with vitexin, particularly on the dose of 2.5 mg/kg (<xref rid=\"T1\" ref-type=\"table\"><bold>Table 1</bold></xref>).</p><fig id=\"f4\" position=\"float\"><label>Figure 4</label><caption><p>Percentages of animals protected against seizures induced by <bold>(A)</bold> kainic acid 150 mg/kg, <bold>(B)</bold> NMDA 150 mg/kg, <bold>(C)</bold> picrotoxin 6 mg/kg and <bold>(D)</bold> PTZ 90 mg/kg in function of different doses of vitexin, diazepam (DZP), and vehicle (VEH) compared with control group (Ctrl-Saline Control). Significantly different from control: *<italic>p</italic> < 0.05 versus saline control group. One-way ANOVA; Tukey <italic>post hoc</italic> test.</p></caption><graphic xlink:href=\"fphar-11-01181-g004\"/></fig><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Latency to onset of seizure (score 7-8) on vitexin treatments.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" rowspan=\"2\" align=\"left\" colspan=\"1\">Convulsant Agent</th><th valign=\"top\" colspan=\"6\" align=\"center\" rowspan=\"1\">Latency<italic> </italic>to<italic> </italic>onset of seizure (seconds)</th></tr><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ctrl</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">VEH</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">DZP (2 mg/kg)</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Vitexin (1.25 mg/kg)</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Vitexin (2.5 mg/kg)</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Vitexin (5 mg/kg)</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NMDA (150 mg/kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4409.50 ± 92.48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4326.30 ± 43.49</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4403.83 ± 98.63</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4514.50 ± 63.59</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4548.50 ± 70.52</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Kainic acid (30 mg/kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4491.33 ± 77.88</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4546.33 ± 77.52</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4693.0 ± 74.59</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4404.83 ± 116.83</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4468.72 ± 124.72</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PTZ<break/>(90 mg/kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">77.4 ± 14.63</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">78.00 ± 7.38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53.66 ± 9.07<sup>#</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">141.2 ± 29.08*<sup>#</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*<sup>#</sup><break/>NS</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Picrotoxin (6 mg/kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1101.0 ± 47.29</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1103.66 ± 47.99</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">745.8 ± 65.35*<sup>#</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1396.6 ± 43.14*<sup>#</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*<sup>#</sup><break/>NS</td></tr></tbody></table><table-wrap-foot><p>∗p < 0.05 versus control group (Ctrl – Saline Control) and <sup>#</sup>p < 0.05 between different doses of vitexin. One-way ANOVA; Tukey post hoc test. NS, no seizure.</p></table-wrap-foot></table-wrap></sec></sec><sec sec-type=\"discussion\" id=\"s6\"><title>Discussion</title><p>In the present study we demonstrated that vitexin presents both anxiolytic and anticonvulsant properties which are possibly mediated by modulation of GABAergic neurotransmission and/or related synaptic pathways. Since those pharmacological activities were achieved after systemic administration (i.p.) of vitexin, it is plausible to affirm that this compound overcomes the blood-brain barrier, making it more attractive in terms of pharmacokinetic or even pharmaceutical perspectives. Vitexin has been found at different medicinal plants with anxiolytic and/or anti-epileptic properties (<xref rid=\"B29\" ref-type=\"bibr\">Nassiri-Asl et al., 2007</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Miroddi et al., 2013</xref>). Based on this and also on our results, it is also possible to suggest that vitexin is at least partially responsible for those pharmacological activities, in most of cases and more probabilistically, acting in a synergistic way with other(s) active compound(s) present in those plants claimed as anxiolytics and/or anti-epileptics.</p><p>In the present study, anxiolytic-like effect of vitexin was confirmed by three different and complementary animal screening tests. This combined arrangement of tests allows a more reliable and complete evaluation of anxiety-related behaviors in rodents (<xref rid=\"B34\" ref-type=\"bibr\">Ramos et al., 2008</xref>). Overall results from different tests confirmed the increase of exploratory performance and the decrease of aversive behaviors of animals, indicating an anxiolytic-like effect promoted by the different doses of vitexin. Anxiolytics agents reduce protected risk assessment behaviors and increase the frequency of unprotected ethological behaviors (<xref rid=\"B3\" ref-type=\"bibr\">Benneh et al., 2018</xref>). Similar to diazepam, pretreatment with vitexin significantly decreased protected head dips and stretch attend postures and increase unprotected behavior suggesting the anxiolytic property of vitexin. In addition, it is possible to reinforce that vitexin did not caused any type of locomotor impairment or even did not produced signals of sedation on animals at same time that the anticonvulsant activity produced by vitexin is not caused by interference with locomotor capacity of the treated animals. Those conclusions are supported by the increased number of crossings at open field test and the increased number of light/dark transitions exhibited by vitexin treated animals in the light/dark box test when compared with the saline control. Locomotor impairment or severe sedation are constantly claimed as important side effects caused by different pharmacological agents used in the treatment of anxiety and epilepsy and should be avoided in the clinical management of those medical conditions (<xref rid=\"B8\" ref-type=\"bibr\">Canevini et al., 2010</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Guina and Merrill, 2018</xref>). Therefore, besides the useful dual pharmacological effect (anxiolytic and anticonvulsant) vitexin presents some advantages in terms of its toxicological profile. The dual effect of vitexin at a very similar range of doses is particularly important for a potential combined and future management of both epilepsy and anxiety, which commonly appears as comorbidities and request the use of several combined drugs. However, this dual pharmacological effect is not surprising or exclusive of vitexin, since epilepsy and anxiety are partially overlapped in terms of neurochemical and neurobiological pathways and some drugs depending on the dose (like benzodiazepines) may act against both disorders despite important observed side effects.</p><p>The effect of acute intraperitoneal injection of the vitexin for the prospect of its anticonvulsant activity was evaluated using different acute seizure models in which the seizures were chemically induced. Given the possibility of false negatives results on drug screening, potential antiepileptic compounds should not be screened solely against one single model but to be tested across a few different seizure models, exploring at same time different neurotransmission pathways involved on initiation and spraying of induced seizures (<xref rid=\"B40\" ref-type=\"bibr\">Yuen and Troconiz, 2015</xref>). These type of animal models are very useful for the rapid and economical screening of new anticonvulsant drugs (<xref rid=\"B24\" ref-type=\"bibr\">Löscher, 2011</xref>). Kainic acid and NMDA have significant convulsant effects, fundamentally by activating glutamate receptors; glutamate is the most prominent excitatory neurotransmitter in the mammalian CNS (<xref rid=\"B5\" ref-type=\"bibr\">Bhandage et al., 2017</xref>). Moreover, in the current study, we explored the therapeutic potential of vitexin using the GABA<sub>A</sub> receptor antagonists PTZ and picrotoxin (<xref rid=\"B9\" ref-type=\"bibr\">Colas et al., 2013</xref>). On the basis of the results presented here, acute treatment with different doses of vitexin applied against the kainic acid and NMDA seizures protocol was not effective when compared with the saline control. In contrast, vitexin exhibited a very clear dose-dependent anticonvulsant effect against seizures induced by PTZ and picrotoxin, reaching the maximum protection (100%) at higher dose of 5 mg/kg. In addition, the lower doses (1.25 and 2.5 mg/kg) delayed the onset of seizures for non-completely protected animals against seizures. The combination of these actions indicated an effective anticonvulsant effect of vitexin at least against PTZ and picrotoxin-induced seizures.</p><p>The underlying molecular mechanisms involved in the anticonvulsant or anxiolytic actions of vitexin were not the central scope of the present study. However, the selective protection offered by vitexin against GABA receptors antagonists at the expense of Glutamate receptor agonists are at least intriguing and may suggest that the modulation of GABA neurotransmission or related pathways may be involved on mode of action of vitexin. It is noteworthy to point out that different natural flavonoids and various synthetic derivatives from natural flavonoids are well known to be positive modulators of GABA<sub>A</sub> receptors (<xref rid=\"B19\" ref-type=\"bibr\">Hanrahan et al., 2011</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Oliveira et al., 2018</xref>). Indeed, many studies agree that vitexin may exert its CNS effects <italic>via</italic> GABA<sub>A</sub> receptor (benzodiazepine site) modulation (<xref rid=\"B1\" ref-type=\"bibr\">Abbasi et al., 2012</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Oliveira et al., 2014</xref>; <xref rid=\"B38\" ref-type=\"bibr\">World Health Organization, 2019b</xref>). However, further neurochemical investigations are required for a more complete understanding by the way vitexin exerts its pharmacological activities. The GABA neurotransmission is a common link between epilepsy and anxiety. In fact, the down regulation of GABA activity is frequently evoked on the neurobiological explanation for both disorders. Also, the up modulation of GABAergic neurotransmission is assumed on the explanation about the mode of action of some drugs such as benzodiazepines, which are well known as a GABA<sub>A</sub> receptors positive allosteric modulators and commonly used for the clinical management for both disorders (<xref rid=\"B4\" ref-type=\"bibr\">Beyenburg et al., 2005</xref>).</p><p>Finally, epilepsy and anxiety frequently appear as comorbidities. Indeed, one of the main psychiatric conditions observed for epileptic patients is anxiety. Anxiety affects approximately 20% of the people diagnosed with epilepsy and is responsible for a decreased quality of life and can worsen epilepsy control (<xref rid=\"B11\" ref-type=\"bibr\">Dworetzky, 2017</xref>; <xref rid=\"B38\" ref-type=\"bibr\">World Health Organization, 2019b</xref>). The actual pharmacological arsenal used in the clinical treatment for both disorders often causes important side effects, such as cognitive/locomotor impairments and sedation, which negatively affects therapeutic adherence (<xref rid=\"B10\" ref-type=\"bibr\">Diniz et al., 2019</xref>). Thus, the prospecting/developing of new pharmacological probes is relevant especially when those new potential drugs exhibit potential advantages in terms of pharmacokinetics, toxicological, and pharmacodynamic features.</p></sec><sec id=\"s7\"><title>Conclusion</title><p>The results of this study showed that vitexin possess both anxiolytic and anticonvulsant properties. Since vitexin has been found in different medicinal plants popularly claimed as anxiolytics and/or anticonvulsants, this work has both ethnopharmacological and pharmaceutical relevance. This work may be useful for the development of new pharmacological probes not only for anxiety and epilepsy patients relieve in the future but also, and indirectly, for the better understanding of pathological events underlying both anxiety and epilepsy themselves.</p></sec><sec sec-type=\"data-availability\" id=\"s8\"><title>Data Availability Statement</title><p>The datasets generated for this study are available on request to the corresponding author.</p></sec><sec id=\"s9\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by University of Ribeirão Preto Ethic Committee (11/2015).</p></sec><sec id=\"s10\"><title>Author Contributions</title><p>Conceived and Designed: DO and RB. Data Collection: DO, CS and BI. Acquisition, Analysis and Interpretation of Data: DO and CS. Article Writing by: DO and RB. All authors contributed to the article and approved the submitted version.</p></sec><sec sec-type=\"funding-information\" id=\"s11\"><title>Funding</title><p>This work was supported by the Coordination for the Improvement of Higher Education Personnel (CAPES) and the Brazilian National Council for Scientific and Technological Development (CNPq).</p></sec><sec id=\"s12\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><title>Acknowledgments</title><p>The authors appreciate the financial support from (CAPES and CNPq) and equally thanking the Biotechnology Department of University of Ribeirão Preto (UNAERP) for the infrastructure, technical support and facilities to carry out this work.</p></ack><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\">\n<person-group person-group-type=\"author\"><name><surname>Abbasi</surname><given-names>E.</given-names></name><name><surname>Nassiri-Asl</surname><given-names>M.</given-names></name><name><surname>Shafeei</surname><given-names>M.</given-names></name><name><surname>Sheikhi</surname><given-names>M.</given-names></name></person-group> (<year>2012</year>). <article-title>Neuroprotective Effects of Vitexin, a Flavonoid, on Pentylenetetrazole-Induced Seizure in Rats</article-title>. <source>Chem. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Cell Dev. Biol.</journal-id><journal-title-group><journal-title>Frontiers in Cell and Developmental Biology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-634X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850870</article-id><article-id pub-id-type=\"pmc\">PMC7431699</article-id><article-id pub-id-type=\"doi\">10.3389/fcell.2020.00779</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cell and Developmental Biology</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>The RUFYs, a Family of Effector Proteins Involved in Intracellular Trafficking and Cytoskeleton Dynamics</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Char</surname><given-names>Rémy</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1002413/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Pierre</surname><given-names>Philippe</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/31239/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Aix Marseille Université, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Centre d’Immunologie de Marseille-Luminy</institution>, <addr-line>Marseille</addr-line>, <country>France</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Institute for Research in Biomedicine and Ilidio Pinho Foundation, Department of Medical Sciences, University of Aveiro</institution>, <addr-line>Aveiro</addr-line>, <country>Portugal</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Shanghai Institute of Immunology, School of Medicine, Shanghai Jiao Tong University</institution>, <addr-line>Shanghai</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Roberto Botelho, Ryerson University, Canada</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Daniel G. S. Capelluto, Virginia Tech, United States; Brian Paul Ceresa, University of Louisville, United States</p></fn><corresp id=\"c001\">*Correspondence: Rémy Char, <email>char@ciml.univ-mrs.fr</email></corresp><corresp id=\"c002\">Philippe Pierre, <email>pierre@ciml.univ-mrs.fr</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Membrane Traffic, a section of the journal Frontiers in Cell and Developmental Biology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>779</elocation-id><history><date date-type=\"received\"><day>11</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>24</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Char and Pierre.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Char and Pierre</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Intracellular trafficking is essential for cell structure and function. In order to perform key tasks such as phagocytosis, secretion or migration, cells must coordinate their intracellular trafficking, and cytoskeleton dynamics. This relies on certain classes of proteins endowed with specialized and conserved domains that bridge membranes with effector proteins. Of particular interest are proteins capable of interacting with membrane subdomains enriched in specific phosphatidylinositol lipids, tightly regulated by various kinases and phosphatases. Here, we focus on the poorly studied RUFY family of adaptor proteins, characterized by a RUN domain, which interacts with small GTP-binding proteins, and a FYVE domain, involved in the recognition of phosphatidylinositol 3-phosphate. We report recent findings on this protein family that regulates endosomal trafficking, cell migration and upon dysfunction, can lead to severe pathology at the organismal level.</p></abstract><kwd-group><kwd>RUFY</kwd><kwd>cancer</kwd><kwd>neurodegenerative diseases</kwd><kwd>immunity</kwd><kwd>RUN</kwd><kwd>FYVE</kwd><kwd>phosphatidylinositol 3-phosphate</kwd><kwd>cytoskeleton</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Fondation pour la Recherche Médicale<named-content content-type=\"fundref-id\">10.13039/501100002915</named-content></funding-source><award-id rid=\"cn001\">DEQ20180339212</award-id></award-group><award-group><funding-source id=\"cn002\">Agence Nationale de la Recherche<named-content content-type=\"fundref-id\">10.13039/501100001665</named-content></funding-source><award-id rid=\"cn002\">DCBIOL Labex ANR-11-LABEX-0043</award-id></award-group><award-group><funding-source id=\"cn003\">Fundação para a Ciência e a Tecnologia<named-content content-type=\"fundref-id\">10.13039/501100001871</named-content></funding-source><award-id rid=\"cn003\">POCI-01-0145-FEDER-016768</award-id></award-group></funding-group><counts><fig-count count=\"4\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"167\"/><page-count count=\"17\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>The organization of cells into multiple membranous compartments with specific biochemical functions requires complex intracellular traffic and sorting of lipids and proteins, to transport them from their sites of synthesis to their functional destination. Intracellular transport involves lipid vesicles or tubules with the capacity to fuse with one another or to be secreted. They collectively participate in the dynamic exchanges necessary for cell homeostasis (<xref rid=\"B114\" ref-type=\"bibr\">Rothman, 2002</xref>; <xref rid=\"B123\" ref-type=\"bibr\">Søreng et al., 2018</xref>). Membrane traffic is tightly coordinated with protein synthesis, signal transduction of environmental stimuli and cytoskeleton organization, allowing the implementation of key cellular functions such as endocytosis, exocytosis, or migration (<xref rid=\"B86\" ref-type=\"bibr\">McMahon and Gallop, 2005</xref>; <xref rid=\"B48\" ref-type=\"bibr\">Habtezion et al., 2016</xref>; <xref rid=\"B142\" ref-type=\"bibr\">Vega-Cabrera and Pardo-López, 2017</xref>; <xref rid=\"B80\" ref-type=\"bibr\">MacGillavry and Hoogenraad, 2018</xref>; <xref rid=\"B82\" ref-type=\"bibr\">Margaria et al., 2019</xref>; <xref rid=\"B136\" ref-type=\"bibr\">Tapia et al., 2019</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Buratta et al., 2020</xref>; <xref rid=\"B125\" ref-type=\"bibr\">Stalder and Gershlick, 2020</xref>).</p><p>Several families of molecular components required for orchestrating membrane vesicle exchange and transport during this process are conserved. They include adaptor and coat proteins, small GTP-binding proteins (GTPases), as well as Synaptosome Associated Protein (SNAP) Receptor (SNARE) proteins and SNARE binding proteins (<xref rid=\"B60\" ref-type=\"bibr\">Juliano, 2018</xref>). The vast superfamily of GTPases is involved in the establishment or regulation of virtually every step of intracellular membrane trafficking. They behave as molecular switches that can alternate between active and inactive states, through GTP binding and hydrolysis into GDP (<xref rid=\"B133\" ref-type=\"bibr\">Takai et al., 2001</xref>; <xref rid=\"B127\" ref-type=\"bibr\">Stenmark, 2009</xref>). The largest group of GTPases involved in intracellular membrane traffic is the Rab proteins family (<xref rid=\"B71\" ref-type=\"bibr\">Lamb et al., 2016</xref>). Rab GTPases specifically localize to different intracellular compartments, regulating vesicle formation and sorting, as well as transport along the cytoskeletal network. Each Rab protein can be recruited to specific membrane subdomains of a defined organelle and is associated to multiple effectors controlling membrane fusion and trafficking. Rab interaction with the membrane fusion complexes and cytoskeleton regulators is therefore crucial for cellular functions, including endocytosis and autophagy (<xref rid=\"B19\" ref-type=\"bibr\">Chen and Wandinger-Ness, 2001</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Bruce et al., 2010</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Geng et al., 2010</xref>; <xref rid=\"B139\" ref-type=\"bibr\">Thomas and Fromme, 2020</xref>; <xref rid=\"B159\" ref-type=\"bibr\">Yuan and Song, 2020</xref>).</p><p>Here, we review the literature concerning a less-well known family of proteins involved in the complex biochemical crosstalk established between the cytoskeleton and intracellular vesicles. This small group of proteins was named RUFY for “<underline>RU</underline>N and <underline>FY</underline>VE domain-containing.” RUFYs share a common structural domain organization, including an N-terminal RUN domain, one or several coiled-coil (CC) repeats and a C-terminal FYVE domain (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). The molecular structures of the different RUFY proteins has been described (<xref rid=\"B31\" ref-type=\"bibr\">Dunkelberg and Gutierrez-Hartmann, 2001</xref>; <xref rid=\"B83\" ref-type=\"bibr\">Mari et al., 2001</xref>; <xref rid=\"B66\" ref-type=\"bibr\">Kukimoto-Niino et al., 2006</xref>; <xref rid=\"B64\" ref-type=\"bibr\">Kitagishi and Matsuda, 2013</xref>), but their function in endocytic regulation and their physiological relevance at the organismal level are still poorly characterized (<xref rid=\"B64\" ref-type=\"bibr\">Kitagishi and Matsuda, 2013</xref>; <xref rid=\"B137\" ref-type=\"bibr\">Terawaki et al., 2016</xref>). We revisit here how the <italic>rufy</italic> gene family was annotated, and propose the addition of a novel member, the <italic>fyco1</italic> (FYVE and Coiled-coil containing domain 1) gene given its sequence and functional similarities with the other <italic>rufy</italic> genes (<xref rid=\"B102\" ref-type=\"bibr\">Pankiv et al., 2010</xref>; <xref rid=\"B138\" ref-type=\"bibr\">Terawaki et al., 2015</xref>). We also highlight recent findings on the implication of RUFY proteins in the regulation of cytoskeleton and endosome dynamics and their contribution to immunity, cancer and neurodegenerative diseases.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>RUN and FYVE domain containing-proteins in the endo-lysosomal pathway. <bold>(A)</bold> Schematic representation of the RUFY proteins family. <bold>(B)</bold> Description of the endo-lysosomal and autophagy pathways and presumed functional locations of RUFY proteins. Extracellular material is ingested by endocytosis or phagocytosis. The action of different endosomes allows cargo to be sorted, recycled or degraded in a complex and regulated process involving fusion, maturation and transport along the cytoskeleton. Alternatively, during autophagy, obsolete components present in cytosol are captured in autophagosomes prior fusion with lysosomes and degradation (macroautophagy) or directly internalized through endosomal invagination (microautophagy). SE, sorting endosome; EE, early endosome; TGN, trans golgi network; LE, late endosome; MVBs, multi vesicular bodies; RE, recycling endosome, MT, microtubule; CT, centrioles; ER, endoplasmic reticulum. The location of PI3P and RUFY proteins known activity is shown. Created with <ext-link ext-link-type=\"uri\" xlink:href=\"https://biorender.com/\">BIoRender.com</ext-link>.</p></caption><graphic xlink:href=\"fcell-08-00779-g001\"/></fig><sec id=\"S1.SS1\"><title>Endocytosis and Autophagy</title><p>Endocytosis and autophagy are membrane traffic pathways required for degradation and recycling of extracellular and intracellular components, respectively (<xref rid=\"B5\" ref-type=\"bibr\">Birgisdottir and Johansen, 2020</xref>). These pathways have a common endpoint at the lysosome, where their cargo is degraded. These both pathways intersect at several stages throughout vesicle formation, transport and fusion and share some of the components of their molecular machineries (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>).</p><p>There are numerous co-existing endocytic pathways, which initiate by the formation of nascent endocytic vesicles formed from plasma membrane invaginations and scissions. These endocytic vesicles undergo homotypic fusion and are rapidly targeted to sorting endosomes (SE). Sorting events initiated in SE determine the fate of internalized cargo molecules, such as recycling to plasma membrane, degradation in lysosomes, or other trafficking events (<xref rid=\"B98\" ref-type=\"bibr\">Naslavsky and Caplan, 2018</xref>; <xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). On their way to degradation, sorted cargo accumulate in early endosomes (EE), that further mature into late endosomes (LE) through multiple events of cargo and lipid sorting. Late endosomes adopt a membrane organization termed multivesicular bodies, that are enriched in lysobisphosphatidic acid and contain intraluminal vesicles (<xref rid=\"B45\" ref-type=\"bibr\">Gruenberg, 2020</xref>). Next, LE potentiate their hydrolytic competence by fusing with lysosomes (<xref rid=\"B107\" ref-type=\"bibr\">Pillay et al., 2002</xref>) resulting in the degradation of their contents, providing nutrients and key factors to the cell (<xref rid=\"B28\" ref-type=\"bibr\">Doherty and McMahon, 2009</xref>; <xref rid=\"B61\" ref-type=\"bibr\">Kaksonen and Roux, 2018</xref>). Notably, endosomes play a role in signal transduction by serving as signaling platforms either for surface activated receptors like Toll-like receptors and epidermal growth factor receptor or metabolic sensors such as mechanistic target of rapamycin complex 1 (mTORC1; <xref rid=\"B2\" ref-type=\"bibr\">Argüello et al., 2016</xref>). Often they promote the degradation of their targets, leading to signal termination (<xref rid=\"B21\" ref-type=\"bibr\">Chung et al., 2010</xref>). The endocytic pathway has also specialized functions in differentiated cells such as neurotransmitter release and recycling in neurons, or antigen processing and presentation in professional antigen presenting cells, like B cells or dendritic cells (<xref rid=\"B2\" ref-type=\"bibr\">Argüello et al., 2016</xref>; <xref rid=\"B122\" ref-type=\"bibr\">Solé-Domènech et al., 2016</xref>; <xref rid=\"B54\" ref-type=\"bibr\">Hinze and Boucrot, 2018</xref>). Endocytosis events and endosomes positioning is highly dependent on the dynamic and spatial re-organization of the different cytoskeleton networks that include actin, intermediate filaments, or microtubules (<xref rid=\"B34\" ref-type=\"bibr\">Fletcher and Mullins, 2010</xref>; <xref rid=\"B105\" ref-type=\"bibr\">Pegoraro et al., 2017</xref>).</p><p>Complementary to endocytosis, autophagy is an intracellular process by which cells degrade and recycle their own cytoplasmic materials (<xref rid=\"B91\" ref-type=\"bibr\">Mizushima and Komatsu, 2011</xref>). Autophagy plays a central role in many physiological processes including stress management, development, immunity and aging (<xref rid=\"B110\" ref-type=\"bibr\">Puleston and Simon, 2014</xref>; <xref rid=\"B165\" ref-type=\"bibr\">Zhong et al., 2016</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Fîlfan et al., 2017</xref>; <xref rid=\"B93\" ref-type=\"bibr\">Moretti et al., 2017</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Doherty and Baehrecke, 2018</xref>). Autophagy is partially controlled though mTORC1 activity and is responsible for degradation and recycling of misfolded proteins, as well as obsolete organelles (<xref rid=\"B39\" ref-type=\"bibr\">Galluzzi et al., 2017</xref>). The endpoint of autophagy is to deliver cytoplasmic material to lysosomes, where like for endocytosed cargo, it is degraded. Several autophagy processes can be distinguished based on the entry mode of the cytosolic components destined for degradation (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). Macroautophagy involves engulfment of cytoplasmic contents into a double membrane vesicle termed the autophagosome. The autophagosome fuses then with lysosomes, becoming an autolysosome, in which its cargo is degraded (<xref rid=\"B39\" ref-type=\"bibr\">Galluzzi et al., 2017</xref>). The presence of specific phosphoinositides lipids, together with Rab GTPases, at a given membrane compartment is often directly correlated with compartment function. One of the common mechanism regulating endocytosis and autophagy is an accumulation of phosphatidylinositol 3-phosphate (PtdIns(3)P) at surface of EE and on intraluminal vesicles of multivesicular endosomes and on autophagosomes (<xref rid=\"B97\" ref-type=\"bibr\">Nascimbeni et al., 2017</xref>; <xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). PtdIns(3)P is also observed at sites of LC3−associated phagocytosis another pathway of internalization used by the cells to ingest large particulate material or microbes. PtdIns(3)P is therefore a beacon used by the cellular machinery to regulate endosomal sorting and autophagy (<xref rid=\"B5\" ref-type=\"bibr\">Birgisdottir and Johansen, 2020</xref>).</p></sec><sec id=\"S1.SS2\"><title>RUN Domains</title><p>The presence of a single copy of a RUN and a FYVE domain at their extremities is the key characteristic defining the RUFY family members. RUN domains were named after three proteins bearing similar peptide motifs, <underline>R</underline>PIP8, <underline>UN</underline>C-14 and <underline>N</underline>ESCA (new molecule containing SH3 at the carboxy−terminus) (<xref rid=\"B100\" ref-type=\"bibr\">Ogura et al., 1997</xref>; <xref rid=\"B84\" ref-type=\"bibr\">Matsuda et al., 2000</xref>). RUN domains are present in multiple proteins (RUN proteins) in a large panel of organisms (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>) and principally allow direct interactions with small GTPases of the Rap and Rab families (<xref rid=\"B14\" ref-type=\"bibr\">Callebaut et al., 2001</xref>; <xref rid=\"B157\" ref-type=\"bibr\">Yoshida et al., 2011</xref>). RUN domains adopt a hydrophobic globular structure bearing six conserved blocks named A to F (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>). These blocks correspond to eight α-helices and some 3<sub>10</sub>-helices. The first helix is crucial to limit hydrophobic exposure and maintain protein solubility of RUN-containing proteins (<xref rid=\"B14\" ref-type=\"bibr\">Callebaut et al., 2001</xref>; <xref rid=\"B66\" ref-type=\"bibr\">Kukimoto-Niino et al., 2006</xref>). In spite of strong conservation among the domains present in RUN-containing proteins, the proteins they interact with, their effectors, are highly variable (<xref rid=\"B83\" ref-type=\"bibr\">Mari et al., 2001</xref>) and the structural features of the RUN domain alone are not sufficient to define binding specificity for one or several members of the GTPase superfamily (<xref rid=\"B38\" ref-type=\"bibr\">Fukuda et al., 2011</xref>). Most RUN domain-bearing proteins bind small GTPases, but interactions with other molecules like kinesin 1 have also been described (<xref rid=\"B8\" ref-type=\"bibr\">Boucrot et al., 2005</xref>). A direct physical link between RUN proteins with actin filaments and microtubules has been also demonstrated (<xref rid=\"B140\" ref-type=\"bibr\">Torti et al., 1999</xref>), reinforcing the idea that these molecules are also critical for cellular functions requiring actin remodeling, such as migration or phagocytosis (<xref rid=\"B109\" ref-type=\"bibr\">Price and Bos, 2004</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Bos, 2005</xref>; <xref rid=\"B89\" ref-type=\"bibr\">Miertzschke et al., 2007</xref>; <xref rid=\"B153\" ref-type=\"bibr\">Xu et al., 2007</xref>; <xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>). Additional functions for RUN domains have been described, for example for the RUN domain present in NESCA, which blocks TRAF6-mediated polyubiquitination of the NF-kappa-B essential modulator and consequently induces NF-kB activation. This is just one of the ways in which RUN proteins can act in signal transduction and the coordination of membrane traffic with actin dynamics upon external stimulation (<xref rid=\"B157\" ref-type=\"bibr\">Yoshida et al., 2011</xref>). As well as promoting endosomal fusion through their binding to Rab or Rap GTPases (<xref rid=\"B14\" ref-type=\"bibr\">Callebaut et al., 2001</xref>; <xref rid=\"B157\" ref-type=\"bibr\">Yoshida et al., 2011</xref>), their interaction with motor proteins, like kinesin or myosin, suggests a role for RUN domains in regulating vesicular and organelle transport (<xref rid=\"B14\" ref-type=\"bibr\">Callebaut et al., 2001</xref>; <xref rid=\"B157\" ref-type=\"bibr\">Yoshida et al., 2011</xref>). Via these different mechanisms, RUN proteins have been implicated in neuronal development (<xref rid=\"B56\" ref-type=\"bibr\">Honda et al., 2017b</xref>), signaling (<xref rid=\"B131\" ref-type=\"bibr\">Sun et al., 2012</xref>), migration (<xref rid=\"B157\" ref-type=\"bibr\">Yoshida et al., 2011</xref>), and regulation of various cellular function like endocytosis or exocytosis (<xref rid=\"B64\" ref-type=\"bibr\">Kitagishi and Matsuda, 2013</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Evolution of RUN and FYVE domain or <italic>rufy</italic> genes among living organisms. Diagram illustrating the evolution of the <italic>rufy</italic> genes. Species representative of various taxonomic groups are listed, data were extracted from the Differential Expression Atlas Genes database (EMBL-EBI). Next to each species studied, the number corresponds to the number of genes having in its sequence a FYVE (green), RUN (blue) or both (red) domain. The “X” corresponds to the appearance of a common <italic>rufy</italic> ancestor gene.</p></caption><graphic xlink:href=\"fcell-08-00779-g002\"/></fig><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Molecular organization of RUN and FYVE domains from the RUFY proteins family. Alignment of the protein sequences of the RUN <bold>(A)</bold> and FYVE <bold>(B)</bold> domains of the RUFY proteins family in human and mouse. <bold>(A)</bold> RUN consensus blocks are represented by segments <bold>(A–F)</bold>. Rpip8 sequence is used as RUN domain reference, <bold>(B)</bold> FYVE conserved motives and zinc fingers are represented by segments. In the alignment, “x” is any amino acid and “+” represents positively charged amino acid. Eea1 sequence is used as FYVE domain reference. For all alignment, amino acids are colored according to their properties: Cyan for hydrophobic positions (A,V,I,L,M), turquoise for aromatic positions (F,Y,W,H), red for basic residues (K,R), purple for acidic residues (D,E), green for polar uncharged (N,Q,S,T), salmon for cysteine (C), orange for glycine (G) and yellow for proline (P). Gray numbers below alignment means the amino acids position after alignment. Black numbers surrounding the alignments represent the start (left) and end (right) positions of the domains in the peptide sequence of each protein. Alignment were realized with Seaviewer analyzer software (<xref rid=\"B44\" ref-type=\"bibr\">Gouy et al., 2010</xref>). Accession numbers for protein are following: human Rpip8 (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_001138297.1\">NP_001138297.1</ext-link>), mouse Rpip8 (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_058039.1\">NP_058039.1</ext-link>), human Eea1 (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_003557.3\">NP_003557.3</ext-link>), mouse Eea1 (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_001001932.1\">NP_001001932.1</ext-link>), human RUFY1 (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_079434.3\">NP_079434.3</ext-link>), mouse RUFY1 (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_766145.1\">NP_766145.1</ext-link>), human RUFY2 (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_060457.4\">NP_060457.4</ext-link>), mouse RUFY2 (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_081701.2\">NP_081701.2</ext-link>), human RUFY3 (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_055776.1\">NP_055776.1</ext-link>), mouse RUFY3 (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_081806.1\">NP_081806.1</ext-link>) human RUFY3XL (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_001032519.1\">NP_001032519.1</ext-link>), mouse RUFY3XL (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_001276703.1\">NP_001276703.1</ext-link>), human RUFY4 (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_940885.2\">NP_940885.2</ext-link>), mouse RUFY4 (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_001164112.1\">NP_001164112.1</ext-link>), human FYCO1 (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_078789.2\">NP_078789.2</ext-link>), mouse FYCO1 (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_001103723.2\">NP_001103723.2</ext-link>).</p></caption><graphic xlink:href=\"fcell-08-00779-g003\"/></fig><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>RUFY proteins are important for intracellular trafficking, signaling and cytoskeleton dynamics. <bold>(A)</bold> Schematic representation of the RUN and FYVE domains activity of RUFY proteins. RUN domains act on signaling, endosomal protein trafficking and cytoskeletal network dynamics via small GTPase proteins. FYVE domains bind PtdIns(3)P and regulates autophagy and endosome trafficking. <bold>(B)</bold> Function of RUFY proteins in homeostatic conditions. <bold>(C)</bold> Consequences of alterations in RUFY proteins functions at the cellular and organismal level.</p></caption><graphic xlink:href=\"fcell-08-00779-g004\"/></fig></sec><sec id=\"S1.SS3\"><title>FYVE Domains</title><p>FYVE-domain-bearing proteins (for <underline>F</underline>ab1, <underline>Y</underline>OTB/ZK632.12, <underline>V</underline>ac1, and <underline>E</underline>EA1) are specifically found in association with membranous organelles enriched in PtdIns(3)P and highly conserved among eukaryotes, including yeast (<xref rid=\"B49\" ref-type=\"bibr\">Hayakawa et al., 2007</xref>; <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). FYVE domains adopt a zinc finger conformation (<xref rid=\"B90\" ref-type=\"bibr\">Misra and Hurley, 1999</xref>; <xref rid=\"B70\" ref-type=\"bibr\">Kutateladze and Overduin, 2001</xref>). In addition to FYVE, ten types of zinc finger folds have been characterized, including conventional, Gal4, GATA-1, TFIIS, MetRS, LIM, RING domain, PKC CRD, and PHD domains. Zinc fingers are structural conformations adopted by peptide chains upon coordination of two Zn<sup>2+</sup> cations within a cysteine rich region (<xref rid=\"B118\" ref-type=\"bibr\">Schwabe and Klug, 1994</xref>; <xref rid=\"B129\" ref-type=\"bibr\">Stenmark et al., 1996</xref>). Unlike most molecules bearing zinc fingers, FYVE proteins display only one copy of the domain located at any position along the peptide chain, highlighting its autonomy as a structural unit. FYVE zinc fingers can stabilize protein-protein or protein-DNA/RNA interactions (<xref rid=\"B31\" ref-type=\"bibr\">Dunkelberg and Gutierrez-Hartmann, 2001</xref>). A “classical” FYVE domain has eight potential zinc coordinating tandem cysteine positions and is characterized by having basic amino acids around the cysteines. Many members of this family also include two histidine residues in a sequence motif including WxxD, CxxC, R+HHC+xCG and RVC where “x” means any amino acid and “+” a positively charged amino acid (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>). Most deviations from this sequence can reduce the domain affinity for zinc and destabilize it (<xref rid=\"B129\" ref-type=\"bibr\">Stenmark et al., 1996</xref>; <xref rid=\"B90\" ref-type=\"bibr\">Misra and Hurley, 1999</xref>; <xref rid=\"B128\" ref-type=\"bibr\">Stenmark and Aasland, 1999</xref>; <xref rid=\"B70\" ref-type=\"bibr\">Kutateladze and Overduin, 2001</xref>). Within this structural framework, specific modifications in the non-conserved residues of the domain can radically affect FYVE protein subcellular localization and function, by forming a “turret loop” and a dimerization interface (<xref rid=\"B50\" ref-type=\"bibr\">Hayakawa et al., 2004</xref>).</p><p>With regard to their affinity for PtdIns(3)P, FYVE domain-containing proteins are mostly found associated to EE or phagosomes (<xref rid=\"B129\" ref-type=\"bibr\">Stenmark et al., 1996</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Gaullier et al., 1998</xref>; <xref rid=\"B128\" ref-type=\"bibr\">Stenmark and Aasland, 1999</xref>; <xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>). The presence of FYVE domains is therefore correlated to the regulation of membrane traffic, through specific recognition of PtdIns(3)P domains by “R+HHC+xCG” motifs (<xref rid=\"B41\" ref-type=\"bibr\">Gaullier et al., 1998</xref>), and modulation by associated phosphatidylinositol kinases. PtdIns(3)P is generated from phosphatidylinositol by Class III PtdIns 3-kinases (PI3K), like Vps34, on target membranes such as nascent autophagosome (omegasomes) (<xref rid=\"B87\" ref-type=\"bibr\">Melia et al., 2020</xref>), or EE (<xref rid=\"B25\" ref-type=\"bibr\">Di Paolo and De Camilli, 2006</xref>; <xref rid=\"B111\" ref-type=\"bibr\">Raiborg et al., 2013</xref>; <xref rid=\"B119\" ref-type=\"bibr\">Scott et al., 2014</xref>; <xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). In turn, accumulation of PtdIns(3)P recruits and activates effector proteins containing FYVE domains, favoring transport or fusion of target organelles (<xref rid=\"B130\" ref-type=\"bibr\">Stenmark and Gillooly, 2001</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Axe et al., 2008</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Burman and Ktistakis, 2010</xref>; <xref rid=\"B117\" ref-type=\"bibr\">Schink et al., 2013</xref>). Affinity for PtdIns(3)P is determined by the pair of histidine residues present in the “R+HHC+XCG” motif of the FYVE domain (<xref rid=\"B124\" ref-type=\"bibr\">Stahelin et al., 2002</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Diraviyam et al., 2003</xref>; <xref rid=\"B73\" ref-type=\"bibr\">Lee et al., 2005</xref>; <xref rid=\"B51\" ref-type=\"bibr\">He et al., 2009</xref>). This affinity can also be harnessed by FYVE proteins to link endosomes with mRNA, ribonucleoprotein particles (mRNP) and associated ribosomes, playing a role in their long-distance transport in the cell (<xref rid=\"B108\" ref-type=\"bibr\">Pohlmann et al., 2015</xref>). Importantly, many FYVE proteins homodimerize. Dimerization multiplies the conserved residues displayed by the different signature motifs present in the FYVE domain and contributes to a network of hydrogen bonding and electrostatic interactions that provides positive selection for binding several PtdIns(3)P head groups. PH-dependent insertion of FYVE domain into cell membranes (<xref rid=\"B51\" ref-type=\"bibr\">He et al., 2009</xref>; <xref rid=\"B102\" ref-type=\"bibr\">Pankiv et al., 2010</xref>) is reinforced by additional hydrophobic membrane interactions with the turret loop and/or tandem lysine residues. These non-specific interactions promote FYVE domain access to phosphate head groups, that are hindered by the close packing of lipid molecules. This bivalent mechanism increases therefore greatly FYVE domains specificity for PtdIns(3)P-enriched domains and discrimination against other mono- or polyphosphorylated PtdIns species (<xref rid=\"B90\" ref-type=\"bibr\">Misra and Hurley, 1999</xref>; <xref rid=\"B128\" ref-type=\"bibr\">Stenmark and Aasland, 1999</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Dumas et al., 2001</xref>; <xref rid=\"B70\" ref-type=\"bibr\">Kutateladze and Overduin, 2001</xref>).</p><p>FYVE proteins are therefore key players in endocytosis and autophagy and mutations in FYVE domains can alter profoundly these functions, as well as cellular homeostasis (<xref rid=\"B62\" ref-type=\"bibr\">Kamentseva et al., 2020</xref>). For example, EEA1 protein (early endosome antigen 1) is known to be crucial for endosome dynamics and any mutation in its conserved residues or the oligomerization site can drastically reduce the affinity between its FYVE domain and PtdIns(3)P (<xref rid=\"B129\" ref-type=\"bibr\">Stenmark et al., 1996</xref>; <xref rid=\"B40\" ref-type=\"bibr\">Gaullier et al., 2000</xref>). In this context, RUFYs proteins, by bearing a N-terminal RUN domain, one or several copies of a coiled-coil domain next to a C-terminal FYVE domain (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>) have all the features required to carry-out specific adaptor functions to regulate endocytosis or autophagy by impacting on organelle fusion and mobility along the cytoskeleton.</p></sec><sec id=\"S1.SS4\"><title>The RUFY Proteins Family</title><p>The RUFY family encompass four genes named <italic>rufy1</italic> to <italic>4</italic>, sharing homologies and displaying specific tissue expression and alternative splicing. <italic>Rufy</italic> genes are relatively conserved genes, absent from prokaryotes and fungi. Upon evolution, the emergence of the common ancestor appeared in vertebrates and arthropods, which possess one ortholog (<italic>CG31064</italic>) (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). No RUFY protein could be detected In <italic>Caenorhabditis elegans</italic> and only a FYVE-bearing protein (<italic>T10G3.5)</italic> considered as an ortholog of human EEA1 shows some sequence similarities with the RUFY family. <italic>T10G3.5</italic> exhibits PtdIns(3)P binding activity and is involved in endocytosis, being mostly expressed in epidermis and intestine of <italic>C. elegans</italic> (<xref rid=\"B49\" ref-type=\"bibr\">Hayakawa et al., 2007</xref>). In chordates, Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein) and FYVE And Coiled-Coil Domain Autophagy Adaptor 1 (FYCO1), display structural and functional features, potentially categorizing them as RUFY proteins. Rubicon was identified as a component of the Class III PI3K complex and a negative regulator of autophagy and endosomal trafficking (<xref rid=\"B85\" ref-type=\"bibr\">Matsunaga et al., 2009</xref>; <xref rid=\"B164\" ref-type=\"bibr\">Zhong et al., 2009</xref>). Like RUFYs, Rubicon contains multiple functional domains that interact with other proteins, including a RUN, a CC and a FYVE-like domains (<xref rid=\"B149\" ref-type=\"bibr\">Wong et al., 2018</xref>). However, despite these similarities, the poor degree of sequence homology and the lack of conservation of its FYVE-like domain, which was found not to bind to PI(3)P (<xref rid=\"B13\" ref-type=\"bibr\">Burman and Ktistakis, 2010</xref>), prevented Rubicon’s integration within the RUFY proteins family, conversely to FYCO1, which we propose here to name RUFY5 and detail the characteristics below.</p></sec></sec><sec id=\"S2\"><title>RUFY1</title><p>RUFY1, previously named Rabip4 is an 80 kDa protein, mainly expressed in the brain, kidney, lung, placenta and testis. There are two RUFY1 isoforms Rabip4, and Rabip4’ that has an additional 108 amino acid upstream of the N-terminal RUN domain (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). They were both shown to interact with the small endosomal GTPases Rab4, Rab5, and Rab14 (<xref rid=\"B35\" ref-type=\"bibr\">Fouraux et al., 2004</xref>; <xref rid=\"B144\" ref-type=\"bibr\">Vukmirica et al., 2006</xref>; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). RUFY1 inactivation inhibits efficient recycling of endocytosed transferrin, implicating RUFY1 in the regulation of EE functions through cooperative interactions with Rab4 and Rab14 (<xref rid=\"B22\" ref-type=\"bibr\">Cormont et al., 2001</xref>; <xref rid=\"B155\" ref-type=\"bibr\">Yamamoto et al., 2010</xref>; <xref rid=\"B96\" ref-type=\"bibr\">Nag et al., 2018</xref>). This was further demonstrated by the alteration of epidermal growth factor receptor endocytic trafficking kinetics in cells depleted of RUFY1 (<xref rid=\"B43\" ref-type=\"bibr\">Gosney et al., 2018</xref>) and the hijacking of RUFY1 by the bacteria <italic>P. gingivalis</italic> to escape lysosomal degradation (<xref rid=\"B134\" ref-type=\"bibr\">Takeuchi et al., 2016</xref>). In melanocytes, RUFY1 was found to form a complex with rabenosyn-5, KIF3A-B, Rab4A and adaptor protein-3 (AP-3) to differentially regulate tyrosinase-related protein-1 and tyrosinase sorting in endosomes, contributing to melanosome maturation (<xref rid=\"B96\" ref-type=\"bibr\">Nag et al., 2018</xref>; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Moreover, silencing the Rabip4’ isoform of RUFY1 was shown to promote outgrowth of plasma membrane protrusions, and to regulate the spatial distribution of lysosomes at their tips, through an interaction with AP-3 (<xref rid=\"B58\" ref-type=\"bibr\">Ivan et al., 2012</xref>; <xref ref-type=\"fig\" rid=\"F1\">Figures 1B</xref>, <xref ref-type=\"fig\" rid=\"F4\">4B</xref>). RUFY1 is also capable of controlling cell migration by regulating integrin trafficking (<xref rid=\"B144\" ref-type=\"bibr\">Vukmirica et al., 2006</xref>), presumably via endocytosis. In full agreement with a role of RUFY1 in regulating endosomal dynamics, a single nucleotide polymorphism (S705A) in the <italic>rufy1</italic> gene was associated with high blood glucose levels and type 2 diabetes mellitus susceptibility in an exome-wide association study (EWAS; <xref rid=\"B154\" ref-type=\"bibr\">Yamada et al., 2017</xref>). This result is consistent with the early finding that Rabip4 expression leads to Glucose transporter-1 (Glut-1) intracellular retention (<xref rid=\"B22\" ref-type=\"bibr\">Cormont et al., 2001</xref>). Interestingly RUFY1 display a SH3-binding motif “PxxPxP” embedded in the FYVE domain and is able to interacting with the epithelial and endothelial tyrosine kinase (ETK), and possibly regulates endocytosis through this interaction (<xref rid=\"B156\" ref-type=\"bibr\">Yang et al., 2002</xref>). Another EWAS, aiming to find early-onset Alzheimer’s Disease (AD) susceptibility genes, identified RUFY1 among genes involved in endo-lysosomal transport and known to be important for the development of AD (<xref rid=\"B69\" ref-type=\"bibr\">Kunkle et al., 2017</xref>; <xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Summary of RUFY proteins functional interactions.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protein (Aliases)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Binding partner</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Functions</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Study</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>RUFY1</bold> (Rabip4; Rabip4’; ZFYVE12)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rab4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recycling endosomal trafficking</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B22\" ref-type=\"bibr\">Cormont et al., 2001</xref></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Etk</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Regulation of endocytosis through its interaction with RUFY1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B156\" ref-type=\"bibr\">Yang et al., 2002</xref></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rab14</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RUFY1’s recruitment, endosome tethering and fusion</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B155\" ref-type=\"bibr\">Yamamoto et al., 2010</xref></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AP-3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Regulates spatial distribution of lysosome</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B58\" ref-type=\"bibr\">Ivan et al., 2012</xref></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rabenosyn-5/KIF3A- B/Rab4A/AP-3 complex</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sorting endosome pathway in endosomal membrane in melanocytes and segregates tyrosinase-related protein-1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B96\" ref-type=\"bibr\">Nag et al., 2018</xref></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PODXL1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Increases cell proliferation, migration and invasion</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B163\" ref-type=\"bibr\">Zhi et al., 2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>RUFY2</bold> (LZ-FYVE; Rabip4r; KIAA1537; FYVE13)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rab33A/Rab4A/Rab6A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Endosome dynamic, Golgi complex-associated Rab33 and autophagosome formation on omegasomes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B38\" ref-type=\"bibr\">Fukuda et al., 2011</xref>; <xref rid=\"B64\" ref-type=\"bibr\">Kitagishi and Matsuda, 2013</xref></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RET</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lead to a fusion of the RET tyrosine kinase domain to a RUN domain and a coiled-coil domain appear to be critical for tumorigenesis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B126\" ref-type=\"bibr\">Staubitz et al., 2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>RUFY3</bold> (Singar-1; RIPX; ZFYVE30; KIAA087)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rap2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Control neuronal polarity</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B59\" ref-type=\"bibr\">Janoueix-Lerosey et al., 1998</xref></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fascin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Control the growth of axons and neuronal growth cone</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B148\" ref-type=\"bibr\">Wei et al., 2014</xref></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rab5/Rab33A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Acts on endosomal trafficking</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B158\" ref-type=\"bibr\">Yoshida et al., 2010</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Fukuda et al., 2011</xref></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GPM6a-Rap2-STEF/Yial2 complex</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Facilitates cell polarity</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B55\" ref-type=\"bibr\">Honda et al., 2017a</xref></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PAK1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Induce cell migration and invasion in gastric cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B145\" ref-type=\"bibr\">Wang et al., 2015</xref></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FOXK1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Increases cells migration RUFY3-mediated with metastasis invasion in colorectal cancer</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B151\" ref-type=\"bibr\">Xie et al., 2017a</xref></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HOXD9</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HOXD9 transactivate RUFY3 and it overexpression induce gastric cancer progression, proliferation and lung metastasis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B166\" ref-type=\"bibr\">Zhu et al., 2019</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>RUFY4</bold> (ZFYVE31)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rab7</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Autophagosome formation and lysosome clustering</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B138\" ref-type=\"bibr\">Terawaki et al., 2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>FYCO1/RUFY5</bold> (ZFYVE7; RUFY3; CTRCT18; CATC2)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MAP1LC3A/B</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Autophagosome formation and elongation</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B20\" ref-type=\"bibr\">Cheng et al., 2016</xref>; <xref rid=\"B101\" ref-type=\"bibr\">Olsvik et al., 2015</xref></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rab7</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Endosomal transport by acting with microtubule plus end-direction transport</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B147\" ref-type=\"bibr\">Wang et al., 2011</xref></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Kinesin-1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Allows translocation from the late endosome, lysosome and autophagosome to the plasma membrane through plus-end microtubule transport</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B65\" ref-type=\"bibr\">Krauß and Haucke, 2015</xref>; <xref rid=\"B113\" ref-type=\"bibr\">Raiborg et al., 2016</xref>, <xref rid=\"B112\" ref-type=\"bibr\">2015</xref></td></tr></tbody></table></table-wrap></sec><sec id=\"S3\"><title>RUFY2</title><p>RUFY2 (or Leucine zipper FYVE-finger protein, LZ-FYVE) is a 75 kDa protein originally identified as an activating transcription factor-2 interactor embryogenesis (<xref rid=\"B31\" ref-type=\"bibr\">Dunkelberg and Gutierrez-Hartmann, 2001</xref>), preferentially located in the nucleus and expressed during. After development, RUFY2 expression remains high in the brain, lung, liver and the gastrointestinal tract (<xref rid=\"B156\" ref-type=\"bibr\">Yang et al., 2002</xref>). RUFY2 displays two N-terminal leucine zipper domains as well as a C-terminal FYVE-finger domain. Although it is likely to have a nuclear function at early stages of embryonic development, the presence of a FYVE domain suggests a cytoplasmic role for RUFY2 in regulating membrane traffic in fully differentiated cells. Importantly, the RUN domain of RUFY2 was shown to associate specifically with the Golgi complex-associated Rab33A (<xref rid=\"B38\" ref-type=\"bibr\">Fukuda et al., 2011</xref>; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Given the reported interaction of Rab33A and Rab33B with Atg16L and its putative role in regulating autophagy (<xref rid=\"B37\" ref-type=\"bibr\">Fukuda and Itoh, 2008</xref>), RUFY2 could contribute to autophagosome formation through a dual interaction with Rab33A and PtdIns(3)P on omegasomes (<xref ref-type=\"fig\" rid=\"F1\">Figures 1B</xref>, <xref ref-type=\"fig\" rid=\"F4\">4B</xref>). Irrespective of its function, <italic>rufy2</italic> expression is subject to modulation by the micro RNA miR-155 (<xref rid=\"B6\" ref-type=\"bibr\">Bofill-De Ros et al., 2015</xref>), which is an important regulator of immune cells development and inflammatory responses (<xref rid=\"B15\" ref-type=\"bibr\">Ceppi et al., 2009</xref>). The <italic>rufy2</italic> gene is also frequently found mutated in cancer cells, with the most frequent mutations converting it into a strong target for nonsense mediated mRNA decay, thereby decreasing considerably its expression (<xref rid=\"B120\" ref-type=\"bibr\">Shin et al., 2011</xref>; <xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>).</p></sec><sec id=\"S4\"><title>RUFY3</title><p>RUFY3, also known as Rap2-interacting protein X (RIPX) (<xref rid=\"B66\" ref-type=\"bibr\">Kukimoto-Niino et al., 2006</xref>) or Single Axon-Related 1 (Singar1) (<xref rid=\"B94\" ref-type=\"bibr\">Mori et al., 2007</xref>), is the best characterized member of the RUFY family. RUFY3, the smallest of the RUFY proteins with a molecular weight of 53 kDa (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>), is mostly expressed in neurons (<xref rid=\"B64\" ref-type=\"bibr\">Kitagishi and Matsuda, 2013</xref>). Neuronal RUFY3 is atypical, since it lacks a FYVE domain and is considered as part of the RUFY family based on strong sequence similarities with the other members, notably in the RUN and coiled-coil domains (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>). RUFY3 is distributed between the cytosol and at the plasma membrane, but not in intracellular vesicles, presumably because it lacks a FYVE domain. In artificial conditions, like following expression of the dominant gain of function mutant form of Rab5 (Q79L) in U937 cells, RUFY3 was found associated in large vesicle structures and to co-immunoprecipitate with Rab5, via an interaction with its carboxyl terminal domain and surprisingly not its RUN domain (<xref rid=\"B158\" ref-type=\"bibr\">Yoshida et al., 2010</xref>). Like RUFY2, RUFY3 was also shown in a 2-hybrid screen and by co-immunoprecipitation to bind Rab33, through its coiled-coil domain 1 (CC1; <xref rid=\"B38\" ref-type=\"bibr\">Fukuda et al., 2011</xref>). In 293T and 3Y1 cell lines however, RUFY3 was shown not to interact with several small GTPases, including Rab2, Rab5, Rab7, Rho, and Ras. This suggests that either RUFY3 requires cell specific partner proteins or post-translation modifications to be able to bind to small GTPases. RUFY3 was first described as interacting with Rap2, a small Ras-like GTPase, via a 173 residue fragment (83–255) located in the RUN domain (<xref rid=\"B59\" ref-type=\"bibr\">Janoueix-Lerosey et al., 1998</xref>; <xref rid=\"B66\" ref-type=\"bibr\">Kukimoto-Niino et al., 2006</xref>; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Together with Rap1, Rap2 interacts with Ras effectors, such as Raf, PI3K, and Ral guanine nucleotide dissociation stimulator, inhibiting activation of their downstream targets, and thus suppressing Ras oncogenic activity (<xref rid=\"B66\" ref-type=\"bibr\">Kukimoto-Niino et al., 2006</xref>; <xref rid=\"B99\" ref-type=\"bibr\">Nussinov et al., 2020</xref>). In the adult nervous system, Rap1 and Rap2 also regulate the maturation and plasticity of dendritic spine and synapses. By forming a complex together with Rap2 and Fascin, RUFY3 interacts with the filamentous actin network and controls the growth of axons and neuronal growth cone (<xref rid=\"B148\" ref-type=\"bibr\">Wei et al., 2014</xref>; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Recent mechanistic studies indicate that RUFY3 accumulates in lipid rafts by forming a Glycoprotein M6A (GPM6a)-RUFY3-Rap2-STEF/Yial2 complex (<xref rid=\"B55\" ref-type=\"bibr\">Honda et al., 2017a</xref>; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). This complex activates the Rac guanine nucleotide exchange factor (<xref rid=\"B56\" ref-type=\"bibr\">Honda et al., 2017b</xref>), impacting actin organization and promoting neuronal polarity and growth (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>). RUFY3 seems therefore to have different axogenic functions in brain (<xref rid=\"B94\" ref-type=\"bibr\">Mori et al., 2007</xref>; <xref rid=\"B56\" ref-type=\"bibr\">Honda et al., 2017b</xref>) and not surprisingly, roles for RUFY3 in amyotrophic lateral sclerosis (<xref rid=\"B3\" ref-type=\"bibr\">Arosio et al., 2016</xref>), major depressive disorder (<xref rid=\"B1\" ref-type=\"bibr\">Aberg et al., 2018</xref>) and AD (<xref rid=\"B160\" ref-type=\"bibr\">Zelaya et al., 2015</xref>) have been reported. Olfactory dysfunction occurs in 90% of AD cases and is correlated with elevated <italic>rufy3</italic> expression in glomerular and mitral layers of the olfactory bulb (<xref rid=\"B160\" ref-type=\"bibr\">Zelaya et al., 2015</xref>). RUFY3 is cleaved by caspase 3 and critically required for caspase-mediated degeneration of tropomyosin receptor kinase A positive sensory axons <italic>in vitro</italic> and <italic>in vivo</italic> (<xref rid=\"B53\" ref-type=\"bibr\">Hertz et al., 2019</xref>; <xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>). Removal of neuronally enriched RUFY3 is able to block caspase 3-dependent apoptosis, while dephosphorylation of RUFY3 at residue S34 appears required for its degradation (<xref rid=\"B53\" ref-type=\"bibr\">Hertz et al., 2019</xref>). Analysis of <italic>rufy3</italic>-deficient mice supports a second distinct function for RUFY3 in neuronal growth and polarity, since mutant embryos show defects in axonal projection patterns. These occur in addition to the prevention of CASP3-dependent apoptosis in dorsal root ganglions. RUFY3 appears therefore to be key for nervous system development, remodeling and function, explaining the embryonic lethality displayed upon <italic>rufy3</italic> genetic inactivation in mouse (<xref rid=\"B53\" ref-type=\"bibr\">Hertz et al., 2019</xref>).</p><p>With the current advance in genomics and single cell RNA sequencing, specific gene expression patterns can be revised and more accurately defined. Analysis of several genomic databases (BioGPS, NCBI, Human Atlas Protein, ImmGen, Ensembl) reveal that, in addition to neurons, RUFY3 expression can be detected in other tissues and cell types. Moreover, the <italic>rufy3</italic> gene appears to have many transcriptional variants, leading to the expression of different protein isoforms. Two of these isoforms display a C-terminal region extended by 150 amino acids, compared to the previously identified neuronal isoform of RUFY3. Importantly, these previously uncharacterized longer isoforms (RUFY3XL) possess the same RUN domain and a putative FYVE domain in their C-terminus (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>), indicating that RUFY3 is a legitimate member of the RUFY family. In contrast to classic FYVE zinc fingers, genomic databases reveal this putative FYVE domain appears to lack the tandem histidine residue cluster that defines affinity for PtdIns(3)P (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>). Interestingly, the SH3 binding site embedded in the RUFY1 and RUFY2 FYVE domains is also present in RUFY3XL, suggesting a potential signal transduction activity for this uncharacterized isoform. The translation of <italic>rufy3xl</italic> mRNA into a functional protein and its capacity to bind PtdIns(3)P remain to be demonstrated. If true, a role for RUFY3 in the coordination of endosome dynamics or organelle transport could be hypothesized. This idea is supported by the observation that RUFY3 is present in Staufen2-containing messenger ribonucleoprotein particles, that are used to transport mRNAs along neuronal dendrites to their site of translation (<xref rid=\"B81\" ref-type=\"bibr\">Maher-Laporte et al., 2010</xref>). FYVE proteins have already been implicated in endosome-mediated transport of mRNP (<xref rid=\"B108\" ref-type=\"bibr\">Pohlmann et al., 2015</xref>) and RUFY3XL could therefore also perform this function. The existence of FYVE domain bearing isoforms, might extend and diversify its function in other specialized cells.</p></sec><sec id=\"S5\"><title>RUFY4</title><p>RUFY4 is a 64 kDa that is atypical among the RUFY family members, since it bears several non-conserved residues in its RUN domain and it lacks the tandem histidine cluster and the SH3 binding domain normally present in the FYVE domain (<xref ref-type=\"fig\" rid=\"F1\">Figures 1A</xref>, <xref ref-type=\"fig\" rid=\"F3\">3A,B</xref>). RUFY4 was shown to interact with PtdIns(3)P enriched membranes (<xref rid=\"B138\" ref-type=\"bibr\">Terawaki et al., 2015</xref>, <xref rid=\"B137\" ref-type=\"bibr\">2016</xref>). Interestingly, EMBL-EBI and SMART genomic databases show that <italic>rufy4</italic> is present only in mammals, suggesting that <italic>rufy4</italic> is the most recently evolved gene in the RUFY family (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). RUFY4 levels are extremely low in most cells and tissues with the exception of lungs and lymphoid organs. RUFY4 was found to be strongly induced <italic>in vitro</italic> in dendritic cells differentiated from bone marrow progenitors in presence of GM-CSF and IL-4. <italic>In vivo</italic>, its expression was confirmed in alveolar macrophages and in lung dendritic cells isolated from asthmatic mice (<xref rid=\"B138\" ref-type=\"bibr\">Terawaki et al., 2015</xref>). RUFY4 interacts with Rab7 through its RUN domain and promotes the generation of large autophagosomes (<xref rid=\"B138\" ref-type=\"bibr\">Terawaki et al., 2015</xref>; <xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref> and <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). RUFY4 over-expression induces the degradation of the autophagy effector LC3/ATG8 and triggers clustering of LAMP1-positive late endosomal compartments. These compartments are distinct from large abnormal autophagosome-like structures positive for RUFY4 and Syntaxin-17, a Qa SNARE involved in autophagosome formation and fusion (<xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>). RUFY4 was also proposed to interact with PLEKHM1 and the HOPS complex, which are implicated in LE and lysosome dynamics and positioning (<xref rid=\"B137\" ref-type=\"bibr\">Terawaki et al., 2016</xref>). RUFY4 seems therefore able to harness the classical autophagy machinery to facilitate autophagosome formation and increase autophagy flux by acting at different biochemical steps (<xref rid=\"B138\" ref-type=\"bibr\">Terawaki et al., 2015</xref>). By optimizing effector protein activity and organelle distribution, RUFY4 expression facilitates the elimination of both damaged mitochondria and intracellular bacteria in phagocytes. RUFY4 expression in HeLa cells can prevent replication of <italic>Brucella abortus</italic> (<xref rid=\"B138\" ref-type=\"bibr\">Terawaki et al., 2015</xref>) and <italic>Salmonella typhimurium</italic> (<xref rid=\"B72\" ref-type=\"bibr\">Lassen et al., 2016</xref>) suggesting that RUFY4 has a key role in anti-bacterial responses in the lung. It also potentially acts to drive immunity though the regulation of endocytosis and autophagy, necessary for the presentation at the cell surface of antigens from intracellular pathogens (<xref rid=\"B138\" ref-type=\"bibr\">Terawaki et al., 2015</xref>).</p></sec><sec id=\"S6\"><title>FYCO1</title><p>FYCO1 is a 150 kDa protein bearing a RUN and a FYVE domains. In several databases, <italic>fyco1</italic> was misidentified as <italic>rufy3</italic>, although these two genes are present on completely distinct chromosomes, in human chromosome 3 and 4, respectively. At the sequence level, although it is larger, FYCO1 appears to be a RUFY4 ortholog gene (<xref ref-type=\"fig\" rid=\"F3\">Figures 3A,B</xref>), suggesting that FYCO1 belongs to the RUFY family. We therefore propose that it could be annotated as RUFY5 to fit the family nomenclature. Separating its N-terminal RUN domain from the FYVE zinc finger, FYCO1 has several CC domains, as well as a LC3/ATG8 Interacting Region (LIR) and a Golgi Dynamic (GOLD) domain in its C-terminus (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). FYCO1 preferentially interacts with MAP1LC3A/B of the Atg8-familly proteins through its LIR (<xref rid=\"B101\" ref-type=\"bibr\">Olsvik et al., 2015</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Cheng et al., 2016</xref>). Coiled-coil domains promote FYCO1 dimerization and have been shown to mediate the formation of a complex with Rab7, via a part of the CC located upstream of the FYVE domain (<xref rid=\"B102\" ref-type=\"bibr\">Pankiv et al., 2010</xref>; <xref rid=\"B147\" ref-type=\"bibr\">Wang et al., 2011</xref>; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Overexpression of FYCO1 was shown to redistribute LC3- and Rab7-positive structures to the cell periphery in a microtubule-dependent manner (<xref rid=\"B102\" ref-type=\"bibr\">Pankiv et al., 2010</xref>). This effect is mediated by the central part of the CC region and suggests a role for FYCO1 in the transport of autophagic vesicles (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>). The capacity of FYCO1 to interact with Rab7 and LC3A/B on the external surface of autophagosomes, and PtdIns3P enriched membranes through its FYVE domain, is likely to be key to its function as an adaptor protein. Indeed, these interactions allow microtubule plus end-directed transport and protrusion of endocytic organelles, including autophagosomes (<xref rid=\"B103\" ref-type=\"bibr\">Pankiv and Johansen, 2010</xref>), LE (<xref rid=\"B112\" ref-type=\"bibr\">Raiborg et al., 2015</xref>, <xref rid=\"B113\" ref-type=\"bibr\">2016</xref>), lysosomes (<xref rid=\"B95\" ref-type=\"bibr\">Mrakovic et al., 2012</xref>; <xref rid=\"B57\" ref-type=\"bibr\">Hong et al., 2017</xref>; <xref rid=\"B75\" ref-type=\"bibr\">Lie and Nixon, 2019</xref>), and phagosomes (<xref rid=\"B79\" ref-type=\"bibr\">Ma et al., 2014</xref>). Endoplasmic reticulum (ER) and endosomes are connected through contact sites, the numbers of which increase as endosomes mature. The functions of such sites include to control the association of endosomes with the minus-end-directed microtubule motor dynein and to mediate endosome fission. Repeated LE–ER contacts promote microtubule-dependent translocation of LEs to the cell periphery and subsequent fusion with the plasma membrane (<xref rid=\"B113\" ref-type=\"bibr\">Raiborg et al., 2016</xref>). Such fusion induces outgrowth of protrusions and neurites in the neuroendocrine cell line PC12, which require the ER-associated protein protrudin on the ER and FYCO1 to interact with LEs and kinesin 1 (<xref rid=\"B65\" ref-type=\"bibr\">Krauß and Haucke, 2015</xref>; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). FYCO1 has been described as a novel mediators of invalopodia formation and function of Protrudin-mediated ER–endosome contact sites (<xref rid=\"B104\" ref-type=\"bibr\">Pedersen et al., 2020</xref>). Multiple studies highlight the critical function of FYCO1 in autophagy and autophagosome/endosome trafficking (<xref rid=\"B26\" ref-type=\"bibr\">Dionne et al., 2017</xref>) with pathological consequences arising when FYCO1 function is altered (<xref ref-type=\"fig\" rid=\"F4\">Figures 4B,C</xref>). Mutations in the <italic>fyco1</italic> gene affect autophagy and cause autosomal-recessive congenital cataracts by altering lens development and transparency in patients (<xref rid=\"B17\" ref-type=\"bibr\">Chen et al., 2011</xref>, <xref rid=\"B18\" ref-type=\"bibr\">2017</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Brennan et al., 2012</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Costello et al., 2013</xref>; <xref rid=\"B16\" ref-type=\"bibr\">Chauss et al., 2014</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Frost et al., 2014</xref>; <xref rid=\"B63\" ref-type=\"bibr\">Khan et al., 2015</xref>; <xref rid=\"B46\" ref-type=\"bibr\">Gunda et al., 2018</xref>; <xref rid=\"B74\" ref-type=\"bibr\">Li et al., 2018</xref>). Sequencing studies of candidate genes potentially involved in several neuromuscular or neurodegenerative diseases have identified rare variants of autophagy related proteins like VCP and SQSTM1. Among these genes, a missense <italic>fyco1</italic> variant was identified to cause sporadic inclusion body myositis (<xref rid=\"B47\" ref-type=\"bibr\">Güttsches et al., 2017</xref>; <xref rid=\"B115\" ref-type=\"bibr\">Rothwell et al., 2017</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Britson et al., 2018</xref>; <xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>). Finally FYCO1 has been implicated in the autophagic clearance of specialized particles or aggregates, like male germ cell-specific RNP ribonucleoprotein granules (<xref rid=\"B24\" ref-type=\"bibr\">Da Ros et al., 2017</xref>), post-mitotic bodies (<xref rid=\"B26\" ref-type=\"bibr\">Dionne et al., 2017</xref>) or α-synuclein aggregates (<xref rid=\"B116\" ref-type=\"bibr\">Saridaki et al., 2018</xref>).</p><sec id=\"S6.SS1\"><title>RUFY Proteins and Cancer</title><p>As describe above, RUFY proteins play a central role in cellular functions by regulating vesicular trafficking and its interactions with the cytoskeleton. Neuronal deficit and neurodegeneration are the most obvious manifestations of RUFY proteins alteration. Not surprisingly, however, given their relatively broad adaptors functions, RUFY proteins have taken center stage in the oncology field.</p><p>The ETK tyrosine kinase has been shown to play a pivotal role in a variety of cellular processes including proliferation, differentiation, motility, and apoptosis (<xref rid=\"B156\" ref-type=\"bibr\">Yang et al., 2002</xref>; <xref rid=\"B68\" ref-type=\"bibr\">Kung, 2011</xref>; <xref rid=\"B167\" ref-type=\"bibr\">Zhuang et al., 2014</xref>; <xref rid=\"B146\" ref-type=\"bibr\">Wang et al., 2018</xref>). Tyrosine phosphorylation of RUFY1 by ETK appears to be important for its endosomal localization and could play an important role promoting tumoral transformation by affecting downstream effectors of PI3-kinase. RUFY1 was also shown to interact with podocalyxin-like protein (PODXL), a transmembrane glycoprotein with anti-adhesive properties associated with poor prognosis of several cancers (<xref rid=\"B135\" ref-type=\"bibr\">Taniuchi et al., 2018</xref>; <xref rid=\"B52\" ref-type=\"bibr\">He et al., 2020</xref>; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Gastric cancer progression is significantly increased upon PODXL expression, a phenotype reduced by concomitant RUFY1 silencing. Depletion of RUFY1 inactivates the PI3K/AKT, NF-κB and MAPK/ERK signaling pathways and reduces drastically migration and invasion of cancer cells <italic>in vitro</italic> (<xref rid=\"B163\" ref-type=\"bibr\">Zhi et al., 2019</xref>). Given the positive correlation between <italic>podxl</italic> and <italic>rufy1</italic> expression in tissues and serum, <italic>rufy1</italic> was proposed as a potential biomarker for gastric cancers stratification (<xref rid=\"B163\" ref-type=\"bibr\">Zhi et al., 2019</xref>; <xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>). Like RUFY1, a role for RUFY2 in various cancer has been reported (<xref rid=\"B120\" ref-type=\"bibr\">Shin et al., 2011</xref>; <xref rid=\"B162\" ref-type=\"bibr\">Zheng et al., 2014</xref>; <xref rid=\"B126\" ref-type=\"bibr\">Staubitz et al., 2019</xref>). <italic>Rufy2</italic> is one of the most frequently mutated genes in high-microsatellite instability tumors and colorectal cancer (<xref rid=\"B120\" ref-type=\"bibr\">Shin et al., 2011</xref>). Gene rearrangement of the proto-oncogene <italic>ret</italic> with <italic>rufy2</italic> have been shown to drive tumorigenesis in lung adenocarcinoma (<xref rid=\"B162\" ref-type=\"bibr\">Zheng et al., 2014</xref>) and papillary thyroid carcinoma (<xref rid=\"B126\" ref-type=\"bibr\">Staubitz et al., 2019</xref>). The gene rearrangement leads to a fusion of the RET tyrosine kinase domain with RUFY2 RUN domain and coiled-coil domain; this appears to be critical for tumorigenesis (<xref rid=\"B126\" ref-type=\"bibr\">Staubitz et al., 2019</xref>; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). <italic>Rufy2</italic> mRNA is the target of several microRNAs, including miR-146a, miR-196a-5p and miR-155 (<xref rid=\"B6\" ref-type=\"bibr\">Bofill-De Ros et al., 2015</xref>). Dysregulated microRNA targeting of RUFY2 expression was found important for the development of human glioblastoma and ovarian cancer, suggesting a tumor suppression role for RUFY2 (<xref rid=\"B77\" ref-type=\"bibr\">Lukács et al., 2019</xref>; <xref rid=\"B161\" ref-type=\"bibr\">Zheng et al., 2020</xref>; <xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>). Given the key role of RUFY3 in cell migration, membrane transport, and cellular signaling, through its interaction with rap2, it is not surprising that RUFY3 dysregulation has been implicated in several cancer processes and metastatic tumor spread. The abnormal expression of RUFY3 is linked to poor prognosis. It can promote growth, invasion and metastasis in lung adenocarcinoma, gastric cancer cells or colorectal cancer (<xref rid=\"B151\" ref-type=\"bibr\">Xie et al., 2017a</xref>, <xref rid=\"B152\" ref-type=\"bibr\">b</xref>; <xref rid=\"B88\" ref-type=\"bibr\">Men et al., 2019</xref>; <xref rid=\"B166\" ref-type=\"bibr\">Zhu et al., 2019</xref>). RUFY3 overexpression and its interaction with P21-activated kinase-1 (PAK1) leads to the formation of F-actin-enriched protrusive structures, increased epithelial-mesenchymal transition and gastric cancer cell migration (<xref rid=\"B67\" ref-type=\"bibr\">Kumar and Vadlamudi, 2002</xref>; <xref rid=\"B141\" ref-type=\"bibr\">Vadlamudi and Kumar, 2003</xref>). Several transcription factors, including Forkhead box k1 (FOXK1) and Homebox D9 (HOXD9) involved in cancer progression (<xref rid=\"B92\" ref-type=\"bibr\">Moens and Selleri, 2006</xref>; <xref rid=\"B132\" ref-type=\"bibr\">Tabuse et al., 2011</xref>; <xref rid=\"B78\" ref-type=\"bibr\">Lv et al., 2015</xref>; <xref rid=\"B106\" ref-type=\"bibr\">Peng et al., 2016</xref>; <xref rid=\"B150\" ref-type=\"bibr\">Wu et al., 2016</xref>; <xref rid=\"B76\" ref-type=\"bibr\">Liu et al., 2018</xref>; <xref rid=\"B166\" ref-type=\"bibr\">Zhu et al., 2019</xref>), have been shown to regulate RUFY3 expression and activity (<xref rid=\"B151\" ref-type=\"bibr\">Xie et al., 2017a</xref>; <xref rid=\"B166\" ref-type=\"bibr\">Zhu et al., 2019</xref>; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). So far, no correlation has been found between RUFY4 and any type of cancer. FYCO1 has also been implicated in colorectal cancer progression (<xref rid=\"B121\" ref-type=\"bibr\">Sillars-Hardebol et al., 2010</xref>) and recent studies have concluded that FYCO1 may serve as a biomarker in bladder cancer (<xref rid=\"B32\" ref-type=\"bibr\">Eissa et al., 2017</xref>) or hepatocellular carcinoma (<xref rid=\"B143\" ref-type=\"bibr\">Vongchan and Linhardt, 2017</xref>; <xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>). Plus, FYCO1 can indirectly associated with cell invasion (<xref rid=\"B104\" ref-type=\"bibr\">Pedersen et al., 2020</xref>).</p></sec></sec><sec id=\"S7\"><title>Conclusion</title><p>Although they have been poorly characterized to date, RUFY proteins play a central role in cellular homeostasis by regulating endocytosis, autophagy and coordinating organelle transport with signal transduction cascades. It is important to note that RUFY proteins also provide a regulatory link between cytoskeletal dynamics and membrane trafficking. Consequently, these proteins have adaptive functions by acting on localized actions (through PtdIns(3)P) and signaling (through small GTPases), which can affect key biological functions in specialized cells, such as migration, tissue repair or targeted secretion. The dysregulated expression of RUFY proteins has therefore severe consequences on cell differentiation and polarization, causing cancers or neurodegenerative diseases. However, further molecular and physiological analyses will be required to understand how these proteins exert their functions in specialized cell types like immune cells or neurons. Immunocytes require endocytosis and migration to perform their functions within primary and secondary lymphoid organs or at sites of infection. The restricted expression of RUFY4, as well as the existence of splicing variants of RUFY3 in alveolar macrophages and dendritic cells, suggest a role for these molecules in phagocytes. Of importance will be the characterization of the different molecules interacting either with their RUN or FYVE domains in a cell specific manner. Identification of these RUFY’s interactors will be crucial to establish the functionality of the domains and their importance for signaling on one end and subcellular targeting at the other end. The coiled-coil structural domains found in the central part of the RUFY proteins should also be scrutinized. CC domains, in addition to support homodimerization and increase affinity for PtdIns(3)P, could also be determinant in promoting RUFY proteins interactions with effector molecules, like Rab7, as observed for FYCO1. The nature and pattern of expression of these effector molecules will allow to sort the different activities displayed by the RUFYs in individual cell types and thereby shed light on their physiological importance in health and diseases.</p></sec><sec id=\"S8\"><title>Author Contributions</title><p>Both authors contributed equally to the design and implementation of the research and writing of the manuscript.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> The PP laboratory is “Equipe de la Fondation de la Recherche Médicale” (FRM) sponsored by the grant DEQ20180339212. The project was supported by grants from l’Agence Nationale de la Recherche (ANR) «DCBIOL Labex ANR-11-LABEX-0043», «INFORM Labex ANR-11-LABEX-0054» funded by the “Investissements d’Avenir” French government program. The research was also supported by the Ilídio Pinho foundation and FCT – Fundaçăo para a Ciência e a Tecnologia – and Programa Operacional Competitividade e Internacionalizaçăo – Compete2020 (FEDER) – references POCI-01-0145-FEDER-016768 and POCI-01-0145-FEDER-030882.</p></fn></fn-group><ack><p>We acknowledge Dr. Jonathan Ewbank for his constructive comments.</p></ack><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aberg</surname><given-names>K. A.</given-names></name><name><surname>Dean</surname><given-names>B.</given-names></name><name><surname>Shabalin</surname><given-names>A. A.</given-names></name><name><surname>Chan</surname><given-names>R. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Physiol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Physiol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Physiol.</journal-id><journal-title-group><journal-title>Frontiers in Physiology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-042X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32848810</article-id><article-id pub-id-type=\"pmc\">PMC7431700</article-id><article-id pub-id-type=\"doi\">10.3389/fphys.2020.00754</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Physiology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Gonad-Specific Transcriptomes Reveal Differential Expression of Gene and miRNA Between Male and Female of the Discus Fish (<italic>Symphysodon aequifasciatus</italic>)</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Fu</surname><given-names>Yuanshuai</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/827067/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Xu</surname><given-names>Zhe</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wen</surname><given-names>Bin</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/489458/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Gao</surname><given-names>Jianzhong</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Zaizhong</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University</institution>, <addr-line>Shanghai</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University</institution>, <addr-line>Shanghai</addr-line>, <country>China</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai Ocean University</institution>, <addr-line>Shanghai</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Zhi Zhou, Hainan University, China</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Changwei Shao, Yellow Sea Fisheries Research Institute (CAFS), China; Xingkun Jin, Hohai University, China</p></fn><corresp id=\"c001\">*Correspondence: Zaizhong Chen, <email>chenzz@shou.edu.cn</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>These authors have contributed equally to this work</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Aquatic Physiology, a section of the journal Frontiers in Physiology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>754</elocation-id><history><date date-type=\"received\"><day>13</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>11</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Fu, Xu, Wen, Gao and Chen.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Fu, Xu, Wen, Gao and Chen</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>The discus fish (<italic>Symphysodon aequifasciatus</italic>) is an ornamental fish that is well-known around the world. In artificial reproduction, they must be matched by one male and one female, whereas phenotype investigation indicated that there are no significant differences in appearance between males and females, which causes great difficulties in the mating during artificial reproduction. So, it is of great importance to establish artificial sex identification methods for the discus fish. The molecular mechanism of the sexual dimorphism of the discus fish was previously unknown. In this study, we constructed six cDNA libraries from three adult testes and three adult ovaries and performed RNA sequencing for identifying sex-biased candidate genes and microRNAs (miRNAs). A total of 50,082 non-redundant genes (unigenes) were identified, of which 18,570 unigenes were significantly overexpressed in testes, and 11,182 unigenes were significantly overexpressed in ovaries. A total of 551 miRNAs were identified, of which 47 miRNAs were differentially expressed between testes and ovaries. Eight differentially expressed unigenes, seven differentially expressed miRNAs and one non-differential miRNA were validated by quantitative real-time polymerase chain reaction. Twenty-four of these differentially expressed miRNAs and their 15 predicted target genes constituted 41 miRNA–mRNA interaction pairs, and some of vital sex-related metabolic pathways were also identified. These results revealed these differentially expressed genes and miRNAs between ovary and testis might be involved in regulating gonadal development, sex determination, gametogenesis, and physiological function maintenance, and there are complex regulatory networks between genes and miRNAs. It can help us understand the molecular mechanism of the sexual dimorphism and obtain a high-efficiency sex identification method in the artificial reproduction process of the discus fish.</p></abstract><kwd-group><kwd><italic>Symphysodon aequifasciatus</italic></kwd><kwd>ovary</kwd><kwd>testis</kwd><kwd>transcriptome</kwd><kwd>mRNAs</kwd><kwd>miRNAs</kwd></kwd-group><counts><fig-count count=\"5\"/><table-count count=\"4\"/><equation-count count=\"0\"/><ref-count count=\"48\"/><page-count count=\"13\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>The discus fish (<italic>Symphysodon aequifasciatus</italic>), one of the most demanded freshwater ornamental fish species around the world, has been widely cultured because of its brilliant colors and pretty disk-shaped body. With the improvement of people’s living standard, the demand for the discus fish is increasing. Before artificial reproduction, the discus fish must be matched by one male and one female. However, the male and female of discus fish cannot be judged according to the external features, which causes great difficulties in the mating between males and females in the artificial reproduction process. As a result, the rapid reproduction of the discus fish is suppressed; its production is limited. Therefore, it is of great importance to establish artificial sex identification techniques that can be used in quick pairing of one male and one female for producing more and more progeny. A comprehensive understanding of developmental mechanism of the sexual dimorphism of the discus fish is urgently needed, including knowledge of the genes and microRNAs (miRNAs) involved in the gonads of both sexes.</p><p>In fish, there is a higher variety of sex determination mechanisms, including genetic sex determination, environmental sex determination, or even interactions of genetic and environmental sex determination (<xref rid=\"B4\" ref-type=\"bibr\">Baroiller and Guiguen, 2001</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Nagahama, 2005</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Baroiller et al., 2009</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Shen and Wang, 2014</xref>). Gonad is an indispensable reproductive organ including testis and ovary; their development is controlled by many genes, which are differentially expressed between testes and ovaries. To date, several master sex-determining genes have been identified and play a key role in regulating sex development as transcription factors. <italic>SRY</italic> in mammals (<xref rid=\"B39\" ref-type=\"bibr\">Sinclair et al., 1990</xref>) and <italic>DMY/dmrt1bY</italic> in medaka (<xref rid=\"B27\" ref-type=\"bibr\">Matsuda et al., 2002</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Nanda et al., 2002</xref>) both initiate male sex determination, are required for testis formation in XY embryos, and are sufficient to induce testis differentiation in XX embryos (<xref rid=\"B16\" ref-type=\"bibr\">Huang et al., 2017</xref>). In females, there exist a number of essential ovary-specific genes, for example, β<italic>-catenin</italic>, <italic>follistatin</italic>, <italic>FOXL2</italic>, <italic>R-spondin</italic>, and <italic>WNT4</italic> (<xref rid=\"B16\" ref-type=\"bibr\">Huang et al., 2017</xref>). Deficiency of these genes may cause ovarian development stasis, and mutation of these genes may result in aberrant ovary development. Gametogenesis can be differentiated in spermatogenesis and oogenesis; there are numerous gene expressions associated with gametogenesis in the reproductive stage in the mature gonads of teleost (<xref rid=\"B10\" ref-type=\"bibr\">Cabas et al., 2013</xref>). Previous studies have indicated that several key genes associated with steroid hormone enzymes are expressed during the stage of gametogenesis in tilapia (<xref rid=\"B36\" ref-type=\"bibr\">Senthilkumaran et al., 2009</xref>).</p><p>MicroRNAs are endogenously expressed, small non-coding RNAs that are approximately 18 to 22 nucleotides (nt) long and that posttranscriptionally regulate gene expression, inhibit targeted gene translation, or degrade target mRNA by partial or complete complementarity binding to the 3′ untranslated region of target genes in both animals and plants (<xref rid=\"B1\" ref-type=\"bibr\">Ambros, 2004</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Bartel, 2009</xref>). The total set of transcripts (mRNA and non-coding RNA) involved in the transcriptome is transcribed at a specific organization during a particular developmental stage (<xref rid=\"B25\" ref-type=\"bibr\">Mardis, 2008</xref>). In organism, one miRNA may control the expression of several genes, or the expression of a single gene requires multiple miRNAs to work simultaneously (<xref rid=\"B5\" ref-type=\"bibr\">Bartel, 2004</xref>). Previous studies showed that miRNA may be an inducible factor to increase the complexity of organism with their roles in regulating gene expression (<xref rid=\"B35\" ref-type=\"bibr\">Sempere et al., 2006</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Niwa and Slack, 2007</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Heimberg et al., 2008</xref>). In miRBase Release 22.1<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup>, there are the identified miRNA information from 16 teleost fish; four of them belong to the cichlid such as <italic>Astatotilapia burtoni</italic> (298 precursors, 236 mature), <italic>Neolamprologus brichardi</italic> (251 precursors, 182 mature), <italic>Oreochromis niloticus</italic> (812 precursors, 695 mature), and <italic>Pundamilia nyererei</italic> (250 precursors, 182 mature) (<xref rid=\"B9\" ref-type=\"bibr\">Brawand et al., 2014</xref>; <xref rid=\"B45\" ref-type=\"bibr\">Xiong et al., 2019</xref>). In tilapia gonads, 111 differentially expressed miRNAs (DEMs) were identified between testes and ovaries, and the targets of these sex-biased miRNAs contained key genes encoding enzymes in steroid hormone biosynthesis pathways (<xref rid=\"B41\" ref-type=\"bibr\">Tao et al., 2016</xref>). In rainbow trout, a total of 13 differential expression miRNAs were observed during stages of oogenesis, which indicated that they might regulate female gamete formation during oogenesis (<xref rid=\"B19\" ref-type=\"bibr\">Juanchich et al., 2013</xref>). There is no available information on miRNAs in the discus fish, so we have carried out preliminary research work about gonad miRNAs and release it in bioRxiv as a preprint (<xref rid=\"B13\" ref-type=\"bibr\">Fu et al., 2018</xref>).</p><p>In this study, we are aiming to screen differentially expressed genes (DEGs) and miRNAs between testes and ovaries through RNA sequencing and identify key genes and miRNAs capable of regulating gonadal development or sex determination. This work will help to further understand the underlying molecular mechanisms of sex differentiation and sex determination in the discus fish.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Sample Collection</title><p>Three 1-year-old healthy males (testicular development completed and visible sperm, average size 18.4 cm, average weight 158.8 g, <italic>n</italic> = 3) and three 1-year-old healthy females (ovarian development completed and visible eggs, average size 17.1 cm, average weight 146.9 g, <italic>n</italic> = 3), were obtained from the Ornamental Aquatic Breeding Laboratory in the College of Fisheries and Life Science of Shanghai Ocean University (Shanghai, China) and maintained in one feed cylinder at 28°C ± 0.5° and fed with beef heart. After 3 days, these six experimental fish were anesthetized in well-aerated water containing a 100 mg/L concentration of MS-222 (3-aminobenzoic acid ethyl ester methane sulfonate; Argent Chemical Laboratories, Redmond, WA, United States) and were killed by decapitation, and then ovaries and testes were collected by dissecting these fish and immersed into liquid nitrogen and finally stored at −80°C until subsequent RNA isolation. Our study was performed in strict accordance with Laboratory Animal–Guideline for Ethical Review of Animal Welfare of China (GB/T 35892-2018). All experimental procedures were approved by the Animal ethics committee of Shanghai Ocean University (SHOU-DW-2017-039).</p></sec><sec id=\"S2.SS2\"><title>RNA Isolation</title><p>Total RNA was extracted using the miRNeasy Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol and treated with RNase-free DNase I (TIANGEN, Beijing, China) to remove genomic DNA contamination. The total RNA quantity and purity were analyzed by Bioanalyzer 2100 (Agilent, Technologies, Santa Clara, CA, United States) with RNA integrity number >7.0. The concentration and quality of the purified RNA samples were determined utilizing a NanoDrop 2000C spectrophotometer (Thermo Scientific, Waltham, MA, United States) and RNA integrity was detected by agarose-gel electrophoresis, and A260/A280 ratios were between 2.0 and 1.9.</p></sec><sec id=\"S2.SS3\"><title>Library Construction and Sequencing for mRNA</title><p>Six gonadal samples for mRNA transcriptome analysis were prepared using a TruSeq<sup>TM</sup> RNA Sample Prep Kit (Illumina, San Diego, CA, United States) according to manufacturer instructions. These mRNAs were isolated from >5 μg of gonadal total RNAs using oligo (dT) magnetic beads. These short fragment RNAs were transcribed to create first-strand cDNAs using random hexamer primers; the second-strand cDNAs were then synthesized using RNase H, buffer, dNTP, and DNA polymerase I. These double-stranded cDNAs were purified using Takara’s polymerase chain reaction (PCR) extraction kit (Takara Bio, Dalian, Liaoning, China), then ligated with sequencing adapters, and resolved by agarose gel electrophoresis. Proper fragments were selected and purified and subsequently amplified by 15 cycles of PCR to create the cDNA libraries. The DSN kit (Evrogen, Moscow, Russia) was used to normalize the cDNA libraries. These normalized cDNA libraries were sequenced on an Illumina HiSeq 2500 sequencing platform, with 125-nt reads length and both end sequencing pattern.</p></sec><sec id=\"S2.SS4\"><title>Library Construction and Sequencing for Small RNA</title><p>Six gonadal samples for miRNA transcriptome analysis were prepared using a TruSeq<sup>TM</sup> Small RNA Sample Prep Kit (Illumina) according to manufacturer instructions. Small RNA was isolated from gonadal total RNA and was ligated with proprietary 5′ and 3′ adapter. Adaptor-ligated small RNAs were then reverse transcribed to create cDNA constructs using Superscript reverse transcriptase (Invitrogen, Carlsbad, CA, United States). These generated small cDNA libraries were amplified by 15 cycles of PCR using Illumina small RNA primer set and Phusion polymerase (New England Lab, United States) and purified on a 6% Novex TBE PAGE gel. The purified PCR libraries were sequenced on an Illumina HiSeq 2500 sequencing platform, with 50 sequencing cycle number, 50-nt reads length, and single end sequencing pattern.</p></sec><sec id=\"S2.SS5\"><title>Bioinformatics Analysis of mRNA and miRNA Transcriptome Data</title><p>The clean reads for mRNA transcriptome were obtained using NGS QC TOOLKIT v2.3.3 software (<xref rid=\"B32\" ref-type=\"bibr\">Patel and Jain, 2012</xref>) by filtering out adapter sequences, low-quality reads (reads with ambiguous bases “N”), and reads with more than 10% <italic>Q</italic> < 25 bases. The clean reads were assembled into non-redundant transcripts using Trinity program<sup><xref ref-type=\"fn\" rid=\"footnote2\">2</xref></sup> (<xref rid=\"B14\" ref-type=\"bibr\">Grabherr et al., 2011</xref>) with default K-mers = 25. The non-redundant transcripts less than 100 bp in length and partially overlapping sequence were removed. To analyze the conservation of the non-redundant transcripts between species, we compared all these transcripts with the NR database<sup><xref ref-type=\"fn\" rid=\"footnote3\">3</xref></sup> using DIAMOND software. Next, the remained non-redundant transcripts were annotated by Blast search against the NR protein, the Gene Ontology (GO), Clusters of Orthologous Group (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) database using an <italic>E</italic> value cutoff of 10<sup>–5</sup>. The functional annotation by GO terms (<xref rid=\"B2\" ref-type=\"bibr\">Ashburner et al., 2000</xref>) was carried out using Blast2GO software; the functional annotation against the COG (<xref rid=\"B42\" ref-type=\"bibr\">Tatusov et al., 2003</xref>) and KEGG database (<xref rid=\"B20\" ref-type=\"bibr\">Kanehisa et al., 2008</xref>) was performed using Blast software.</p><p>The raw reads from miRNA transcriptome were subjected to initially filter and remove low-quality reads (including reads shorter than 18 nt) and adapter sequences. The clean reads with a length of ∼18 to 26 nt were subsequently aligned to Rfam 11.0 and the NCBI database searching, and these reads similar to rRNA, tRNA, snRNA, snoRNA, and scRNA were removed. The remnant reads were aligned against correlation sequences in miRBase 21 (<xref rid=\"B22\" ref-type=\"bibr\">Kozomara and Griffiths-Jones, 2014</xref>) and the reference genome of <italic>S. aequifasciatus</italic> non-redundant transcripts of this study, allowing length variation at both 3′ and 5′ ends and one mismatch inside of the sequence (<xref rid=\"B12\" ref-type=\"bibr\">Fu et al., 2011</xref>). The unmapped sequences were BLASTed against the specific genomes, and the hairpin RNA structures containing sequences were predicated from the flank 80-nt sequences using RNAfold software<sup><xref ref-type=\"fn\" rid=\"footnote4\">4</xref></sup>.</p><p>Two computational target prediction algorithms (TargetScan and miRanda) were used to predict the target genes of miRNA from blast matching against the <italic>S. aequifasciatus</italic> mRNA transcriptome sequence of this study. miRanda was used to match the entire miRNA sequences. The TargetScan (<xref rid=\"B38\" ref-type=\"bibr\">Shi et al., 2017</xref>) parameters were set as a context score percentile >50. The miRanda parameters (<xref rid=\"B18\" ref-type=\"bibr\">John et al., 2004</xref>) were set as free energy <−10 kcal/mol and a score >50. All miRNA targets were categorized into functional classes using the GO terms and KEGG pathway. And the results predicted by the two algorithms were combined, and the overlaps were calculated.</p></sec><sec id=\"S2.SS6\"><title>Expression Analysis of mRNAs and miRNAs</title><p>mRNA expression level between different mRNA transcriptome were measured by RPKM (reads per kb per million reads) using RSEM software (<xref rid=\"B24\" ref-type=\"bibr\">Li et al., 2010</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Li and Dewey, 2011</xref>). All transcript sequences obtained by splicing in Trinity were used as reference sequences. Each sample’s valid data alignment was quantified to the reference sequence. The main parameters were as follows: no-mixed, no-discordant gbar 1000, and end-to-end-k200. The mRNAs with log-fold difference ratios (log<sub>2</sub> ratio) ≥1 and false discovery rate (<0.05) were considered to be significantly differentially expressed.</p><p>MicroRNA differential expression based on normalized deep-sequencing counts was analyzed by <italic>t</italic> test. Comparisons between testes and ovaries were made to identify significantly DEMs [|log<sub>2</sub>(fold change)| >1 and <italic>P</italic> ≤ 0.05]. Data normalization followed the procedures as described in a previous study (<xref rid=\"B11\" ref-type=\"bibr\">Cer et al., 2014</xref>).</p></sec><sec id=\"S2.SS7\"><title>Quantitative Real-Time PCR Validation for miRNA and mRNA Expression</title><p>To validate the RNA-Seq data, eight mRNAs and eight miRNAs were randomly selected from differentially expressed mRNAs and miRNAs, and specific primers for mRNAs, miRNAs, 18S rRNA, and 5S rRNA (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table S1</xref>) were designed to quantify their expression levels between ovaries and testes using quantitative real-time PCR (qRT-PCR). Total RNAs were reverse transcribed to cDNAs using M-MLV Reverse Transcriptase (Promega), with an equal amount of mixed reverse primer of the Oligo(dT)<sub>18</sub> (Takara) and random primer [hexadeoxyribonucleotide mixture; pd(N)6] (Takara) for the quantification of mRNAs, and stem-loop RT primer (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table S1</xref>) for the quantification of miRNAs.</p><p>Quantitative RT-PCR was performed using PowerUp<sup>TM</sup> SYBR<sup>TM</sup> Green Master Mix (Thermo Fisher Scientific Inc., Rockford, IL, United States) on a CFX96 Touch<sup>TM</sup> Real-Time PCR Detection System (Bio-Rad, Heracles, CA, United States). The 18s rRNA that was not differentially expressed between testes and ovaries was used as an internal control for mRNA expression levels, and the 5s rRNA without differential expression between testes and ovaries was used as the internal control for the normalization of miRNA expression levels. Quantitative RT-PCR reactions of 20 μL contained 10.0 μL SYBR Green Master Mix, 1.0 μL forward primer (10 μM), and 1.0 μL reverse primer (10 μM), 1.0 μL cDNA, and 7.0 μL DEPC H<sub>2</sub>O, and the amplification procedure was carried out at 95°C for 2 min, 40 cycles of 95°C for 10 s, and 60°C for 20 s followed by disassociation curve analysis. Samples were replicated two times for each run, and the average Ct value was used to calculate gene expression levels. The relative expression levels were determined using the 2<sup>–ΔΔCt</sup> method. All of data are expressed as the mean ± SEM. Comparisons between the two groups were performed using Student <italic>t</italic> tests. <italic>P</italic> < 0.05 was considered to indicate significant difference. All primers are listed in <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table S1</xref>.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title><italic>De novo</italic> Assembly and Functional Annotation of mRNA Transcriptome</title><p>Six cDNA libraries from three testes and three ovaries were sequenced using Illumina HiSeq 4000 sequencing. A total of 437,621,708 reads were obtained from six cDNA libraries (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). After quality filtering using the Trinity <italic>de novo</italic> assembly method, we obtained 50,082 non-redundant genes (unigenes) with an average length of 885 bp and 65,496 transcripts with an average length of 1,077 bp (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). All of the raw mRNA transcriptome sequencing data have been submitted to the SRA database<sup><xref ref-type=\"fn\" rid=\"footnote5\">5</xref></sup>.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Summary of Illumina HiSeq 4000 sequence reads.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Sample</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Raw data reads</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Valid data reads</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Q20 percentage</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Q30 percentage</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>GC percentage</bold></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Testis_1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">88,682,568</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">86,322,782</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">97.26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92.56</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50.25</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Testis_2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">82,098,772</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">79,904,932</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">97.39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92.72</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50.72</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Testis_3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">54,797,130</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53,361,960</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">97.28</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92.51</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">49.13</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ovary_1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">69,045,830</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">66,995,128</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">96.93</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91.72</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50.51</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ovary_2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">62,969,370</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">61,204,582</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">97.27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92.41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">51.22</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ovary_3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80,028,038</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">77,880,556</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">97.38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92.70</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">49.97</td></tr></tbody></table></table-wrap><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p><italic>De novo</italic> assembly statistics of the discus fish transcriptomic sequences.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Category</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>All</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Median GC%</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Mean GC%</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Mean length</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>N50 of contigs</bold></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gene</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50,082</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">47.80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">47.28</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">885</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,540</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Transcript</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">65,496</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">48.10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">47.64</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,077</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,896</td></tr></tbody></table></table-wrap><p>The conservative analysis of the non-redundant transcripts showed that the transcripts were mainly distributed in fish, such as <italic>Larimichthys crocea</italic> (19%), <italic>Lates calcarifer</italic> (15.03%), <italic>Seriola dumerili</italic> (10.23%), <italic>Seriola lalandi</italic> (4.08%), <italic>Stegastes partitus</italic> (4.83%), and <italic>Mastacembelus armatus</italic> (4.25%) (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1</xref>). To annotate and analyze the putative functional roles of unigenes, all unigenes were compared with the Swiss-Prot database, Pfam database, and NCBI non-redundant nucleotide sequences (Nr) in the priority order of GO, eukaryotic Orthologous Groups (KOG) database, and KEGG database. A total of 25,026 unigenes (24.51%) were annotated in the NR database, and 22,739 unigenes in the Swiss-Prot, whereas the other unannotated unigenes represent novel genes of unknown functions (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). A structured and controlled vocabulary to describe gene products was obtained using GO and KOG analysis. A total of 20,176 unigenes (40.29%) were assigned to the GO database (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). Three distinct GO categories were characterized with 10 molecular function groups, 15 cellular component groups, and 25 biological process groups (<xref ref-type=\"supplementary-material\" rid=\"FS2\">Supplementary Figure S2</xref>). A total of 21,311 (42.55%) unigenes were assigned to the KOG database and classified into 25 functional categories (<xref ref-type=\"supplementary-material\" rid=\"FS3\">Supplementary Figure S3</xref>). Kyoto Encyclopedia of Genes and Genomes analysis was performed to identify potential candidate unigenes in biological pathways. A total of 15,702 (31.35%) unigenes were assigned to the KEGG database (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>) and mapped onto 36 predicted pathways (<xref ref-type=\"supplementary-material\" rid=\"FS4\">Supplementary Figure S4</xref>).</p><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>Annotation statistics of the discus fish transcriptome sequencing.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Gene_number</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Swiss-Prot</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Pfam</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Nr</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>GO</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>KOG</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>KEGG</bold></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">50,082</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22,739</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20,450</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23,371</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20,176</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21,311</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15,702</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">100%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">45.40%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40.83%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">46.67%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40.29%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">42.55%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.35%</td></tr></tbody></table></table-wrap></sec><sec id=\"S3.SS2\"><title>Differentially Expressed mRNAs</title><p>A total of 29,752 DEGs were detected by comparing DEGs between testis and ovary transcriptomic sequencing (<xref ref-type=\"supplementary-material\" rid=\"TS2\">Supplementary Table S2</xref>). A total of 18,570 of DEGs were higher expressed and 11,182 lower expressed in the testis compared to the ovary (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). There were 3,151 genes that were specifically expressed in the testis, 222 in the ovary, and 26,379 in both tissues (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). We can visually find the DEGs by a volcano plot (<xref ref-type=\"supplementary-material\" rid=\"FS5\">Supplementary Figure S5</xref>). The <italic>x</italic> axis shows the differences in expression given as the log values, whereas the <italic>y</italic> axis shows the significant differences in expression as negative log values. Differentially expressed genes are indicated by red dots, and non-DEGs are indicated by blue dots.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Identification, GO, and pathway of differentially expressed genes between testis and ovary. <bold>(A)</bold> Number of up-/down-expressed mRNAs in testis versus ovary. “Up-regulated” means that these unigenes were higher expressed in the testes comparing to the ovaries, and “down-regulated” means that these unigenes were lower expressed in the testes comparing to the ovaries; <bold>(B)</bold> the Venn diagram of testis-specific and ovarian-specific genes; <bold>(C)</bold> GO scatter diagram different expression genes between the testis and ovary; <bold>(D)</bold> KEGG scatter diagram different expression genes between the testis and ovary.</p></caption><graphic xlink:href=\"fphys-11-00754-g001\"/></fig><p>To further determine and compare the functions of these DEGs, a total of 122 GO terms including these DEGs were classified into three GO categories (cellular component, biological process, and molecular function) (<xref ref-type=\"supplementary-material\" rid=\"TS3\">Supplementary Table S3</xref> and <xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref>). The DEGs were compared to the KEGG pathway to gain an overview of the gene pathway networks. A total of 1,251 DEGs involved in 243 pathways were predicted in the pairwise comparison of testis versus ovary (<italic>p</italic> < 0.05) (<xref ref-type=\"supplementary-material\" rid=\"TS4\">Supplementary Table S4</xref> and <xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>).</p></sec><sec id=\"S3.SS3\"><title>Differentially Expressed miRNAs</title><p>In this study, a total of 551 miRNAs were identified from the gonad tissues, and they ranged from 18 to 26 nt in length (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). By comparing miRNA expression levels between testes and ovaries, a total of 47 DEMs were identified between testis and ovary (<xref ref-type=\"supplementary-material\" rid=\"TS5\">Supplementary Table S5</xref>). Thirty-one miRNAs were significantly higher expressed and 16 miRNAs significantly lower expressed in the testes comparing to the ovaries (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS6\">Supplementary Figure S6</xref>). We found that four miRNAs were specifically expressed in the testis, and they were dre-miR-124-5p, ccr-miR-7133, PC-5p-60222, and aca-miR-726, whereas three miRNAs were specifically expressed in the ovaries. Using the heat map of the DEMs, one can intuitively see the changes in miRNA expression (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). These DEMs were regarded as candidate sex-related miRNAs.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Differential expressed miRNAs between testis and ovary. <bold>(A)</bold> Number of up-/down-expressed miRNAs in testis and ovary. “Up” means that these miRNAs were higher expressed in the testes comparing to the ovaries, and “down” means that these miRNAs were lower expressed in the testes comparing to the ovaries; <bold>(B)</bold> heat map of the differentially expressed miRNAs in testis and ovary.</p></caption><graphic xlink:href=\"fphys-11-00754-g002\"/></fig><table-wrap id=\"T4\" position=\"float\"><label>TABLE 4</label><caption><p>Distribution of these identified miRNAs in length.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Length</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Unique miRNA</bold></td><td valign=\"top\" align=\"right\" rowspan=\"1\" colspan=\"1\"><bold>Percentage</bold></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"right\" rowspan=\"1\" colspan=\"1\">4.36</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td valign=\"top\" align=\"right\" rowspan=\"1\" colspan=\"1\">6.35</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td valign=\"top\" align=\"right\" rowspan=\"1\" colspan=\"1\">6.35</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">93</td><td valign=\"top\" align=\"right\" rowspan=\"1\" colspan=\"1\">16.88</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">22</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">266</td><td valign=\"top\" align=\"right\" rowspan=\"1\" colspan=\"1\">48.28</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">76</td><td valign=\"top\" align=\"right\" rowspan=\"1\" colspan=\"1\">13.79</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13</td><td valign=\"top\" align=\"right\" rowspan=\"1\" colspan=\"1\">2.36</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">25</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"right\" rowspan=\"1\" colspan=\"1\">1.09</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"right\" rowspan=\"1\" colspan=\"1\">0.54</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">All</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">551</td><td valign=\"top\" align=\"right\" rowspan=\"1\" colspan=\"1\">100.00</td></tr></tbody></table></table-wrap></sec><sec id=\"S3.SS4\"><title>Integrated Analysis of DEMs and DEGs</title><p>A total of 9,311 DEGs under the control of 47 miRNAs were identified, and of these, 5,600 were positively regulated, and 3,711 were negatively regulated. In the KEGG functional enrichment, by percentage, we identified the KEGG pathway that needs to be focused on in the joint analysis (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>). Approximately 41 DEGs were assigned to 37 signal pathways (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>), and DEMs were involved in 23 signal pathways (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>). We identified 41 miRNA–mRNA interaction pairs with 24 DEMs targeting 15 DEGs (<xref ref-type=\"supplementary-material\" rid=\"TS7\">Supplementary Table S7</xref>).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Integrated analysis results of DEMs and DEGs. <bold>(A)</bold> Percent of up-/down-expressed mRNAs associated with differentially expressed miRNAs in KEGG enrichment. “Up-regulated” means that these unigenes were higher expressed in the testes comparing to the ovaries, and “down-regulated” means that these unigenes were lower expressed in the testes comparing to the ovaries; <bold>(B)</bold> clustering and pathway distribution of 41 differentially expressed mRNAs; <bold>(C)</bold> clustering and pathway distribution of 41 differentially expressed miRNAs.</p></caption><graphic xlink:href=\"fphys-11-00754-g003\"/></fig><p>Different expression of miRNAs with their predicted target genes was explored for cognate mRNA targets in their respective unigenes list in order to profile miRNA–mRNA functional interactions. These identified 41 miRNA–mRNA pairs were involved in sex differentiation and steroid hormone biosynthesis (<xref ref-type=\"supplementary-material\" rid=\"TS7\">Supplementary Table S7</xref>). Among these candidate miRNA–mRNA interaction pairs, each miRNA can target one or more genes, whereas each gene can be targeted by one or more miRNAs, which indicated that there were complex regulatory networks between miRNAs and gene mRNAs (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). For example, miR-2187-3p, miR-24-3p, miR-181a, miR-181-5p, miR-101a, miR-218–5p, miR-217, and miR-7133 can target <italic>Hsd11</italic>β<italic>2</italic>, whereas <italic>Dmrt1</italic> was targeted by miR-129-5p, miR-27b-3p, miR-27e, miR-152, miR-27b-3p, miR-17-3p, and miR-460.</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Proposed network of putative interactions between miRNAs and mRNAs in the gonad development. The regulation network of miRNAs and mRNAs involved in sexual development and maintenance is illustrated by Cytoscape. Pink represents miRNAs, and blue indicates their target genes.</p></caption><graphic xlink:href=\"fphys-11-00754-g004\"/></fig></sec><sec id=\"S3.SS5\"><title>Validation of miRNA and mRNA Expression Using qRT-PCR</title><p>To verify the reliability and accuracy of the RNA-Seq results, we randomly selected eight genes and eight miRNAs to examine their expression levels between testis and ovary using qRT-PCR (<xref ref-type=\"supplementary-material\" rid=\"TS6\">Supplementary Table S6</xref> and <xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>). Expression results of genes indicated that <italic>Amh</italic>, <italic>Cyp11a1</italic>, <italic>Cyp17a1</italic>, <italic>Dmrt1</italic>, <italic>Dmrt2</italic>, <italic>Hira</italic>, and <italic>Hsd11b2</italic> were more highly expressed in the testis, whereas <italic>Sox3</italic> was more highly expressed in the ovary. Expression analysis of miRNAs indicated that miR-196a, miR-26a-5p, and miR-375-3p were up-regulated, and miR-129b-5p, miR-18a-5p, miR-30d-5p, and let-7i-3p were down-regulated. The qRT-PCR results for these eight miRNAs and eight mRNAs were similar to the RNA-Seq data.</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Validation of miRNAs and mRNAs expression between testis and ovary. Quantitative RT-PCR analysis of eight gene mRNAs and eight miRNAs selected randomly from RNA-Seq results between testis and ovary. All data were shown as mean ± SD. Student <italic>t</italic> test was used to identify significant differences. *<italic>P</italic> < 0.05 represents a statistically significant difference.</p></caption><graphic xlink:href=\"fphys-11-00754-g005\"/></fig></sec></sec><sec id=\"S4\"><title>Discussion</title><p>In this study, using a high-throughput sequencing approach, we performed RNA-Seq analysis to study the expression of miRNAs and mRNAs in the gonads. The aim of the present work was to identify differentially expressed mRNAs and miRNAs between testes and ovaries and to predict miRNA possible targets, as well as to discover possible mechanisms responsible for sexual dimorphism in gonads.</p><p>In this study, <italic>de novo</italic> assembly revealed 50,082 non-redundant genes in the gonad. Based on the functional annotation of non-redundant genes, several sex-related metabolic pathways were summarized from other species. The main metabolic pathways of steroid hormone biosynthesis and oocyte meiosis were identified. Then, a total of 29,752 DEGs were identified between the ovary and testis tissue, including 18,570 up-regulated genes and 11,182 down-regulated genes in the testis compared with the ovary in the discus fish. Among these DEGs, some of the male-enhanced genes included the following the genes. <italic>Dmrt1</italic> (double-sex and mab-3–related transcription factor), which has been demonstrated to be the duplicated homologs of the medaka (<italic>Oryzias latipes</italic>). The <italic>DMY</italic> gene (<xref rid=\"B26\" ref-type=\"bibr\">Matsuda, 2003</xref>), which was first found as a sex-determination gene in non-mammalian vertebrates, has a conservative role in sex determination and differentiation of vertebrates as an ancestral function. <italic>Dmrt2</italic> gene may play a functional role in gonadal differentiation/development and germ cell maturation in the testis (<xref rid=\"B48\" ref-type=\"bibr\">Zhu et al., 2019</xref>). <italic>Cyp17a1</italic> is the qualitative regulator of steroidogenesis (<xref rid=\"B28\" ref-type=\"bibr\">Missaghian et al., 2009</xref>), and its expression level in the testis was higher compared to the ovary, which indicates that <italic>CYP17a1</italic> may be involved in testicular formation during sex differentiation (<xref rid=\"B34\" ref-type=\"bibr\">Sakurai et al., 2008</xref>). <italic>Amh</italic>, a male-specific gene, is derived from the Sertoli cells at the initiation of testis differentiation, participates in steroidogenic pathway to produce testosterone, or negatively controls estrogen production (<xref rid=\"B33\" ref-type=\"bibr\">Poonlaphdecha et al., 2011</xref>). Their expression levels were significantly higher in the testis than in the ovary using the RNA-Seq and qRT-PCR methods, which indicates that they may play key roles in regulating testis development in the discus fish.</p><p>Additionally, some of the female-enhanced genes were identified to be various ovary marker genes. These include the following several genes. <italic>Sox3</italic>, which is highly conserved in fish sex differentiation pathways (<xref rid=\"B40\" ref-type=\"bibr\">Takehana et al., 2014</xref>), plays a key role in regulating gametogenesis and gonad differentiation of vertebrates, and previous studies have shown that it might have more important effect in oogenesis than in spermatogenesis (<xref rid=\"B8\" ref-type=\"bibr\">Bo et al., 2007</xref>). <italic>Cyp19a1</italic>, a cytochrome P450 aromatase, which can catalyze the conversion of androgens to estrogens, plays dual roles in regulating testicular development during the initial period of sexual differentiation and later in ovarian development during the natural sex change in the protandrous black porgy (<italic>Acanthopagrus schlegeli</italic>) (<xref rid=\"B44\" ref-type=\"bibr\">Wu et al., 2008</xref>); In pejerrey (<italic>Odontesthes bonariensis</italic>), the tissue distribution analysis of <italic>cyp19a1</italic> mRNA in adult fish revealed high expression in the ovary, which is involved in the process of ovarian formation (<xref rid=\"B21\" ref-type=\"bibr\">Karube et al., 2007</xref>). <italic>Dnd</italic>, as an ovarian marker, plays an essential role during female gametogenesis and embryo development in pigs (<xref rid=\"B46\" ref-type=\"bibr\">Yang et al., 2012</xref>). However, <italic>Sox9</italic> expressed no difference between the testis and ovary in the discus fish. A possible reason is that <italic>Sox9</italic> participates in an early stage of gonadal development and is identified as an early signal of ovarian differentiation (<xref rid=\"B7\" ref-type=\"bibr\">Berbejillo et al., 2013</xref>).</p><p>We performed a comprehensive annotation and comparative analysis of miRNA using high-throughput sequencing and bioinformatics methods and identified 47 DEMs between the testis and the ovary in the discus fish, including 31 significantly up-regulated miRNAs and 16 significantly down-regulated miRNAs with testis compared to the ovary. In this study, the results from miRNA RNA-Seq sequencing indicated that miR-7641, miR-205a, miR-181a-5p, miR-143-3p, miR-145-3p, and miR-129-5p were differentially expressed between testis and ovary in the discus fish. MicroRNAs play crucial roles in a variety of biological processes via regulating expression of their target genes at the mRNA level. There is increasing evidence that miRNAs play an important role in regulating biological process, whereas miRNAs targeting mRNA are a key part of understanding their role in gene regulation networks (<xref rid=\"B17\" ref-type=\"bibr\">Jing et al., 2014</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Zhang et al., 2016</xref>). In this study, 41 miRNA–mRNA interaction pairs were identified with 15 DEGs targeted by 24 DEMs.</p><p>MicroRNAs play crucial roles in a variety of biological processes via regulating expression of their target genes at the mRNA level. The miRNA–mRNA interaction pair with miRNA targeting mRNA is a key part of understanding miRNA function. In this study, 41 miRNA–mRNA interaction pairs were identified with 15 DEGs targeted by 24 DEMs using miRNA target prediction method. In these pairs, miR-2187-3p, miR-24-3p, miR-181a, miR-181-5p, miR-101a, miR-218–5p, miR-217, and miR-7133 can target <italic>Hsd11</italic>β<italic>2</italic>, which is involved in the steroid hormone biosynthesis pathway, indicating that these miRNAs might play an important role in regulating steroid hormone biosynthesis; miR-181a can also target <italic>Hsd17</italic>β<italic>10</italic>, which is involved in the steroid hormone biosynthesis pathway, and both were down-regulated in testes, indicating that miR-181a might exert different functions on regulating steroid hormone biosynthesis between ovaries and testes. Otherwise, previous studies showed that <italic>Dmrt1</italic> was predominantly expressed in spermatogonia, spermatocytes, and spermatids, as well as in Sertoli cells, indicating that <italic>Dmrt1</italic> plays an important role in spermatogenesis in <italic>Halobatrachus didactylus</italic> (<xref rid=\"B43\" ref-type=\"bibr\">Ubeda-Manzanaro et al., 2014</xref>). In the discus fish, <italic>Dmrt1</italic> was mainly expressed in the testes, whereas it was weakly expressed in the ovaries, and the <italic>Dmrt1</italic> gene was targeted by miR-129-5p, miR-27b-3p, miR-27e, miR-152, miR-27b-3p, miR-17-3p, and miR-460 in our miRNA target prediction data, which indicates that these might be involved in regulating spermatogenesis and testis development of the discus fish. Additionally, miR-129-5p was highly expressed in the ovary, was weakly expressed in the testis, and could target <italic>Dmrt1</italic>, GHR (growth hormone receptor), and RERG (ras-related and estrogen-regulated growth inhibitor), indicating that miR-129-5p might play important roles in regulating growth development and physiological function of the ovary in the discus fish. Previous studies have shown that <italic>zp3</italic> is found on the extracellular matrix of oocytes and acts as a receptor for mammalian sperm binding. In this study, we found that <italic>zp3</italic> can be targeted by miR-7641 and miR-205a in the miRNAs target prediction data, and miR-7641 and miR-205a were lowly expressed in the ovaries of the discus fish, whereas they were highly expressed in the testes, which indicates that lower expression of the two miRNAs in the ovaries might be necessary for expression of the <italic>zp3</italic> gene, playing an important role in regulating the biological process of the ovary binding sperm. Therefore, in the discus fish, miRNAs can play important roles in regulating steroid hormone synthesis, gonadal physiological function and sex development by regulating their target genes. However, the real function of these miRNAs needs to be confirmed in the gonad of the discus fish in the further studies.</p></sec><sec id=\"S5\"><title>Conclusion</title><p>In our study, we found some DEGs and miRNAs between the ovary and testis and predicted target relations between genes and miRNAs using miRNA target prediction methods, and these DEGs and miRNAs were involved in regulating gonadal development, gametogenesis, and physiological function of ovary and testis. These results can help us further understand the mechanism of gonad development between female and male discus fish and provide data for further study of the sex development of the discus fish in the future.</p></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>The datasets generated for this study can be found in the Sequence Read Archive database (SRP148426).</p></sec><sec id=\"S7\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by Laboratory animal—Guideline for ethical review of Animal Welfare of China (GB/T 35892-2018) and Animal Ethics Committee of Shanghai Ocean University (SHOU-DW-2017-039).</p></sec><sec id=\"S8\"><title>Author Contributions</title><p>ZC and JG designed the study. YF and ZX performed the bioinformatics analysis and verification experiments, and wrote the manuscript. BW provided important suggestions about the manuscript writing and collected the samples. All authors reviewed the manuscript.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> The Illumina HiSeq 2500 sequencing analysis was supported by the Key Project of Developing Agriculture through Science and Technology of Shanghai Municipal Agricultural Commission (2015–2019), China; the qRT-PCR analysis for mRNAs and miRNAs was supported by the China Postdoctoral Science Foundation (2017M621433), and the Doctoral Scientific Research Foundation of Shanghai Ocean University (A2-0203-17-100306). Funding agencies played no role in study design, data collection, analysis, interpretation of results, or writing the manuscript.</p></fn></fn-group><ack><p>We thank LC-Bio (Hangzhou, China) for carrying out the Illumina RNA-Seq sequencing of miRNAs and mRNA libraries of the testes and ovaries in the discus fish. This manuscript has been released as a pre-print at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.biorxiv.org/content/10.1101/492264v1\">https://www.biorxiv.org/content/10.1101/492264v1</ext-link> (YF, ZX, ZC, BW, and JG).</p></ack><fn-group><fn id=\"footnote1\"><label>1</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.mirbase.org/cgi-bin/browse.pl\">http://www.mirbase.org/cgi-bin/browse.pl</ext-link></p></fn><fn id=\"footnote2\"><label>2</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://trinityrnaseq.sourceforge.net/\">http://trinityrnaseq.sourceforge.net/</ext-link></p></fn><fn id=\"footnote3\"><label>3</label><p><ext-link ext-link-type=\"ftp\" xlink:href=\"ftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/nr.gz\">ftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/nr.gz</ext-link></p></fn><fn id=\"footnote4\"><label>4</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi\">http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi</ext-link></p></fn><fn id=\"footnote5\"><label>5</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/sra/SRP148426\">https://www.ncbi.nlm.nih.gov/sra/SRP148426</ext-link></p></fn></fn-group><sec id=\"S11\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fphys.2020.00754/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fphys.2020.00754/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"FS1\"><label>FIGURE S1</label><caption><p>Conservative analysis of the non-redundant transcripts between species by comparing to the NR database.</p></caption><media xlink:href=\"Image_1.tif\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS2\"><label>FIGURE S2</label><caption><p>Functional classification of assembled unique sequences based on gene ontology (GO) terms: molecular function, cellular component, and biological process.</p></caption><media xlink:href=\"Image_2.tif\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS3\"><label>FIGURE S3</label><caption><p>Functional categories of KOG database in discus fish.</p></caption><media xlink:href=\"Image_3.tif\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS4\"><label>FIGURE S4</label><caption><p>KEGG Pathway Classification of the gonad in discus fish.</p></caption><media xlink:href=\"Image_4.tif\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS5\"><label>FIGURE S5</label><caption><p>Volcano plot of different expression genes between testis and ovary.</p></caption><media xlink:href=\"Image_5.tif\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS6\"><label>FIGURE S6</label><caption><p>Venn diagram of different expression genes between testis and ovary. The 31 and 16 showed that they were expressed highly in testis and ovary, respectively.</p></caption><media xlink:href=\"Image_6.tif\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS1\"><label>TABLE S1</label><caption><p>miRNA and mRNA Primers used in this study for qRT-PCR validation.</p></caption><media xlink:href=\"Table_1.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS2\"><label>TABLE S2</label><caption><p>The differential expression mRNA between testis and ovary.</p></caption><media xlink:href=\"Table_1.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS3\"><label>TABLE S3</label><caption><p>GO functional annotation of DEGs for testis and ovary.</p></caption><media xlink:href=\"Table_1.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS4\"><label>TABLE S4</label><caption><p>KEGG enrichment annotation of DEGs for testis and ovary.</p></caption><media xlink:href=\"Table_1.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS5\"><label>TABLE S5</label><caption><p>The differential expression miRNA between testis and ovary.</p></caption><media xlink:href=\"Table_1.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS6\"><label>TABLE S6</label><caption><p>The data for qRT-PCR and RNA-Seq of mRNAs and miRNA.</p></caption><media xlink:href=\"Table_1.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS7\"><label>TABLE S7</label><caption><p>41 miRNA-mRNA interaction pairs involved in sex development and maintenance between testis and ovary comparisons, respectively.</p></caption><media xlink:href=\"Table_1.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ambros</surname><given-names>V.</given-names></name></person-group> (<year>2004</year>). <article-title>The functions of animal microRNAs.</article-title>\n<source><italic>Nature</italic></source>\n<volume>431</volume>\n<fpage>350</fpage>–<lpage>355</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature02871</pub-id>\n<pub-id pub-id-type=\"pmid\">15372042</pub-id></mixed-citation></ref><ref id=\"B2\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ashburner</surname><given-names>M.</given-names></name><name><surname>Ball</surname><given-names>C. 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] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Clin Pract Epidemiol Ment Health</journal-id><journal-id journal-id-type=\"iso-abbrev\">Clin Pract Epidemiol Ment Health</journal-id><journal-id journal-id-type=\"publisher-id\">CPEMH</journal-id><journal-title-group><journal-title>Clinical Practice and Epidemiology in Mental Health : CP & EMH</journal-title></journal-title-group><issn pub-type=\"epub\">1745-0179</issn><publisher><publisher-name>Bentham Science Publishers</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32874190</article-id><article-id pub-id-type=\"pmc\">PMC7431701</article-id><article-id pub-id-type=\"publisher-id\">CPEMH-16-125</article-id><article-id pub-id-type=\"doi\">10.2174/1745017902016010125</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Clinical Practice Epidemiology in Mental Health</subject></subj-group></article-categories><title-group><article-title>Implementing WHO-Quality Rights Project in Tunisia: Results of an Intervention at Razi Hospital</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Carta</surname><given-names>Mauro Giovanni</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref><xref rid=\"cor1\" ref-type=\"corresp\">*</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ghacem</surname><given-names>Rym</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Milka</surname><given-names>Myriam</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Moula</surname><given-names>Olfa</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Staali</surname><given-names>Nidhal</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Uali</surname><given-names>Uta</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Bouakhari</surname><given-names>Ghassene</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mannu</surname><given-names>Monica</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Refrafi</surname><given-names>Rym</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yaakoubi</surname><given-names>Souha</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Moro</surname><given-names>Maria Francesca</given-names></name><xref ref-type=\"aff\" rid=\"aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Baudel</surname><given-names>Marie</given-names></name><xref ref-type=\"aff\" rid=\"aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>Vasseur-Bacle</surname><given-names>Simon</given-names></name><xref ref-type=\"aff\" rid=\"aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Drew</surname><given-names>Natalie</given-names></name><xref ref-type=\"aff\" rid=\"aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>Funk</surname><given-names>Michelle</given-names></name><xref ref-type=\"aff\" rid=\"aff5\">5</xref></contrib><aff id=\"aff1\"><label>1</label>Department of Medical Sciences and Public Health, <institution>University of Cagliari</institution>, Cagliari, <country>Italy</country></aff><aff id=\"aff2\"><label>2</label>Razi Hospital, <addr-line><city>Tunis</city></addr-line>, <country>Tunisia</country></aff><aff id=\"aff3\"><label>3</label>Mental Health Departement ,University Hospital Mongi Slim, Tunis, Tunisia</aff><aff id=\"aff4\"><label>4</label>Mailman School of Public Health, Columbia University, New York, NY, USA</aff><aff id=\"aff5\"><label>5</label>Department of Public Health, WHO, Geneva, Switzerland</aff><aff id=\"aff6\"><label>6</label><institution>WHO Collaborating Centre for Mental Health Lille</institution>, Lille, France</aff></contrib-group><author-notes><corresp id=\"cor1\"><label>*</label>Address correspondence to this author at the Department of Medical Sciences and Public Health, University of Cagliari, Cagliari, Italy; Tel: +39 070609349; Fax: +39 0706093498; E-mails: <email xlink:href=\"mgcarta@tiscali.it\">mgcarta@tiscali.it</email>, <email xlink:href=\"maurogcarta@mgcarta.com\">maurogcarta@mgcarta.com</email></corresp></author-notes><pub-date pub-type=\"epub\"><day>30</day><month>7</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>16</volume><issue>Suppl-1</issue><fpage>125</fpage><lpage>133</lpage><history><date date-type=\"received\"><day>23</day><month>2</month><year>2020</year></date><date date-type=\"rev-recd\"><day>04</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>04</day><month>4</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 Carta <italic>et al</italic>.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Carta</copyright-holder><license license-type=\"open-access\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/legalcode\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: <uri xlink:href=\"https://creativecommons.org/licenses/by/4.0/legalcode\">https://creativecommons.org/licenses/by/4.0/legalcode</uri>. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract><sec><title>Background:</title><p>The aims were: 1) to measure the attitudes of learners (and future trainers) before and after a course on WHO-Quality Rights (QR); 2) to evaluate a psychiatric ward, by previously trained staff on QR, comparing it with a previous evaluation and discussing an improvement plan.</p></sec><sec><title>Methods:</title><p>1) Training sample: 19 subjects (8 males), 41.4±10.6 years, including jurists/lawyers, health professionals, and experts.</p><p>The QR team developed the 26-item tool to assess the knowledge and attitudes of participants.</p><p>2) Evaluation of quality of care and respect for human rights in the ward was carried out on 20 staff representatives, 20 family members and 20 users with QRToolkit.</p></sec><sec><title>Results:</title><p>1) Learning in QR has partially changed the knowledge and attitudes of trained people.</p><p>2) The evaluation shows significant delays in the implementation of the rights advocated by the United Nations Convention on the Human Rights of Persons with Disabilities (CRPD). In Themes 1, 3, 4 and 5, the evaluation shows no differences compared to 2014, but in Theme 2, the level was lower than four years before.</p></sec><sec><title>Conclusion:</title><p>The scarcity of resources due to the economic crisis that Tunisia is going through, cannot be considered the only cause of the delays highlighted. However, it is likely that in a context of uncertainty for the future, scarcity of resources and a decrease in staff (<italic>i.e</italic>., professionals dedicated to psychosocial intervention) may have demotivated the team towards recovery. The improvement in knowledge and attitudes of many staff members after the training may open future positive scenarios.</p></sec></abstract><kwd-group kwd-group-type=\"author\"><title>Keywords</title><kwd>WHO Quality rights project</kwd><kwd>Human rights</kwd><kwd>Mental health</kwd><kwd>Psychosocial intervention</kwd><kwd>Disabilities</kwd><kwd>Degrading treatment</kwd></kwd-group></article-meta></front><body><sec sec-type=\"intro\" id=\"sec1\"><label>1</label><title>INTRODUCTION</title><p>The WHO Quality Rights project (QR) [<xref rid=\"r1\" ref-type=\"bibr\">1</xref>] aims to implement the United Nations Convention on the Human Rights of Persons with Disabilities (CRPD) (UN 2006) [<xref rid=\"r2\" ref-type=\"bibr\">2</xref>, <xref rid=\"r3\" ref-type=\"bibr\">3</xref>], in the field of psychosocial disability. Its purpose is “to improve access to quality mental health and social services and to promote the rights of people with mental health conditions, and psychosocial, intellectual and cognitive disabilities” [<xref rid=\"r1\" ref-type=\"bibr\">1</xref>].</p><p>Tunisia signed (2007) and ratified (2008) the CRPD [<xref rid=\"r4\" ref-type=\"bibr\">4</xref>], aiming to improve the level of respect for the human rights of people with disabilities (including psychosocial ones).</p><p>The project “Tunisia and Sardinia in support of the quality of human rights of people with psychosocial disabilities”, funded by the Sardinian Cooperation, aims to support the application of the QR program in Tunisia. This project conducted a pilot intervention to train a group of professionals, human rights experts and people with experience in psychosocial disability, with the principles of CRPD and the use of instruments of the QR project (“ToolKit”) [<xref rid=\"r5\" ref-type=\"bibr\">5</xref>] through an intensive one-week course in Tunisia. The purpose was to create the first group of trainers available for future experience.</p><p>The participants of the intensive course completed a questionnaire, before and at the end of the course; the questionnaire evaluated the participant's knowledge and attitudes regarding human rights in mental health and the CRPD. The group of training participants then conducted a standardized assessment of Quality of Care focused on compliance with human rights in a Razi Hospital ward in accordance with QR rules and application of the QR ToolKit. The group then discussed the results with the staff of the ward and hypothesized possible implementation plans.</p><p>The aim of the study was to measure the knowledge and attitudes of the training participants (and future trainers) before and after the course, present the results of the evaluation of the psychiatric ward, compare them with a similar evaluation conducted four years earlier and discuss the results and differences between the two assessments over time in the light of the state of the country’s public health system and socio-economic conditions.</p></sec><sec sec-type=\"methods\" id=\"sec2\"><label>2</label><title>METHODS</title><sec><label>2.1</label><title>Setting</title><p>The Razi hospital is the only psychiatric hospital in Tunisia. It guarantees 6 beds x 100,000 inhabitants of the country (WHO 2009). It is estimated that Razi Hospital is supported by about 50% of total spending on mental health, which in turn would represent only 1% of total health expenditure, representing 6.4% of the Gross Domestic Product (GDP) [<xref rid=\"r4\" ref-type=\"bibr\">4</xref>]. Tunisia's mental health policies were established in 1992 and have undergone some changes thanks to a specific law of 2004 [<xref rid=\"r4\" ref-type=\"bibr\">4</xref>]. The guidelines prefigured two components of care, one in the hospital and the other integrating mental health care into primary health care, to guarantee fair access to mental health services for the majority of citizens in the community. The law provides for the development of human resources, protection of users' human rights, support and promotion, quality improvement and a monitoring system in the field of mental health. However, it involves neither users nor families (in contrast to the CRPD), nor does it refer to the methods of financing. Also, the Tunisian legislator has not addressed the problem of reducing the part of psychiatric inpatient care in favor of developing community care, nor have adequate resources been allocated for this purpose.</p><p>According to the 2009 WHO report [<xref rid=\"r4\" ref-type=\"bibr\">4</xref>], there are 16 public outpatient mental health facilities in the country, of which 13% are for children and adolescents only. These facilities treat around 1.000 users per 100,000 inhabitants of the community (only considering public services) in a year. All outpatient facilities, with the exception of the outpatient clinic connected to the psychiatric hospital, provide follow-up assistance in the community, while there are no mobile mental health clinic teams. In terms of available interventions, few users (less than 20%) have received one or more psychosocial interventions in the last year, given the high number of patients involved and the limited number of services. The data collected in 2009 does not seem to have currently improved. In recent years, staff involved in rehabilitation therapy in Razi Hospital also progressively decreased, including the mental health facilities; it was not sometimes available one psychotropic drug of each principal therapeutic class (antipsychotic, antidepressant, mood stabilizer, anxiolytic and antiepileptic drugs) nor in a nearby pharmacy. With regards to the accessibility to pharmacotherapy (in general), the Tunisian media often complained of shortcomings in the months before the assessment.</p><p>Professional staff complains of a gradual decrease in resources that has led to a reduction in the number of interventions in recent years.</p><p>There are no community residential facilities available in the country, but only protected homes for people with mental disabilities without family support, whose capacity (two hundred beds) has long been insufficient.</p><p>Organizations of professionals and scientific societies have repeatedly expressed the need for a renewal low related to mental health as well as the recruitment of more human resources in mental health and better availability of medications.</p><p>For this reason, it appeared useful to compare the data of this study with the results of an evaluation carried out in 2014 in the same hospital [<xref rid=\"r5\" ref-type=\"bibr\">5</xref>].</p></sec><sec id=\"sec2.2\"><label>2.2</label><title>Design of the Study</title><p>The study adopts an observational methodology. Improvement of knowledge about CRPD and possible modifications of the attitude of those who participated in the intensive training on CRPD and human rights in mental health was measured with a before-after comparison.</p><p>Evaluation of the Razi hospital using the Quality Rights Tool Kit was compared with that obtained by the evaluation conducted in 2014 of the same Razi hospital using the same QualityRight Tool Kit [<xref rid=\"r6\" ref-type=\"bibr\">6</xref>].</p></sec><sec id=\"sec2.3\"><label>2.3</label><title>Phases and Timing of the Action</title><p>The general timing of the project was established during a preliminary meeting in November 2017, between the team of the University of Cagliari and the team of the two Tunisian units, representing the Razi University Hospital of Manouba - Tunis and the CHU Mongi Slim La Marsa Hospital.</p><p>The World Health Organization, as an external partner to the project, was to provide two trainers (SVB from the WHO Collaborative Center in Lille, and MB, a WHO intern in Geneva) who would conduct the training together MGC and one expert for the discussion of the results (MFM from Columbia University).</p><p>The first operational phase of the study was the implementation of training in Tunis (from 12 to 17 February 2018).</p><p>The course had the general purpose of training participants on the principles of the CRPD and on the legal, social and health implications of applying the same convention. Also proposed on a practical level was the dissemination of knowledge on the use of tools (WHO-Quality Rights Tool Kit [<xref rid=\"r5\" ref-type=\"bibr\">5</xref>]) developed by WHO for the implementation of plans to improve the quality of rights in the practice of care services in mental health. This would allow the implementation of the subsequent phases of the program. As scheduled, 19 learners participated in the course: professionals (psychiatrists, psychologists, nurses, occupational therapists, speech therapists) from the two collaborative centers (Razi and La Marsa) as well as two jurists and two forensics from the University of Tunis, and two people who had experience in treating psychosocial disability.</p><p>The next phase of the study was the completion of the evaluation of an inpatient unit of Razi Hospital, which took place through an accreditation visit of the structure and interviews with staff, users and family members. The evaluation phase was prepared from 2 to 7 June 2018. The visit to the facilities was conducted by the project leader (MGC) with WHO staff (SVB and MFM) in collaboration with staff and family representatives. Data collection through the QualityRights Tool interview was conducted in the period from 1 July to 7 July 2018.</p><p>From 12 to 17 August, during a new work meeting, the results were codified, and two discussion sessions were held with staff, Tunisian assessment experts, WHO staff and family members. In the following months, the report was prepared and the discussion continued via Skype meetings and e-mails.</p></sec><sec id=\"sec2.4\"><label>2.4</label><title>Tools</title><p>The tool used to verify the knowledge and attitudes of participants in the training in human rights and the CRPD was a questionnaire developed by the team of the World Health Organization that deals with the QualityRights study [<xref rid=\"r5\" ref-type=\"bibr\">5</xref>]. The questionnaire investigates the knowledge of the CRPD in general and the realization of the same in the field of mental health through 26 multiple-choice questions, The answers to the questionnaire were: I totally agree, I agree, I am neutral, I disagree and I totally disagree. The questionnaire was administered before the start of the training and at the end, so as to highlight the impact that the training had on learners’ knowledge and attitude.</p><p>As for the assessment of the unit of the Razi Hospital, the tool used was the QualityRights Toolkit [<xref rid=\"r5\" ref-type=\"bibr\">5</xref>]. The QualityRights Toolkit aims to support countries in assessing and improving the quality and respect for human rights in mental health and social care facilities. The QualityRights Toolkit contains 5 themes, taken from the CRPD, which are:</p><p>(1) The right to an adequate standard of living (Article 28 of the CRPD).</p><p>(2) The right to enjoy the highest achievable standard of physical and mental health (Article 25 of the CRPD).</p><p>(3) The right to exercise the legal capacity and the right to freedom and security of the person (Articles 12 and 14 of the CRPD).</p><p>(4) The right not to be subjected to torture or cruel, inhuman or degrading treatment or punishment, or to exploitation, violence, and abuse (Articles 15 and 16 of the CRPD).</p><p>(5) The right to live independently and be included in the community (Article 19 of the CRPD).</p><p>Each of the themes/rights in the toolkit is then divided into a series of standards, which in turn are divided into a series of criteria. The criteria are the basis for the quality assessment and respect for human rights. It is by means of criteria that the situation in the structures is assessed, through interviews, observations and reviews of the documentation. The evaluation of each criterion allowed those who carried out the evaluation to determine if a certain standard was reached. The standards, in turn, helped to determine if the general theme was implemented. The QualityRights Toolkit also provides detailed instructions on how to carry out the evaluation and how to report the results obtained; in fact, it provides the evaluation tools (the WHO QualityRights interview tool and the WHO QualityRights tool for document review and observation) and the tabs for the report. The interviews carried out in Razi hospital during 2018 took place in French with an operator who, if necessary, acted as a French / Arab interpreter. The interviews carried out in 2014 assessment took place in Arabic only.</p></sec><sec id=\"sec2.5\"><label>2.5</label><title>Sample</title><p>The training sample was composed of 8 males and 11 females, whose average age was 41.4 ± 10.6 years. Participants included: 2 jurists/lawyers, 1 trainer, 2 managers, 8 health professionals, 4 university teachers, 2 attenders with both health professional and university teacher profiles. Each participant was free not to complete the questionnaire without having to present any justifications.</p><p>As regards the sample related to the evaluation of the Razi hospital, 20 random staff representatives (doctors, nurses, cleaning staff), 20 representatives of family members and 20 users were interviewed, for a total of 60 people. In the evaluation carried out in 2014, the sample was composed of 35 users, 18 representatives of family members and 35 representatives of the staff, for a total of 88 people.</p></sec><sec id=\"sec2.6\"><label>2.6</label><title>Data Analysis</title><p>The analysis of the results on the questionnaire administered to participants in the training was performed with a one-way ANOVA statistical analysis. This analysis made it possible to calculate which questions showed a difference between the answers to the first administration (before training) with respect to the second (after training) and their statistical significance. The threshold value for the significance level was set at 0.05. The analysis was carried out with the Bonferroni correction since the high number of measures increased the probability of alpha errors.</p><p>As for the analysis of the results obtained from the evaluation of the Razi hospital with the QualityRights Toolkit, this was reported in the grid provided by the Toolkit.</p><p>The assessments relating to criteria, standards and topics were reported in the results grids with a rating scale divided into 4 levels:</p><list list-type=\"bullet\" id=\"L2\"><list-item><p>Completely achieved (A / F-Achieved Fully): it is evident that the criterion/standard/theme has been totally achieved.</p></list-item><list-item><p>Partially achieved (A / P-Achieved Partially): it is clear that the criterion/standard/theme has been achieved, but improvements are needed.</p></list-item><list-item><p>Problem that is starting to be addressed (A / I-Achievement initiated): it is clear that there has been a commitment to the realization of the criterion/standard/theme, but substantial improvements are needed.</p></list-item><list-item><p>Not started (N / I-Not initiated): there is no evidence that something has been put in place for the realization of the criterion/standard/theme.</p></list-item></list><p>The results obtained in the 2014 evaluation were analyzed in the same way.</p></sec><sec id=\"sec2.7\"><label>2.7</label><title>Ethical Aspects</title><p>The board of the Razi hospital in Tunisia approved this project. Informed consent was obtained from those who agreed to take part in the project.</p></sec></sec><sec sec-type=\"results\" id=\"sec3\"><label>3</label><title>RESULTS</title><p>For each of the 26 items of the questionnaire, Table <bold><xref rid=\"T1\" ref-type=\"table\">1</xref></bold> shows the average score and the standard deviation of answers reported at time T0 (pre-training) and T1 (post-training). The questionnaire was completed by 19 participants before the training, and by 15 participants at the end of the training.</p><p>Participants under training showed a general tendency to greater sensitivity towards the patient's point of view and against the use of coercive practices near the end of the training. Most items show a modification of the scores in this sense. However, a statistically significant before-after difference was achieved only in items: J – People who use mental health services should have the power to decide on their treatments (increased score P=0.004); M – People with psychosocial disabilities need someone to plan all their activities (decreased score P=0.049); N – The opinions of people with psychosocial disabilities should have more weight with regard to their treatments than the views of health professionals (increased score P=0.007); O – It is unacceptable to put pressure on users of a mental health service to take treatment they would not like (increased score P=0.04); T – Controlling users of mental health services is necessary to maintain order (decreased score P=0.014).</p><p>Tables <bold><xref rid=\"T2\" ref-type=\"table\">2</xref></bold>-<bold><xref rid=\"T6\" ref-type=\"table\">6</xref></bold> show the results, divided by theme, obtained following the evaluation of Razi hospital, carried out during 2018. Based on the results obtained in the individual criteria, it was possible to evaluate the score for the standards, and from the scores of the standards, it was possible to obtain the overall score for each theme, <italic>i.e</italic>., the score for specific rights of the CRPD related to each theme. With regards to Theme 1 (Table <bold><xref rid=\"T2\" ref-type=\"table\">2</xref></bold>) “The right to an adequate standard of living” (Article 28 of the United Nations Convention on the Rights of Persons with Disabilities, CRPD), only standard 1.4 (“Service users are given food, safe drinking water and clothing that meet their needs and preferences“) resulted as partially achieved, all other standards resulted initially achieved, except standard 1.7 (“Service users can enjoy fulfilling social and personal lives and remain engaged in community life and activities“) that was ‘not started’. With regards to Theme 2 (Table <bold><xref rid=\"T3\" ref-type=\"table\">3</xref></bold>) “The right to the enjoyment of the highest attainable standards of physical and mental health“ (Article 25 of the CRPD), standard 2.1 (“Facilities are available to everyone who requires treatment and support“) resulted fully achieved. The other standards were initially achieved; standard 2.3 (“Treatment, psychosocial rehabilitation and links to support networks and other services are elements of a service user-driven recovery plan and contribute to a service user’s ability to live independently in the community“) resulted in ‘not started’. Concerning the theme “The right to exercise legal capacity and the right to personal liberty and the security of persons“ (Articles 12 and 14 of the CRPD) (Table <bold><xref rid=\"T4\" ref-type=\"table\">4</xref></bold>), standard 3.4 (“Service users have the right to confidentiality and access to their personal health information“) was found partially achieved, while all the other standards were ‘not initiated’ except for standard 3.3 (“Service users can exercise their legal capacity and are given the support they may require to exercise their legal capacity“) which was partially achieved. As regards Theme 4, “Freedom from torture or cruel, inhuman or degrading treatment or punishment and from exploitation, violence and abuse“ (Articles 15 and 16 of the CRPD) (Table <bold><xref rid=\"T5\" ref-type=\"table\">5</xref></bold>), standard 4.4 (“No service user is subjected to medical or scientific experimentation without his or her informed consent“) was totally achieved, standard 4.3 (“Electroconvulsive therapy, psychosurgery and other medical procedures that may have permanent or irreversible effects, whether performed at the facility or referred to another facility, must not be abused and can be administered only with the free and informed consent of the service user“) was partially achieved; standards 4.1 (“Service users have the right to be free from verbal, mental, physical and sexual abuse and physical and emotional neglect“) and 4.5 (“Safeguards are in place to prevent torture or cruel, inhuman or degrading treatment and other forms of ill-treatment and abuse“) were partially achieved and standard 4.2 (“Alternative methods are used in place of seclusion and restraint as means of de-escalating potential crises“) was ‘not initiated’. All standards included in Theme 5 (“The right to live independently and be included in the community“ (Article 19 of the CRPD) (Table <bold><xref rid=\"T6\" ref-type=\"table\">6</xref></bold>) resulted ‘not initiated’.</p><p>Table <bold><xref rid=\"T7\" ref-type=\"table\">7</xref></bold> shows the synthetic score by theme and the comparison between the scores of 2014 and 2018 at Razi Hospital. In four themes, the scores were identical over time, but in Theme 2 (“The right to enjoyment of the highest attainable standards of physical and mental health“ - Article 25 of the CRPD), the score decreased from partially achieved to initiated.</p></sec><sec sec-type=\"discussion\" id=\"sec4\"><label>4</label><title>DISCUSSION</title><p>Participants of the intensive one-week training on CRPD and human rights in mental health showed a significant improvement in knowledge or attitude, as reflected in a significant change in scores in 5 questions out of 26 of the questionnaire. These 5 questions have in common the participants’ 'beliefs about user control and users' freedom of choice. Although the difference reached statistical significance only in 19% of the questions, an improvement (in some cases at the limits of statistical significance) was also observed in many other questions, or in the clear majority, and it can be said that those who participated in the training are more willing to leave freedom of choice to those who use mental health services. It also appears to have changed to the belief that it is important not to try to control or replace the user since this can often prove harmful.</p><p>The data obtained from the 2018 assessment of the Razi hospital in Tunisia in accordance with QualityRights Toolkit show a condition in which the achievement of the rights declared by the CRPD is only partial or insufficient. Concerning the implementation of Article 28 of the CRPD, that is, the right to an adequate standard of living, the results clearly demonstrate that most of the standards show deficiencies and / or an initial level of achievement. The only standard that has been partially achieved relates to food, water, and clothing provided to users of the facility, while as regards, the standard on social life and participation of users in the community, it has been found that no efforts have yet been made to achieve it. Concerning the implementation of article 25 of the CRPD, that is, the right to enjoy the highest achievable level of physical and mental health, most of the standards are classified as objectives that are starting to be implemented. Only the standard regarding the availability of facilities for all those who require care and support is classified as fully achieved, while the standard of the presence of a plan for recovery and user participation in the drafting of the latter is classified as uninitiated. The achieving of rights provided for in Articles 12 and 14 of the CRPD, <italic>i.e</italic>., the right to exercise legal capacity and the right to personal freedom and personal security, show some delay. In this “theme”, the two standards, the first concerning preferences of users regarding their treatment and the second concerning procedures put in place to avoid detention and treatment without consent, are classified as unrealized. The third standard, regarding legal capacity, is classified as starting to be achieved. Only the last standard, which concerns the right to privacy and access to one's personal health information, is classified as partially achieved. The results concerning Articles 15 and 16 of the CRPD, <italic>i.e</italic>., the right to freedom from torture or from cruel, inhuman or degrading treatment or punishment and from exploitation, violence and abuse, are not univocal. In this theme, the first and last standards are classified as objectives that are being started. There is then a totally realized standard; No user is exploited for medical or scientific experimentation without his or her consent. The standard requiring alternative methods to insulation and restraint in the structure are unrealized. The standard concerning electroconvulsive therapy and its non-abuse is classified as partially realized. Article 19 of the CRPD, <italic>i.e</italic>., the right to live independently and be included in the community, is the one that shows the most negative results. In fact, all standards and all criteria have been classified as unrealized objectives.</p><p>Taken together, the evaluation shows significant delays in the implementation of the rights advocated by the CRPD. If in Themes 1, 3, 4 and 5, the evaluation shows no differences compared to 2014, it showed in Theme 2, an even lower level than four years before; from “achieved partially” in 2014 to “achievement initiated” in 2018 [<xref rid=\"r6\" ref-type=\"bibr\">6</xref>].</p><p>It must be kept in mind that the evaluation team in 2014 was composed in a different way. The evaluation was then carried out only by the structure’s staff, while in 2018, external experts were also involved and, above all, they were able to directly evaluate users and family members. This was not the case during the 2014 assessment and the different compositions may have increased the level of severity of the judgments.</p><p>However, the worsening specifically concerned items that the team members themselves complained of, especially in relation to the difficulty in obtaining drugs of primary necessity and to the decrease in staff that mainly involved people employed in rehabilitation and networking. Hence, the difficulty in the opening to the outside world and problems in the work of social inclusion.</p><p>It cannot be said that the scarcity of resources related to the serious economic and political crisis that Tunisia [<xref rid=\"r7\" ref-type=\"bibr\">7</xref>] is going through can be considered the only cause of the delays highlighted. However, it is likely that in a context of general crisis and uncertainty for the future, the scarcity of resources and the decrease in staff, in particular of professionals dedicated to psychosocial intervention [<xref rid=\"r8\" ref-type=\"bibr\">8</xref>, <xref rid=\"r9\" ref-type=\"bibr\">9</xref>] may have been one of the factors that demotivated the team towards recovery and social inclusion [<xref rid=\"r10\" ref-type=\"bibr\">10</xref>-<xref rid=\"r12\" ref-type=\"bibr\">12</xref>].</p><p>An indirect demonstration of this demotivation emerges from the fact that the staff of Razi hospital has seen in recent years an impoverishment due to the departure of many professionals for jobs abroad.</p><p>An important fact is that the evaluation conducted in 2014 does not appear to have led to any improvement and it may be a demonstration that conducting an assessment without staff training and carrying out improvement plans may be ineffective.</p></sec><sec sec-type=\"conclusions\" id=\"sec5\"><title>CONCLUSION</title><p>The evaluation conducted in 2018 reveals significant delays in the implementation of the rights advocated by the CRPD. In themes 1, 3, 4 and 5, the evaluation shows no differences compared to 2014, but as concerns Theme 2, we find an even lower level than four years before.</p><p>The scarcity of resources related to the serious economic crisis that Tunisia is going through cannot be considered the only cause of the delays highlighted. It is likely that in a context of general crisis and uncertainty for the future, the scarcity of resources and the decrease in staff (in particular of professionals dedicated to psychosocial intervention) may have been one of the factors demotivating the teamwork towards recovery and social inclusion.</p><p>However, the training on Quality Rights appears, at least in part, to have changed the knowledge and attitudes of many staff members, and this may open positive scenarios for the future. The commitment that staff and users have made in this action is another element that shows a desire for improvement.</p><p>The paper does not imply that we were expecting a change in the quality of care of the service, which is not the case because we are aware that assessments can only help to understand the level of the respect of human rights in a done facility and limited training, alone, does not allow to begin a process of change. The results and the needs emerged indicate that there is need of consolidated training throughout Tunisia (with the inclusion of all stakeholders) and specific transformation plans for the services would be required to see the change in human rights respect and quality of care – as was done in Gujarat and as is happening in other countries [<xref rid=\"r13\" ref-type=\"bibr\">13</xref>].</p></sec></body><back><ack><title>ACKNOWLEDGEMENTS</title><p>The authors thank the staff, the users and the family of users of the ward of Razi Hospital involved in the study for their collaboration and helpfulness.</p><p>The authors thank Regione Autonoma di Sardegna for supporting the research.</p></ack><sec sec-type=\"competing-interests\"><title>AUTHORS' CONTRIBUTIONS</title><p>MGC and MFM conceived the project that was immediately shared with RG and RR; MF and ND revised and approved the project.</p><p>All authors have offer substantial contributions to design of the work; to conduction of the training course (except MFM, MM, MF and ND), the intervention at Razi Hospital (except MM, MF and ND), the analysis and interpretation of data; and have drafted and revise the work and approved the last version.</p></sec><sec><title>ETHICS APPROVAL AND CONSENT TO PARTICIPATE</title><p>The study was approved by the Board of The Razi Hospital ,Tunisia with approval no. 11-11-19.</p></sec><sec sec-type=\"competing-interests\"><title>HUMAN AND ANIMAL RIGHTS</title><p>No animals were used in this research. All human research procedures followed were in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2013.</p></sec><sec sec-type=\"competing-interests\"><title>CONSENT FOR PUBLICATION</title><p>Informed consent was obtained from those who agreed to take part in the project.</p></sec><sec sec-type=\"financial-disclosure\"><title>AVAILABILITY OF DATA AND MATERIALS</title><p>The anonymous data base support the Department of Medical Sciences and Public Health of University of Cagliari in responsibility of the Project Leader Mauro Giovanni Carta it can be available upon request and approval by the board of the Razi Hospital, Tunis.</p></sec><sec sec-type=\"competing-interests\"><title>FUNDING</title><p>Regione Autonoma di Sardegna, supported the study (funds by Regional Law 11 April 1996, n. 19 “Rules on cooperation with developing countries and international collaboration”. 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Br J Psychiatry</source><year>2019</year><pub-id pub-id-type=\"pmid\">31218972</pub-id></element-citation></ref></ref-list></back><floats-group><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><title>Results on QualityRights Questionnaire pre and post-training</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>Item</bold>\n</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">Mean±SD <bold>t0</bold></th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">Mean±SD <bold>t1</bold></th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>F</bold>\n</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>P</bold>\n</th></tr></thead><tbody><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">a- Knowledge and understanding of human rights can improve the quality of care in mental health services</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.94±0.22 (N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.93±0.24<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.016<break/>df 1,32,33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.900</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">B – Mental health workers can do a lot to improve the rights of people with mental disorders</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.70±0.45<break/>(N=17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.86±0.33<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,328<break/>df 1,32,33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.258</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">C – People with severe mental disorders should consult their doctor before getting married</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.63±1.17<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.46±0.95<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.208<break/>df 1,32,33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.651</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">D - Much can be improved in mental health services without additional resources</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.00±0.66<break/>(N=18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.07±0.96<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.063<break/>df 1,31,32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.803</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">E – People with dementia should live in structures where people could take care of them</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.15±1.53<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.33±1.19<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.911<break/>df 1,32,33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.098</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">F – People with psychosocial disabilities should not be employed in jobs that require contact with the public</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.00±1.07<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.80±0.90<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.336<break/>df 1,32,33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.566</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">G – Medicines are the most important factor in enabling people with mental disorders to get better</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.31±1.07<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.35±1.28<break/>(N=14)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.010<break/>df 1,31,32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.923</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">H – Taking your medicine is the most important factor in helping people with mental disorders get better</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.61±1.06<break/>(N=18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.94±1.28<break/>(N=13)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.615<break/>df 1,29,30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.339</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">I – We only need to inspire hope once a person has recovered</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.55±1.06<break/>(N=18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.93±0.85<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.072<break/>df 1,31,32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.090</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">J – People who use mental health services should have the power to decide on their treatments</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.26±1.01<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.20±0.65<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.763<break/>df 1,32,33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.004</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">K – Following the advice of other people who have experienced mental disorders is too risky</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.47±0.81<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.93±0.85<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.568<break/>df 1,32,33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.088</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">L – It is important to take tough positions with users of mental health services in order not to be manipulated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.10±1.11<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.53±0.88<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.639<break/>df 1,32,33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.114</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">M – People with psychosocial disabilities need someone to plan all their activities</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.83±0.89<break/>(N=18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.20±0.90<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>4.170</bold><break/>df 1,31,32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.049</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">N – The opinions of people with psychosocial disabilities should have more weight in regards to their treatments than the views of health professionals</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.55±0.68<break/>(N=18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.46±1.14<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>8.377</bold><break/>df 1,32,33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.007</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">O – It is unacceptable to put pressure on users of a mental health service to take treatment they would not like</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.26±1.16<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.93±0.57<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>4.185</bold><break/>df 1,31,32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.049</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">P – People with mental disorders should not have important responsibilities</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.73±1.20<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.20±0.97<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>1.927</bold><break/>df 1,31,32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.175</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Q – When people are unable to communicate, you have to make a decision based on what you think is best for them.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.73±1.20<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.73±1.18<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0001<break/>df 1,31,32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.9999</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">R – Health workers are in the best perspective to understand what they are capable of doing in life</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.57±1.09<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.20±0.97<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>1.063</bold><break/>df 1,31,32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.310</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">S – People with psychosocial disabilities have the right to make their decisions even if I don't agree with them</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.89±0.55<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.13±0.80<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>1.073</bold><break/>df 1,31,32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.308</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">T – Controlling users of mental health services is necessary to maintain order</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.26±1.06<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.33±1.01<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>6.723</bold><break/>df 1,31,32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.014</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">U – The use of isolation and restraint is necessary if users become threatening</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.10±1.16<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.46±1.20<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>2.476</bold><break/>df 1,31,32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.125</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">V – Isolation is not the ideal solution to manage a crisis</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.42±1.26<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.13±1.02<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>3.134</bold><break/>df 1,31,32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.086</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">W- The use of isolation and restraint negatively affects the therapeutic relationship between users of mental health services and staff</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.89±1.11<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.26±0.99<break/>(N=15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>1.023</bold><break/>df 1,31,32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.319</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">X - Locking up a person in a room is acceptable if the person presents a risk of injury or injury to others</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.36±0.92<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.86±1.30<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>1.724</bold><break/>df 1,31,32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.199</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Y – Most people are not harmed if they are put on sedatives to defuse a tense situation</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.89±1.11<break/>(N=19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.20±1.10<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>3.265</bold><break/>df1,31, 32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.080</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Z – Involuntary hospitalization does more good than harm</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.61±1.20<break/>(N=18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.46±1.08<break/>(N=15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.143</bold><break/>df 1,31,32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.708</td></tr></tbody></table></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><title>Theme 1. - The right to an adequate standard of living (Article 28 of the United Nations Convention on the Rights of Persons with Disabilities, CRPD).</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">Standard</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\nScore\n</th><th colspan=\"6\" valign=\"top\" align=\"center\" scope=\"colgroup\" rowspan=\"1\">\nCriterion\n</th></tr></thead><tbody><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">1.1 The building is in good physical condition.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.1.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.1.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.1.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.1.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>N/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">1.2 The sleeping conditions of service users are comfortable and allow sufficient privacy.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.2.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.2.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.2.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.2.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.2.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.2.6</td></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>N/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>N/I</italic>\n</td></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">1.3 The facility meets hygiene and sanitary requirements.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.3.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.3.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.3.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.3.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">1.4 Service users are given food, safe drinking water and clothing that meet their needs and preferences.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.4.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.4.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.4.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.4.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">1.5 Service users can communicate freely, and their right to privacy is respected.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.5.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.5.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.5.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.5.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.5.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>N/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>N/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">1.6 The facility provides a welcoming, comfortable, stimulating environment conducive to active participation and interaction.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.6.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.6.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.6.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.6.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>N/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>N/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">1.7 Service users can enjoy fulfilling social and personal lives and remain engaged in community life and activities.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>N/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.7.1<break/><italic>N/I</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.7.2<break/>A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.7.3<break/><italic>N/I</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.7.4<break/><italic>N/I</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.7.5<break/><italic>N/I</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></table-wrap><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><title>Theme 2. The right to enjoyment of the highest attainable standards of physical and mental health (Article 25 of the CRPD)</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">Standard</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\nScore\n</th><th colspan=\"5\" valign=\"top\" align=\"center\" scope=\"colgroup\" rowspan=\"1\">\nCriterion\n</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\"/></tr></thead><tbody><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">2.1 Facilities are available to everyone who requires treatment and support.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.1.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.1.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.1.3</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">2.2 The facility has skilled staff and provides good-quality mental health services.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.2.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.2.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.2.3</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.2.4</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.2.5</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.4.6</italic></bold>\n</td></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">2.3 Treatment, psychosocial rehabilitation and links to support networks and other services are elements of a service user-driven recovery plan and contribute to a service user’s ability to live independently in the community.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.3.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.3.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.3.3</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.3.4</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.3.5</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.3.6</italic></bold>\n</td></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/I</td></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">2.4 Psychotropic medication is available, affordable and used appropriately.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.4.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.4.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.4.3</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.4.4</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.4.5</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">2.5Adequate services are available for general and reproductive health.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.5.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.5.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.5.3</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.5.4</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.5.5</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>2.5.6</italic></bold>\n</td></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td></tr></tbody></table></table-wrap><table-wrap id=\"T4\" orientation=\"portrait\" position=\"float\"><label>Table 4</label><caption><title>Theme 3. The right to exercise legal capacity and the right to personal liberty and the security of person (Articles 12 and 14 of the CRPD)</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">Standards</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\nScore\n</th><th colspan=\"7\" valign=\"top\" align=\"center\" scope=\"colgroup\" rowspan=\"1\">\nCriterion\n</th></tr></thead><tbody><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">3.1 Service users’ preferences regarding the place and form of treatment are always a priority.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.1.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.1.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.1.3</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">3.2 Procedures and safeguards are in place to prevent detention and treatment without free and informed consent.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.2.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.2.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.2.3</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.2.4</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.2.5</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.2.6</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">3.3 Service users can exercise their legal capacity and are given the support they may require to exercise their legal capacity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.3.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.3.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.3.3</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.3.4</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.3.5</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.3.6</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.3.7</italic></bold>\n</td></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/</td></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">3.4 Service users have the right to confidentiality and access to their personal health information.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.4.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.4.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.4.3</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.4.4</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></table-wrap><table-wrap id=\"T5\" orientation=\"portrait\" position=\"float\"><label>Table 5</label><caption><title>Theme 4. Freedom from torture or cruel, inhuman or degrading treatment or punishment and from exploitation, violence and abuse (Articles 15 and 16 of the CRPD)</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">Standard</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\nScore\n</th><th colspan=\"6\" valign=\"top\" align=\"center\" scope=\"colgroup\" rowspan=\"1\">\nCriterion\n</th></tr></thead><tbody><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">4.1 Service users have the right to be free from verbal, mental, physical and sexual abuse and physical and emotional neglect.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.1.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.1.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.1.3</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.1.4</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.1.5</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">4.2 Alternative methods are used in place of seclusion and restraint as means of de-escalating potential crises.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.2.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.2.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.2.3</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.2.4</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.2.5</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">4.3 Electroconvulsive therapy, psychosurgery and other medical procedures that may have permanent or irreversible effects, whether performed at the facility or referred to another facility, must not be abused and can be administered only with the free and informed consent of the service user.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.3.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.3.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.3.3</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.3.4</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.3.5</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.3.6</italic></bold>\n</td></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">4.4 No service user is subjected to medical or scientific experimentation without his or her informed consent.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.4.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.4.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.4.3</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.4.4</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">4.5 Safeguards are in place to prevent torture or cruel, inhuman or degrading treatment and other forms of ill-treatment and abuse.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.5.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.5.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.5.3</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.5.4</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.5.5</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>4.5.6</italic></bold>\n</td></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td></tr></tbody></table></table-wrap><table-wrap id=\"T6\" orientation=\"portrait\" position=\"float\"><label>Table 6</label><caption><title>Theme 5. The right to live independently and be included in the community (Article 19 of the CRPD)</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><tbody><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">5.1 Service users are supported in gaining access to a place to live and have the financial resources necessary to live in the community.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>5.1.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>5.1.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>5.1.3</italic></bold>\n</td></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">\n5\n.2 Service users can access education and employment opportunities.\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>5.2.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>5.2.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>5.2.3</italic></bold>\n</td></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">5.3 The right of service users to participate in political and public life and to exercise freedom of association is supported.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>5.3.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>5.3.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>5.3.3</italic></bold>\n</td></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td></tr><tr><td rowspan=\"2\" valign=\"top\" align=\"center\" scope=\"row\" colspan=\"1\">5.4 Service users are supported in taking part in social, cultural, religious and leisure activities</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>5.4.1</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>5.4.2</italic></bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>5.4.3</italic></bold>\n</td></tr><tr><td valign=\"top\" colspan=\"1\" align=\"center\" scope=\"row\" rowspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/I</td></tr></tbody></table></table-wrap><table-wrap id=\"T7\" orientation=\"portrait\" position=\"float\"><label>Table 7</label><caption><title>Synthetic score by theme: comparison 2014-2018 at Razi Hspital</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>Theme</bold>\n</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>Description</bold>\n</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>SCORE 2018</bold>\n</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<bold>SCORE</bold>\n<break/>\n<bold>2014</bold>\n</th></tr></thead><tbody><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\nThe right to an adequate standard of living (Article 28 of CRPD)\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\nThe right to the enjoyment of the highest attainable standards of physical and mental health (Article 25 of the CRPD)\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/I</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/P</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\nThe right to exercise legal capacity and the right to personal liberty and the security of person (Articles 12 and 14 of the CRPD)\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A/I</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\nThe right to exercise legal capacity and the right to personal liberty and the security of person (Articles 12 and 14 of the CRPD)\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>A/I</italic>\n</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\nThe right to live independently and be included in the community (Article 19 of the CRPD)\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>N/I</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>N/I</italic>\n</td></tr></tbody></table></table-wrap></floats-group></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Clin Pract Epidemiol Ment Health</journal-id><journal-id journal-id-type=\"iso-abbrev\">Clin Pract Epidemiol Ment Health</journal-id><journal-id journal-id-type=\"publisher-id\">CPEMH</journal-id><journal-title-group><journal-title>Clinical Practice and Epidemiology in Mental Health : CP & EMH</journal-title></journal-title-group><issn pub-type=\"epub\">1745-0179</issn><publisher><publisher-name>Bentham Science Publishers</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32874193</article-id><article-id pub-id-type=\"pmc\">PMC7431702</article-id><article-id pub-id-type=\"publisher-id\">CPEMH-16-180</article-id><article-id pub-id-type=\"doi\">10.2174/1745017902016010180</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Clinical Practice Epidemiology in Mental Health</subject></subj-group></article-categories><title-group><article-title>Arabic Version of the Personality Inventory for the DSM-5 (PID-5) in a Community Sample of United Arab Emirates Nationals</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-0700-5467</contrib-id><name><surname>Coelho</surname><given-names>Olga</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref><xref ref-type=\"corresp\" rid=\"cor1\">*</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0003-1019-7794</contrib-id><name><surname>Pires</surname><given-names>Rute</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref><xref ref-type=\"aff\" rid=\"aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-3582-4179</contrib-id><name><surname>Ferreira</surname><given-names>Ana Sousa</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">2</xref><xref ref-type=\"aff\" rid=\"aff3\">3</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0003-3355-4539</contrib-id><name><surname>Gonçalves</surname><given-names>Bruno</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref><xref ref-type=\"aff\" rid=\"aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0001-6371-3911</contrib-id><name><surname>AlJassmi</surname><given-names>Maryam</given-names></name><xref ref-type=\"aff\" rid=\"aff4\">4</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-5654-0378</contrib-id><name><surname>Stocker</surname><given-names>Joana</given-names></name><xref ref-type=\"aff\" rid=\"aff4\">4</xref></contrib><aff id=\"aff1\">\n<label>1</label>CICPSI, Faculdade de Psicologia, Universidade de Lisboa, <institution>Alameda da Universidade</institution>, <addr-line><postal-code>1649-013 </postal-code><city>Lisboa</city></addr-line>, <country>Portugal</country></aff><aff>\n<target id=\"aff2\" target-type=\"aff\"><sup>2</sup></target>Faculdade de Psicologia, Universidade de Lisboa, <institution>Alameda da Universidade</institution>, <addr-line><postal-code>1649-013 </postal-code><city>Lisboa</city></addr-line>, <country>Portugal</country></aff><aff>\n<target id=\"aff3\" target-type=\"aff\"><sup>3</sup></target>Instituto Universitário de Lisboa (ISCTE-IUL), Business Research Unit, Av. das Forças Armadas, <addr-line><postal-code>1649-026 </postal-code><city>Lisboa</city></addr-line>, <country>Portugal</country></aff><aff id=\"aff4\">\n<label>4</label>College of Natural and Health Sciences, Zayed University, P.O. Box 19282 Dubai, U.A.E</aff></contrib-group><author-notes><corresp id=\"cor1\"><label>*</label>Address correspondence to this author at the CICPSI, Faculdade de Psicologia, Universidade de Lisboa, <institution>Alameda da Universidade</institution>, <addr-line><postal-code>1649-013 </postal-code><city>Lisboa</city></addr-line>, <country>Portugal</country>, E-mail: <email xlink:href=\"ocoelho@edu.ulisboa.pt\">ocoelho@edu.ulisboa.pt</email></corresp></author-notes><pub-date pub-type=\"epub\"><day>30</day><month>7</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>16</volume><fpage>180</fpage><lpage>188</lpage><history><date date-type=\"received\"><day>11</day><month>3</month><year>2020</year></date><date date-type=\"rev-recd\"><day>30</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>31</day><month>5</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 Coelho <italic>et al</italic>.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Coelho</copyright-holder><license license-type=\"open-access\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/legalcode\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (<uri xlink:href=\"https://creativecommons.org/licenses/by/4.0/legalcode\">https://creativecommons.org/licenses/by/4.0/legalcode</uri>). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract><sec><title>Background:</title><p>Section III of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) proposes a model for conceptualizing personality disorders in which they are characterized by impairments in personality functioning and maladaptive personality traits. The Personality Inventory for DSM-5 (PID-5) is a self-report measure that assesses the presence and severity of these maladaptive personality traits.</p></sec><sec><title>Objective:</title><p>The current study examined the reliability and validity of the Arabic version of the Personality Inventory for DSM-5 (PID-5) to measure maladaptive personality traits in the Emirati population of the United Arab Emirates.</p></sec><sec><title>Methods:</title><p>The Arabic version of the PID-5 was administered to a community sample of 1,090 United Arab Emirates nationals (89.5% female and 10.5% male, mean age<italic> = </italic>22.44 years old). The descriptive measures, internal consistency, test-retest reliability, convergent validity with NEO – Five Factor Inventory, as well as PID-5’s factor structure, were all addressed.</p></sec><sec><title>Results:</title><p>The PID-5facets and domains mean scores were higher in the Emirati sample compared to the original US sample. Internal consistency of the PID-5 scales was acceptable to high and test-retest coefficients ranged from 0.84 (facets) to 0.87 (domains). As expected, the five domains of the Arabic version of the PID-5 correlated significantly with all Five-Factor Model domains of personality. Additionally, the Arabic version of the PID-5 confirmed a five-factor structure that resembles the PID-5 domains.</p></sec><sec><title>Conclusion:</title><p>The findings of this study provided initial support for the use of the Arabic version of the PID-5 to assess maladaptive personality traits in the Emirati population of the United Arab Emirates.</p></sec></abstract><kwd-group kwd-group-type=\"author\"><title>Keywords</title><kwd>Personality</kwd><kwd>DSM-5,Personality trait model</kwd><kwd>PID-5</kwd><kwd>United Arab Emirates</kwd><kwd>Psychometric properties</kwd></kwd-group></article-meta></front><body><sec sec-type=\"intro\" id=\"sec1\"><label>1</label><title>INTRODUCTION</title><p>The Diagnostic and Statistical Manual of Mental Disorders (APA) and the International Classification of Mental and Behavioural Disorders (WHO) are currently shifting towards a more evidence-based dimensional conceptualization of Personality Disorders (PDs), as the traditional categorical paradigm has proven to be conceptually and empirically problematic [<xref rid=\"r1\" ref-type=\"bibr\">1</xref>, <xref rid=\"r2\" ref-type=\"bibr\">2</xref>] with limited clinical utility [<xref rid=\"r3\" ref-type=\"bibr\">3</xref>]. This has resulted in many patients being undiagnosed, receiving multiple Personality Disorder (PD) diagnoses, or, most commonly, diagnosed with a PD not otherwise specified [<xref rid=\"r4\" ref-type=\"bibr\">4</xref>].</p><p>A reflection of this was the inclusion of the Alternative DSM-5 Model for Personality Disorders (AMPD) in Section III of the DSM-5 [<xref rid=\"r5\" ref-type=\"bibr\">5</xref>] and more than 200 publications on its main diagnostic criteria: the assessment of impairment in personality function (Criterion A) and the presence of maladaptive personality traits (Criterion B), that followed its publication. The primary measure for the assessment of the AMPD [<xref rid=\"r5\" ref-type=\"bibr\">5</xref>] mal-adaptive traits is provided by The Personality Inventory for the DSM-5 (PID-5) [<xref rid=\"r6\" ref-type=\"bibr\">6</xref>], which is a self-rated inventory that characterizes 25 trait facets organized into five high order domains of personality variation (Negative Affectivity, Detachment, Antagonism, Disinhibition, and Psychoticism).</p><p>The PID-5 psychometric properties have been extensively examined and review studies have consistently shown it to be a reliable measure with internal consistency coefficients ranging from acceptable at the trait facets level to high at the domain trait level [<xref rid=\"r7\" ref-type=\"bibr\">7</xref>], and with the ability to capture individual differences that were stable during four weeks up to four months intervals [<xref rid=\"r8\" ref-type=\"bibr\">8</xref>, <xref rid=\"r9\" ref-type=\"bibr\">9</xref>]. Furthermore, in regards to its factor structure, the PID-5 confirmed a five-factor structure similar to the Five Factor Model (FFM), both in clinical and non-clinical studies and across different countries [<xref rid=\"r10\" ref-type=\"bibr\">10</xref>]. However, researchers also reported that the loading pattern of some trait facets appeared to deviate from the model, such as Suspiciousness that belongs to the Detachment domain, but was more often loaded in Negative affectivity, or Hostility that belongs to domain Negative affectivity, but frequently loaded in the Antagonism domain [<xref rid=\"r11\" ref-type=\"bibr\">11</xref>].</p><p>The PID-5 facets and domains had conceptually and meaningfully converged with other established measures of personality and personality pathology [<xref rid=\"r12\" ref-type=\"bibr\">12</xref>-<xref rid=\"r15\" ref-type=\"bibr\">15</xref>], including The Personality Inventory for the ICD-11 [<xref rid=\"r16\" ref-type=\"bibr\">16</xref>]. Also, a vast body of research has conceptualized the PID-5 trait domains as mal-adaptive extensions of general personality traits and supports the continuum between adaptive and mal-adaptive personality trait models [<xref rid=\"r17\" ref-type=\"bibr\">17</xref>, <xref rid=\"r18\" ref-type=\"bibr\">18</xref>], established by the association between Negative affectivity with Neuroticism, Detachment with Extraversion, Antagonism with Agreeableness and Disinhibition with Consciousness. The relation between Psychoticism and Openness is less clear and debatable [<xref rid=\"r19\" ref-type=\"bibr\">19</xref>].</p><p>Additionally, the PID-5 has proven its ability to capture the DSM-5 Section II PDs categories and symptoms [<xref rid=\"r20\" ref-type=\"bibr\">20</xref>], and other studies claimed its utility for treatment planning [<xref rid=\"r21\" ref-type=\"bibr\">21</xref>], as well as predicting psychosocial impairment [<xref rid=\"r22\" ref-type=\"bibr\">22</xref>].</p><p>The PID-5 has been translated into different languages and cultures and can be found in Arabic [<xref rid=\"r23\" ref-type=\"bibr\">23</xref>], Czech [<xref rid=\"r24\" ref-type=\"bibr\">24</xref>], Danish [<xref rid=\"r25\" ref-type=\"bibr\">25</xref>], Dutch [<xref rid=\"r26\" ref-type=\"bibr\">26</xref>], French [<xref rid=\"r27\" ref-type=\"bibr\">27</xref>], German [<xref rid=\"r28\" ref-type=\"bibr\">28</xref>], Indonesian [<xref rid=\"r29\" ref-type=\"bibr\">29</xref>], Italian [<xref rid=\"r30\" ref-type=\"bibr\">30</xref>], Norwegian [<xref rid=\"r31\" ref-type=\"bibr\">31</xref>], Persian [<xref rid=\"r32\" ref-type=\"bibr\">32</xref>], Polish [<xref rid=\"r33\" ref-type=\"bibr\">33</xref>], Portuguese [<xref rid=\"r8\" ref-type=\"bibr\">8</xref>], Brazilian-Portuguese [<xref rid=\"r34\" ref-type=\"bibr\">34</xref>], Russian [<xref rid=\"r35\" ref-type=\"bibr\">35</xref>], Spanish [<xref rid=\"r36\" ref-type=\"bibr\">36</xref>], and Swedish [<xref rid=\"r37\" ref-type=\"bibr\">37</xref>].</p><p>The translation study of the Arabic PID-5 [<xref rid=\"r23\" ref-type=\"bibr\">23</xref>] was conducted with college students in three Middle-East countries (Bahrain, Kuwait, and Qatar) and is written in Modern Standard Arabic (MSA), which is the formal written expression used in the literature, as well as in the translation of psychological tests, common to all the Arabic speaking countries [<xref rid=\"r38\" ref-type=\"bibr\">38</xref>, <xref rid=\"r39\" ref-type=\"bibr\">39</xref>]. However, the Arabic language is a <italic>diglossic</italic> language [<xref rid=\"r40\" ref-type=\"bibr\">40</xref>, <xref rid=\"r41\" ref-type=\"bibr\">41</xref>] that, beyond the MSA derived from the Classic Arabic, is also comprised of colloquial forms used to orally communicate ideas, feelings, and emotions, but for which there is no written form of expression, resulting in the inability to use it in the translation of psychological tools. The MSA, although useful as a standard form of the Arabic language, carries some limitations such as the use of outdated terms that are no longer used colloquially, and some MSA words might have different meanings across countries [<xref rid=\"r40\" ref-type=\"bibr\">40</xref>, <xref rid=\"r41\" ref-type=\"bibr\">41</xref>]. In a recent lexical study on personality traits, using the MSA in the Arab Levant, the authors reported an under representation of terms to describe some dimensions of general personality, such as Openness [<xref rid=\"r42\" ref-type=\"bibr\">42</xref>], which is related with Fantasy, Aesthetics, Feelings, Actions, Ideas, and Values [<xref rid=\"r43\" ref-type=\"bibr\">43</xref>]. These findings are not surprising considering that these topics, although extremely relevant for the psychological assessment, are more often communicated using the colloquial Arabic forms. Therefore, assuming the generalizability of the Arabic PID-5 [<xref rid=\"r23\" ref-type=\"bibr\">23</xref>], or other translated tests, to all Arabic speaking countries could carry important reliability and validity issues that might be minimized by validity studies, in Arabic speaking clinical and non-clinical samples, for which this study aimed to contribute through the following objectives: (a) to test possible cultural variations between Western and non-Western cultures by comparing the Emirati community sample results as well as the ones obtained in the PID-5 Arabic translation study [<xref rid=\"r23\" ref-type=\"bibr\">23</xref>], with the original test data, (b) to address the PID-5 scales’ internal consistency and test-retest reliability, as the PID-5 traits stability was not addressed in the Arabic translation study [<xref rid=\"r23\" ref-type=\"bibr\">23</xref>], (c) to explore the association between the PID-5 domains with the FFM, measured by the Arabic NEO – Five Factor Inventory (NEO-FFI), [<xref rid=\"r4\" ref-type=\"bibr\">4</xref>, <xref rid=\"r5\" ref-type=\"bibr\">5</xref>] and (d) to examine the PID-5’s factor structure in the Emirati community sample.</p></sec><sec sec-type=\"methods\" id=\"sec2\"><label>2</label><title>METHODS</title><sec id=\"sec2.1\"><label>2.1</label><title>Sample</title><p>The participants were a total of 1,090 volunteers aged between 18 and 57 years old (<italic>M</italic> = 22.44, <italic>SD</italic> = 6.63, 89.5% female, 10.5% male) recruited from Zayed University students and their acquaintances. Test-retest reliability was studied with a sample of 28 students, 85.7% females, 14.3% males, <italic>M<sub>age</sub></italic>= 28.6, <italic>SD </italic>= 9.64. The inclusion criteria were Emirati native Arabic speakers aged 18 years old and above who have completed primary school or higher.</p></sec><sec id=\"sec2.2.\"><label>2.2</label><title>Procedures</title><p>Participation in this study was voluntary and all respondents signed a written informed consent form requesting their participation in the study, the possibility of giving up at any time, and that the data would be used exclusively in a scientific study. The experimental sessions were held collectively and conducted at Zayed University after obtaining approval from the Research Ethics Committee of Zayed University. In the temporal stability study, the interval between the 1<sup>st</sup> and the 2<sup>nd</sup> application was four weeks and data was matched through a code given to the participants in the first session.</p></sec><sec id=\"sec2.3.\"><label>2.3</label><title>Measures</title><p>The Personality Inventory for the DSM-5 (Krueger <italic>et al</italic> [<xref rid=\"r6\" ref-type=\"bibr\">6</xref>], Arabic version by Al-Attiyah <italic>et al</italic>. [<xref rid=\"r23\" ref-type=\"bibr\">23</xref>])</p><p>The PID-5 is a self-report measure composed of 220 items, rated on a four-point Likert scale ranging from 0 (very false or often false) to 3 (very true or often true), that characterizes 25 empirically derived lower level facets grouped into five major domains of mal-adaptive personality variation. Data from the Al-Attiyah <italic>et al</italic>. [<xref rid=\"r23\" ref-type=\"bibr\">23</xref>] study showed that the <italic>Cronbach’s</italic> alphas of the PID-5 scales were moderate to high, ranging from .70 (Manipulativeness) to .93 (Attention seeking) at the facet level, and .92 (Antagonism) to .96 (Detachment) at the domain level.</p><p>NEO-Five Factor Inventory (NEO-FFI, Costa & McCrae [<xref rid=\"r44\" ref-type=\"bibr\">44</xref>], Arabic version by Alansari [<xref rid=\"r45\" ref-type=\"bibr\">45</xref>])</p><p>The NEO-FFI is a measure of the five basic personality factors (Neuroticism, Extraversion, Openness to Experiences, Agreeableness, and Conscientiousness) composed by 60 items rated on a five-point Likert response format, ranging from 0 (strongly disagree) to 4 (strongly agree). The Arabic version of the NEO-FFI [<xref rid=\"r45\" ref-type=\"bibr\">45</xref>] was used, and to prevent validity issues and ensure conceptual equivalence of the measure, a preliminary study was conducted in the Emirati population. Results confirmed a five-factor structure supporting the universality of the FFM. <italic>Cronbach’s</italic> alphas ranged from acceptable .65 (Openness) to high .85 (Neuroticism), in line with the results reported in the US sample, which ranged from .68 to .86 [<xref rid=\"r44\" ref-type=\"bibr\">44</xref>].</p></sec><sec id=\"sec2.4\"><label>2.4</label><title>Data Analysis</title><p>Analysis was conducted with the IBM SPSS Statistics (v.25, SPSS Inc., Chicago, IL). <italic>Cohen’s d</italic> was used as a measure of effect size, in order to study the mean score differences between the Emirati and the original sample [<xref rid=\"r6\" ref-type=\"bibr\">6</xref>].The effect size was considered small when <italic>d</italic> ≤ .20, medium when .20 <<italic> d</italic> ≤ .50, large when .50 <<italic> d</italic> ≤ 1.0, and very large when <italic>d </italic>> 1.0. The internal consistency was measured by <italic>Cronbach’s</italic> alpha, while test-retest and convergent validity analyses were conducted by the <italic>Pearson</italic> coefficient, or <italic>Spearman’s</italic> rank coefficient if the dataset did not follow a normal distribution. Due to the complexity of the personality structure, in which traits present several cross-loadings, the PID-5 structure in the United Arab Emirates national population was examined through exploratory factor analyses (EFA), using <italic>Equamax</italic> oblique rotation, and the number of factors to be extracted and interpreted was based on the <italic>Kaiser’s</italic>, <italic>Velicer’s</italic> minimum average partial test (MAP), and Parallel Analysis criteria.</p></sec></sec><sec sec-type=\"results\" id=\"sec3\"><label>3</label><title>RESULTS</title><sec id=\"sec3.1\"><label>3.1</label><title>Descriptive Statistics</title><p>Descriptive statistics for the five domains and 25 facets were compared with the data from the original study [<xref rid=\"r6\" ref-type=\"bibr\">6</xref>] through <italic>Cohen’s d</italic> (Table <bold><xref rid=\"T1\" ref-type=\"table\">1</xref></bold>). Small to medium effect sizes would reveal greater similarities between the original study and the Emiratis’ response style. The domains Negative affectivity, Detachment, and Disinhibition showed medium effect sizes (≤ .50), and large effect sizes were obtained for Psychoticism (.60) and Antagonism (.95). At the facets level, medium effect sizes (.20 - .50) were found for 13 of the facets, with nine facets showing large effect sizes (> .50).The smaller effect sizes (≤ .20) were found on Anhedonia, Rigid perfectionism, and Withdrawal, while the larger effect sizes (≥ .80) were displayed in Cognitive and Perceptual dysregulation and Irresponsibility.</p></sec><sec id=\"sec3.2\"><label>3.2</label><title>Reliability</title><p>The internal consistency of the Arabic PID-5 scales in the Emirati sample showed moderate (≥ .70 for 13 of the 25 facets) to high (≥ .80 for 11 of the 25 facets) coefficients, with a mean alpha of 0.74 (Table <bold><xref rid=\"T1\" ref-type=\"table\">1</xref></bold>). One facet showed a poor reliability coefficient of .37 (Suspiciousness). At the domain level, the alphas ranged from .81 (Antagonism) to .92 (Psychoticism) with a mean of .86. These results showed that the majority of the facets and the five domains were reliable, although with coefficients slightly lower than the ones previously found with other Arabic-speaking samples [<xref rid=\"r23\" ref-type=\"bibr\">23</xref>] and in the original study [<xref rid=\"r6\" ref-type=\"bibr\">6</xref>].</p></sec><sec><label>3.3</label><title>\nTest-retest Reliability\n</title><p>The results of the test-retest reliability are displayed in Table <bold><xref rid=\"T2\" ref-type=\"table\">2</xref></bold>. At the domain level, the correlation coefficients values ranged from .79 (<italic>p</italic> < .01) for Detachment to .92 (<italic>p </italic><.01) for the Antagonism domain. At the facets level, the correlation coefficients values were higher than ≥ .80 for 19 of the 25 facets, ranging from .73 (<italic>p </italic>< .01) for Restricted affectivity to .94 (<italic>p </italic>< .01) for the Attention seeking scale.</p></sec><sec id=\"sec3.4\"><label>3.4</label><title>Convergent Validity</title><p>The convergent validity of the Arabic PID-5 in the Emirati sample was investigated by correlating the five domains of the PID-5 with the five factors of NEO-FFI (Table <bold><xref rid=\"T3\" ref-type=\"table\">3</xref></bold>). As expected, the domain Negative affectivity correlated moderate and positively with Neuroticism (<italic>r</italic> = .57, <italic>p</italic> < .01), Detachment correlated moderate and negatively with Extraversion (<italic>r</italic> = -.49, <italic>p</italic> < .01) as well as Antagonism with Agreeableness (<italic>r</italic> = -.36, <italic>p</italic> < .01), and Disinhibition with Conscientiousness (<italic>r</italic> = -.50, <italic>p</italic> < .01). The domain Psychoticism displayed a low positive relationship with the factor Openness to Experience (<italic>r</italic> = .24, <italic>p </italic>< .01).</p></sec><sec id=\"sec3.5\"><label>3.5</label><title>Structure of the PID-5</title><p>The structure of the Arabic PID-5 in the Emirati community sample was tested through EFA of the 25 facets and the <italic> Kaiser,</italic> MAP, and Parallel analysis criteria were considered to evaluate the number of factors to be extracted and interpreted. A five-factor solution was supported by the <italic>Kaiser</italic> and Parallel analysis. The model showed excellent fit indices (KMO=.906), with a total explained variance of 61.21%. Communalities showed that the percentage of variance explained by the extracted factors was above 50% for all but four facets (Hostility, Risk taking, Submissiveness, and Suspiciousness), as can be seen in Table <bold><xref rid=\"T4\" ref-type=\"table\">4</xref></bold>.</p><p>Factor 1 was composed of the facets Anxiousness, Emotional lability, Hostility, Perseveration, Separation insecurity, Submissiveness, and Suspiciousness and matched the Negative affectivity domain structure.</p><p>Factor 2 was similar to Detachment and was composed of Anhedonia, Depressivity, Intimacy avoidance, Restricted affectivity, and Withdrawal. The only exception was the facet Suspiciousness, which loaded onto Factor 1. However, according to the DSM-5 personality model, this facet together with Depressivity and Restricted affectivity, simultaneously characterizes the domains Negative affectivity and Detachment.</p><p>The third Factor aggregated the facets Distractibility, Impulsivity, and Risk taking and resembled the Disinhibition domain, with the majority of the domain facets loaded. The only exception was the facet Irresponsibility that loaded primarily in the fourth Factor (.43) but had its secondary load in (.43) Factor three.</p><p>The fourth Factor mirrored the Antagonism domain, with all the facets of the domain primarily loaded in this factor. The exception was the facet Grandiosity (a facet of Antagonism), which unexpectedly also loaded primarily in Factor five.</p><p>Finally, the factor that most deviated from the personality domain structure of the AMPD [<xref rid=\"r5\" ref-type=\"bibr\">5</xref>], was the fifth one, onto the facets Grandiosity, Rigid perfectionism, and Unusual beliefs and experiences mainly weighted. However, both the facets Cognitive and perceptual dysregulation and Eccentricity (≥ .30) loaded on a second level in this factor, which might suggest that the fifth Factor is similar to the Psychoticism domain.</p><p>Ultimately, the Arabic PID-5 in the Emirati population revealed a five-factor solution similar to the DSM-5 AMPD [<xref rid=\"r5\" ref-type=\"bibr\">5</xref>], although not entirely overlapped. Moreover, the internal consistency of the new factors was calculated based on all the facets loaded onto each factor. The mean reliability coefficient varied from 0.81 for the first Factor (Negative affectivity) to 0.68 for the fifth Factor (Psychoticism), being this last factor the outlier of the original structure and consequently less interpretable. Although the three facets are considered loaded in the fifth Factor in conjunction with the other two facets of Psychoticism, namely the Cognitive and perceptual dysregulation and Eccentricity (loaded secondarily onto it), an alpha of .75 is obtained.</p></sec></sec><sec sec-type=\"discussion\" id=\"sec4\"><label>4</label><title>DISCUSSION</title><p>The current study aimed to examine the psychometric properties of the PID-5 in an Emirati community sample and addressed the cross-cultural replicability of its factor structure in a non-Western culture.</p><p>The findings in the Emirati sample were comparable to the original US study [<xref rid=\"r6\" ref-type=\"bibr\">6</xref>], in terms of the PID-5 internal consistency, convergent validity with the NEO-FFI and factor structure. However, significant differences were identified in the mean scores, with higher scores in most of the facets and domains, similar to the results found in the Arabic translation study [<xref rid=\"r23\" ref-type=\"bibr\">23</xref>]. The facets Cognitive and perceptual dysregulation and the domain Antagonism showed the larger effect size (≥.90). These results might suggest that the response style obtained could reflect situational factors or cultural specificities as if a certain numerical score represents the same absolute trait level in different cultures, and if the intensity or difficulty of a given item changes across languages [<xref rid=\"r43\" ref-type=\"bibr\">43</xref>, <xref rid=\"r46\" ref-type=\"bibr\">46</xref>]. Nevertheless, the PID-5 has demonstrated that it is a reliable measure and perhaps some specific items are compensated by the scales’ overall sum.</p><p>Moreover, the Arabic PID-5, beyond adequate internal consistency at the facet (mean alpha .74) and domain level (mean alpha .86), also demonstrated good temporal reliability, in line with previous studies (for a review see Al-Dajani, Gralnick, and Bagby [<xref rid=\"r7\" ref-type=\"bibr\">7</xref>]).</p><p>As expected, the five domains of the Arabic PID-5 displayed meaningful associations with the five domains of the Arabic NEO-FFI [<xref rid=\"r23\" ref-type=\"bibr\">23</xref>, <xref rid=\"r47\" ref-type=\"bibr\">47</xref>, <xref rid=\"r48\" ref-type=\"bibr\">48</xref>]. Nonetheless the positive relationship between Psychoticism and Openness to experience was rather small [<xref rid=\"r14\" ref-type=\"bibr\">14</xref>, <xref rid=\"r49\" ref-type=\"bibr\">49</xref>], which might be related to the conceptual nature of these domains and how they are assessed. Openness is mostly an adaptive domain of personality (measured by the NEO-FFI) whereas Psychoticism is entirely a mal-adaptive domain (measured by the PID-5), which might decrease the probability of both domains load in the same direction and in the same factor, once they have opposite functions, as one is adaptative and the other is mal-adaptive [<xref rid=\"r50\" ref-type=\"bibr\">50</xref>].</p><p>With regards to the Arabic PID-5 factor structure in the Emirati sample, these findings confirmed a five-factors solution similar to the one displayed by Krueger <italic>et al.</italic> [<xref rid=\"r6\" ref-type=\"bibr\">6</xref>] and by Al-Attiyah <italic>et al.</italic> [<xref rid=\"r23\" ref-type=\"bibr\">23</xref>]. The first four factors featured the domains Negative affectivity, Detachment, Distractibility, and Antagonism. Although the loading patterns of some facets deviated from the original structure, particularly in the fifth Factor, where Grandiosity, Rigid perfectionism, and Unusual beliefs and experiences were primarily loaded, resembling an imperfect conjunction of the fifth (Compulsivity) and sixth (Schizotypy) domains, initially proposed by the AMPD [<xref rid=\"r5\" ref-type=\"bibr\">5</xref>]. However, if it is considered that the facets Cognitive and perceptual dysregulation and Eccentricity loaded secondarily in this factor, perhaps it might be also considered that this factor is similar to the Psychoticism domain.</p><p>One possible reason for this deviant factor could be that Psychoticism, beyond encompassing the tendency to have unusual beliefs and experiences, behave eccentrically, and manifest cognitive dysregulation, might also enclose some aspects of Antagonism and low Disinhibition, such as being self-centered or superior and having the need to impose a rigid and dogmatic order towards others and their environment [<xref rid=\"r51\" ref-type=\"bibr\">51</xref>]. In this regard, some studies have found evidence for an association between some features of Obsessive-Compulsive PD with Schizotypal PD [<xref rid=\"r52\" ref-type=\"bibr\">52</xref>]. In fact, although the domain Psychoticism primarily emerged from features of Negative affectivity, Disinhibition, and Detachment [<xref rid=\"r53\" ref-type=\"bibr\">53</xref>, <xref rid=\"r54\" ref-type=\"bibr\">54</xref>], it has been pointed as heterogeneous, and some studies found deviant facet loading in this domain [<xref rid=\"r29\" ref-type=\"bibr\">29</xref>, <xref rid=\"r55\" ref-type=\"bibr\">55</xref>]. Others even reported its absence from their factor structure in a clinical sample [<xref rid=\"r56\" ref-type=\"bibr\">56</xref>]. Furthermore, studies that tried to harmonize the DSM-5 trait model with the ICD-11 personality model stated that in order to facilitate the communication between clinicians, the domain Psychoticism should not be conceptualized in terms of personality pathology, as it is considered under the spectrum of schizophrenia disorder by the World Health Organization [<xref rid=\"r57\" ref-type=\"bibr\">57</xref>, <xref rid=\"r58\" ref-type=\"bibr\">58</xref>]. However, a trait profile does not correspond to arbitrary diagnose categories or syndromes, but instead denotes stylistic dimensions that contribute to the expression of the personality dysfunction under the umbrella of a more general factor of psychopathology [<xref rid=\"r59\" ref-type=\"bibr\">59</xref>]. On this note, a recent study by Bastiaens <italic>et al</italic>. [<xref rid=\"r60\" ref-type=\"bibr\">60</xref>], which claimed the PID-5 clinical utility to discriminate between patients with and without a psychotic disorder, concluded that the patients significantly differed on all PID-5 domains, except for Antagonism, and that lower Detachment, lower Negative Affect, lower Disinhibition, and higher Psychoticism were the trait profiles that best discriminated patients with a psychotic disorder from patients with other diagnoses.</p><p>Considering the findings, future studies in non-Western countries should try to establish normative values for the general population in order to better identify the presence of mal-adaptive traits, and examine how the facet traits could help to discriminate between what is normal and abnormal in a given culture or language.</p><p>This study has several limitations that should be considered in future research. First, the sample was predominantly composed by female college students and their acquaintances, which might have biased the results considering that women often report a higher level of Neuroticism compared to men [<xref rid=\"r61\" ref-type=\"bibr\">61</xref>, <xref rid=\"r62\" ref-type=\"bibr\">62</xref>] and that gender roles and expectations tend to be more clearly demarcated in Arabic cultures when compared to Western cultures [<xref rid=\"r63\" ref-type=\"bibr\">63</xref>]. Also, data was collected from a Governmental University in only two of the seven Emirates (Abu Dhabi and Dubai), and most of the participants had medium to high economic status as well as high educational levels, which might have influenced the response to the test. Second, the test-retest sample size was small due to many losses between the 1<sup>st</sup> and the 2<sup>nd</sup> data collection sessions.</p><p>\nFinally, given that the PID-5 is a clinical diagnostic measure, the expansion of this research to clinical Emirati samples is a crucial\nendeavor\n, that will bridge the current\nstudy\nlimitations with future developments and provide relevant data on the PID-5’s predictive validity.\n</p></sec><sec sec-type=\"conclusions\"><title>CONCLUSION</title><p>Notwithstanding the aforementioned, this study concluded that the Arabic version of the PID-5 is a valid measure to describe pathological personality traits in the Emirati population of the United Arab Emirates, and provides additional evidence for the generalizability of the AMPD [<xref rid=\"r5\" ref-type=\"bibr\">5</xref>] to other Arab countries.</p></sec></body><back><ack><title>ACKNOWLEDGEMENTS</title><p>We would like to thank all the volunteer students and staff from Zayed University Dubai and Abu Dhabi that collaborated on this research.</p></ack><glossary><title>\nLIST OF ABBREVIATIONS\n</title><def-list><def-item><term>AMPD</term><def><p> = Alternative DSM-5 Model for personality Disorders</p></def></def-item><def-item><term>APA</term><def><p> = American Psychiatric Association</p></def></def-item><def-item><term><italic>d</italic></term><def><p> = \n<italic>Cohen’s d</italic>\n</p></def></def-item><def-item><term>EFA</term><def><p> = Exploratory Factor analyses</p></def></def-item><def-item><term>DSM-5</term><def><p> = Diagnostic and Statistical Manual of Mental disorders - 5<sup>th</sup> Edition</p></def></def-item><def-item><term>FFM</term><def><p> = Five Factor Model</p></def></def-item><def-item><term>IBM SPSS statistics</term><def><p> = IBM Statistical Package for Social Sciences</p></def></def-item><def-item><term>ICD-11</term><def><p> = International Classification of Mental and Behavioural Disorders – 11<sup>th</sup> Edition</p></def></def-item><def-item><term>KMO</term><def><p> = Kaiser-Meyer-Olkin test of sampling adequacy</p></def></def-item><def-item><term><italic>M</italic></term><def><p> = Media</p></def></def-item><def-item><term>MAP</term><def><p> = \n<italic>Velicer’s</italic> minimum average partial test</p></def></def-item><def-item><term>MSA</term><def><p> = Modern Standard Arabic</p></def></def-item><def-item><term><italic>N</italic></term><def><p> = number of participants</p></def></def-item><def-item><term>NEO</term><def><p> = FFI – NEO Five Factor Inventory</p></def></def-item><def-item><term><italic>p</italic></term><def><p> = Value of significance</p></def></def-item><def-item><term>PDs</term><def><p> = Personality Disorders</p></def></def-item><def-item><term>PD</term><def><p> = Personality Disorder</p></def></def-item><def-item><term>PID-5</term><def><p> = Personality Inventory for DSM-5</p></def></def-item><def-item><term><italic>r</italic></term><def><p> = Pearson coefficient</p></def></def-item><def-item><term><italic>r<sub>s</sub></italic></term><def><p> = Spearman’s rank coefficient</p></def></def-item><def-item><term><italic>SD</italic></term><def><p> = Standard deviation</p></def></def-item><def-item><term>UAE</term><def><p> = United Arab Emirates</p></def></def-item><def-item><term>WHO</term><def><p> = World Health Organization</p></def></def-item><def-item><term>α</term><def><p> = Cronbach alpha</p></def></def-item></def-list></glossary><sec sec-type=\"competing-interests\"><title>ETHICS APPROVAL & CONSENT TO PARTICIPATE</title><p>The experimental sessions were held collectively and conducted at Zayed University after obtaining approval from the Research Ethics Committee of Zayed University, UAE.</p></sec><sec sec-type=\"competing-interests\"><title>HUMAN AND ANIMAL RIGHTS</title><p>Not applicable.</p></sec><sec sec-type=\"competing-interests\"><title>\nCONSENT FOR PUBLICATION\n</title><p>Participation in this study was voluntary and all respondents signed a written informed consent form requesting their participation in the study.</p></sec><sec sec-type=\"competing-interests\"><title>\nAVAILABILITY OF DATA AND MATERIALS\n</title><p>The data that support the findings of this study are available from the corresponding author [O.C.] upon reasonable request.</p></sec><sec sec-type=\"competing-interests\"><title>FUNDING</title><p>None.</p></sec><sec sec-type=\"competing-interests\"><title>CONFLICT OF INTEREST</title><p>The authors declare no conflict of interest, financial or otherwise.</p></sec><ref-list><title>REFERENCES</title><ref id=\"r1\"><label>1</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krueger</surname><given-names>R.F.</given-names></name><name><surname>Markon</surname><given-names>K.E.</given-names></name></person-group><article-title>The role of the DSM-5 personality trait model in moving toward a quantitative and empirically based approach to classifying personality and psychopathology.</article-title><source>Annu. 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domains.</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\"/><th colspan=\"3\" valign=\"top\" align=\"center\" scope=\"colgroup\" rowspan=\"1\">Study 1</th><th colspan=\"3\" valign=\"top\" align=\"center\" scope=\"colgroup\" rowspan=\"1\">Study 2</th><th colspan=\"3\" valign=\"top\" align=\"center\" scope=\"colgroup\" rowspan=\"1\">Study 3</th><th colspan=\"2\" valign=\"top\" align=\"center\" scope=\"colgroup\" rowspan=\"1\"/></tr></thead><tbody><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\"/><td colspan=\"3\" valign=\"top\" align=\"center\" rowspan=\"1\">Krueger <italic>et al.</italic>, 2012 (<italic>N</italic> = 264)</td><td colspan=\"3\" valign=\"top\" align=\"center\" rowspan=\"1\">Al-Attiyah <italic>et al.</italic>, 2017 (<italic>N</italic> = 710)</td><td colspan=\"3\" valign=\"top\" align=\"center\" rowspan=\"1\">UAE data<break/>(<italic>N</italic> = 1090)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Studies 1 & 2</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Studies 1 & 3</italic>\n</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">-</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>α</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>M</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SD</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>α</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>M</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SD</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>α</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>M</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>SD</italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>d<sub>1,2</sub></italic>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>d<sub>1,3</sub></italic>\n</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Anhedonia</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.88</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.64</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.88</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.52</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.77</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.90</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.51</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.02</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Anxiousness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.91</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.73</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.52</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.60</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.84</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.60</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.64</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Attention seeking</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.81</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.65</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.93</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.66</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.83</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.05</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.85</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.40</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Callousness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.91</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.92</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.71</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.73</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.54</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.62</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.37</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Cognitive dysregulation</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.86</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.44</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.71</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.46</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.91</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.98</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Deceitfulness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.85</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.52</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.54</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.88</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.54</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.71</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.87</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.44</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.91</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.76</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Depressivity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.53</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.62</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.92</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.85</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.53</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.87</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.70</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.49</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.33</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Distractibility</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.91</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.82</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.69</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.88</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.79</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.51</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.59</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.53</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Eccentricity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.96</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.82</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.76</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.92</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.63</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.46</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.90</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.96</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.34</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.23</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Emotional lability</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.94</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.74</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.86</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.75</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.28</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.53</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.57</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Grandiosity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.72</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.82</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.82</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.67</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.52</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.56</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Hostility</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.91</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.67</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.57</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.75</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.60</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.54</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Impulsivity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.77</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.77</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.57</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.87</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.62</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.75</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.04</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.57</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.82</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.47</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Intimacy avoidance</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.84</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.61</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.65</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.77</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.71</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.85</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.54</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.59</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.43</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Irresponsibility</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.81</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.49</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.84</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.99</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.53</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.66</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.77</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.46</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.82</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Manipulativeness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.81</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.67</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.70</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.54</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.67</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.37</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Perseveration</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.88</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.82</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.62</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.85</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.49</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.70</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.44</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.54</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Restricted affectivity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.73</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.97</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.56</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.81</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.61</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.47</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.41</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Rigid perfect.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.90</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.05</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.68</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.90</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.45</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.57</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.77</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.44</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.67</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.06</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Risk taking</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.85</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.05</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.51</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.92</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.22</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.52</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.79</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.22</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.44</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.37</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Separation insecurity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.85</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.68</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.87</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.56</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.76</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.98</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.56</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.47</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.31</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Submissiveness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.66</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.84</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.67</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.96</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.57</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.36</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Suspiciousness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.73</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.47</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.46</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Unusual beliefs</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.83</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.64</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.63</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.90</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.45</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.45</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.74</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.91</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.52</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.50</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Withdrawal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.93</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.72</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.90</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.07</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.53</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.51</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.13</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Negative affectivity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.93</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.07</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.44</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.94</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.25</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.87</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.45</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.36</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Detachment</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.96</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.54</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.96</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.86</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.94</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.37</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Antagonism</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.61</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.46</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.92</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.96</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.81</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.36</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.95</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Disinhibition</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.84</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.06</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.85</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.97</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.23</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Psychoticism</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.96</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.64</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.57</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.79</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.92</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.93</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.46</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.60</td></tr></tbody></table><table-wrap-foot><p>Krueger <italic>et al.</italic>, 2012 [<xref rid=\"r6\" ref-type=\"bibr\">6</xref>]; Al-Attiyah <italic>et al.</italic>, 2017 [<xref rid=\"r23\" ref-type=\"bibr\">23</xref>]; Small effect <italic>d </italic> ≤ .20, medium effect size .20 <<italic> d</italic> ≤ .50, large .50 <<italic> d</italic> ≤ 1.0, and very large <italic>d </italic>> 1.0</p></table-wrap-foot></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><title>Stability coefficients of the Arabic version of the PID-5 facets and domains in the UAE sample.</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">PID-5A Scales</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<italic>r</italic> (<italic>N</italic> = 28)</th></tr></thead><tbody><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Anhedonia<sup>1</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.84**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Anxiousness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.89**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Attention seeking</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.94**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Callousness<sup>1</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.82**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Cognitive and perceptual dysregulation</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.78**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Deceitfulness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.91**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Depressivity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.76**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Distractibility</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.85**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Eccentricity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.95**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Emotional lability</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.87**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Grandiosity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.80**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Hostility</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.92**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Impulsivity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.84**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Intimacy avoidance</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.78**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Irresponsibility<sup>1</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.76**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Manipulativeness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.92**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Perseveration</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.77**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Restricted affectivity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.73**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Rigid perfectionism</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.88**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Risk taking</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.87**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Separation insecurity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.82**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Submissiveness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.80**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Suspiciousness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.83**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Unusual beliefs and experiences</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.84**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Withdrawal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.83**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Negative affectivity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.88**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Detachment</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.79**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Antagonism</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.92**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Disinhibition</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.91**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Psychoticism</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.87**</td></tr></tbody></table><table-wrap-foot><p><italic>r Pearson</italic> correlation coefficient; <sup>1</sup>\n<italic>Spearman</italic> correlation coefficient (<italic>r<sub>s</sub></italic>); **Significant correlations <italic>p </italic>˂ .01. Four weeks interval between applications</p></table-wrap-foot></table-wrap><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><title>Correlations r of the Arabic version of the PID-5 with the NEO-FFI in the UAE sample.</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">PID Domains</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\nNeuroticism\n</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\nExtraversion\n</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\nOpenness\n</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\nAgreeableness\n</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\nConsciousness\n</th></tr></thead><tbody><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Negative affectivity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.57**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.05</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.04</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.17**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.11**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Detachment</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.34**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.49**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.07*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.29**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.27**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Antagonism</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.08**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.15**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.03</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.36**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.02</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Disinhibition</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.38**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.17**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.37**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.50**</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Psychoticism</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.32**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.04</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.24**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.37**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.11**</td></tr></tbody></table><table-wrap-foot><p> **Significant correlations <italic>p</italic> ˂ .01; *Significant correlations <italic>p</italic> ˂ .05</p><p><italic>r Pearson</italic> correlation coefficient</p></table-wrap-foot></table-wrap><table-wrap id=\"T4\" orientation=\"portrait\" position=\"float\"><label>Table 4</label><caption><title>Exploratory factor analysis with Equamax rotation solution in an UAE community sample.</title></caption><table frame=\"border\" rules=\"all\" width=\"100%\"><thead><tr><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\"/><th colspan=\"5\" valign=\"top\" align=\"center\" scope=\"colgroup\" rowspan=\"1\">\n<italic>Factors</italic>\n</th><th valign=\"top\" align=\"center\" scope=\"col\" rowspan=\"1\" colspan=\"1\">\n<italic>Communalities</italic>\n</th></tr></thead><tbody><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">PID-5 facets</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Anhedonia</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.66</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.09</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.68</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Anxiousness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.73</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.68</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Attention seeking</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.59</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.62</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Callousness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.46</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.63</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.64</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Cognitive dysregulation</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.62</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.69</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Deceitfulness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.09</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.74</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.69</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Depressivity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.58</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.04</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.72</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Distractibility</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.51</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.03</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.63</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Eccentricity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.03</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.62</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.68</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Emotional lability</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.54</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.05</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.22</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.62</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Grandiosity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.09</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.60</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.55</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Hostility</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.43</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.34</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.49</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Impulsivity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.67</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.60</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Intimacy avoidance</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.09</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.70</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.06</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.52</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Irresponsibility</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.43</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.43</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.62</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Manipulativeness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.03</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.72</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.36</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.67</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Perseveration</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.50</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.36</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.61</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Restricted affectivity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.63</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.56</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Rigid perfectionism</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.04</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.04</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.79</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.70</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Risk taking</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.56</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.22</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.48</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Separation insecurity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.70</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.09</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.57</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Submissiveness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.60</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.09</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.07</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.44</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Suspiciousness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.39</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.36</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.03</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.42</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Unusual beliefs</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.47</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.59</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.66</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Withdrawal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>.75</bold>\n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.07</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.66</td></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">Eigenvalues</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"center\" scope=\"row\" rowspan=\"1\" colspan=\"1\">% variance explained</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.51</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.69</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></table-wrap></floats-group></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Psychol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Psychol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Psychol.</journal-id><journal-title-group><journal-title>Frontiers in Psychology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-1078</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849120</article-id><article-id pub-id-type=\"pmc\">PMC7431703</article-id><article-id pub-id-type=\"doi\">10.3389/fpsyg.2020.01911</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Psychology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Predictors of High-School Dropout Among Ultraorthodox Jewish Youth</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Itzhaki-Braun</surname><given-names>Yael</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/947023/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Itzhaky</surname><given-names>Haya</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Yablon</surname><given-names>Yaacov B.</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1004193/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>The Gershon Gordon Faculty of Social Sciences, The Bob Shapell School of Social Work, Tel Aviv University</institution>, <addr-line>Tel Aviv</addr-line>, <country>Israel</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Faculty of Social Sciences, School of Social Work, Bar-Ilan University</institution>, <addr-line>Ramat Gan</addr-line>, <country>Israel</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Faculty of Social Sciences, School of Education, Bar-Ilan University</institution>, <addr-line>Ramat Gan</addr-line>, <country>Israel</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Pei Sun, Tsinghua University, China</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Avi Assor, Ben-Gurion University of the Negev, Israel; Ying Wang, Tsinghua University, China</p></fn><corresp id=\"c001\">*Correspondence: Yael Itzhaki-Braun, <email>yaelitbr@tauex.tau.ac.il</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Educational Psychology, a section of the journal Frontiers in Psychology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1911</elocation-id><history><date date-type=\"received\"><day>06</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>10</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Itzhaki-Braun, Itzhaky and Yablon.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Itzhaki-Braun, Itzhaky and Yablon</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Focusing on the unique social characteristics of closed communities, the current study examined the predictors of high-school dropout among Ultraorthodox Jewish youth, focusing on background variables [i.e., individual’s age, family’s religious group affiliation, and other high-school dropout(s) in family]; social resources (i.e., self-esteem and mastery); and parental conditional regard (PCR) and societal conditional regard (SCR), with reference to both positive regard and negative regard. The study was conducted in Israel with the participation of 261 Ultraorthodox Jewish males, ages 14–21 (<italic>M</italic> = 17, <italic>SD</italic> = 1.17), who were at different stages in the dropout process. Path analysis modeling indicated that being a member of a newly religious family, or of a family in which another member had already dropped out of school, was a predictor of dropout. Surprisingly, personal resources were not found to be a predictor of dropout, whereas parental conditional regard and societal conditional negative regard (SCNR) were found to be the most significant predictors. Findings highlight the unique predictors of high-school dropout among youth from the Ultraorthodox Jewish community, and the role of PCR and SCR in this process.</p></abstract><kwd-group><kwd>high school dropout</kwd><kwd>the Ultraorthodox Jewish community</kwd><kwd>parental conditional regard</kwd><kwd>societal conditional regard</kwd><kwd>newly religious family</kwd></kwd-group><counts><fig-count count=\"1\"/><table-count count=\"4\"/><equation-count count=\"0\"/><ref-count count=\"70\"/><page-count count=\"11\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Dropping out of high school is a risk situation for youth, with negative personal and social consequences, as discussed in the research literature at length (<xref rid=\"B62\" ref-type=\"bibr\">Rumberger and Lim, 2008</xref>; <xref rid=\"B67\" ref-type=\"bibr\">Staff and Kreager, 2008</xref>; <xref rid=\"B48\" ref-type=\"bibr\">Makarova and Birman, 2015</xref>). Thus, many studies have been conducted in order to identify the predictors of high-school dropout among youth at risk (<xref rid=\"B40\" ref-type=\"bibr\">Janosz et al., 2008</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Archambault et al., 2009</xref>), under the assumption that recognizing at-risk youth would be helpful in the prevention of their dropping out (<xref rid=\"B56\" ref-type=\"bibr\">Ricard and Pelletier, 2016</xref>). Yet, there is very little research or specific data, if any, regarding the predictors of high-school dropout among youth from closed religious communities, despite the fact that this phenomenon, and the percentage of students from these communities in society at large, is growing (<xref rid=\"B33\" ref-type=\"bibr\">Israeli Central Bureau of Statistics, 2016</xref>). Given that in these communities, dropping out of school reflects a violation of community norms (<xref rid=\"B42\" ref-type=\"bibr\">Kelly, 2014</xref>), the underlying rationale of this research was the expectation that there would be unique predictors for high-school dropout among youth in this population. As such, the aim of the current study was to identify possible predictors of high-school dropout in a closed religious community. Specifically, we focused on three groups of students from the Ultraorthodox Jewish community in Israel, who represent three different stages in the dropout process. These three groups were defined as (1) students who were at risk of dropping out of high school, (2) students who had dropped out of high school and enrolled in a program for dropouts, and (3) students who had dropped out of school and did not enrolled in any educational framework at all. Based on social capital theory (<xref rid=\"B15\" ref-type=\"bibr\">Coleman, 1988</xref>), we used an ecological perspective and investigated the contribution of background variables, personal resources, family resources, and communal resources, to high-school dropout in this community.</p><sec id=\"S1.SS1\"><title>Dropping Out of School in Religious Communities</title><p>Youth who dropped out from school can engage in many risk situations. These include unemployment, poverty, health issues, early death, and crime (<xref rid=\"B7\" ref-type=\"bibr\">Belfield and Levin, 2007</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Brekke, 2014</xref>). Young people who drop out of high school are therefore considered to be an at-risk group. For youths from religious communities, the repercussions of dropout are even greater, as the individuals in these communities live in accordance with strict norms. For instance, individuals are expected to study in the community’s schools, which “multitask:” that is, they educate, provide a framework for socializing, and indoctrinate. However, when individuals leave this framework, they effectively reject communal norms, potentially distancing themselves from their communities (<xref rid=\"B25\" ref-type=\"bibr\">Finkelman, 2011</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Cates and Weber, 2013</xref>). Conflicts between the youths and their surrounded systems, the parents, and the community (<xref rid=\"B70\" ref-type=\"bibr\">Yogev, 2012</xref>) are therefore created when a young person drops out of school in these settings.</p><p>The current study focused on Israel’s Ultraorthodox Jewish sector, a sector that constitutes 9% of Israel’s overall Jewish population (<xref rid=\"B35\" ref-type=\"bibr\">Israeli Central Bureau of Statistics, 2019</xref>). These individuals live in closed-off neighborhoods (away from secular neighborhoods), have specified dress codes for men and women, and strictly observe Jewish law. The youth of these communities attend state-certified private educational institutions, with content uniquely intended for them. The schools are single-sex. In high school, boys focus on Jewish subjects at their “yeshivas” and continue studying in this manner until marriage (<xref rid=\"B25\" ref-type=\"bibr\">Finkelman, 2011</xref>). The yeshiva high school plays many roles – religious, educational, social – and its goal is to mold the boys’ behavior to be in line with the tenets of Ultraorthodox Judaism. Ultraorthodox Jewish parents send their sons to yeshiva high schools for reasons similar to those of parents who send their children to other kinds of private religious schools (e.g., Christian, Mormon, Amish): to maintain their children’s observance of the religion, faith to community values, and distance from the secular world (<xref rid=\"B32\" ref-type=\"bibr\">Irwin, 2002</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Cates and Weber, 2013</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Bertrand, 2015</xref>).</p><p>Surprisingly, there is a relatively high rate of high-school dropout in the Ultraorthodox Jewish community as compared with the rate of high-school dropout in the state education framework (<xref rid=\"B34\" ref-type=\"bibr\">Israeli Central Bureau of Statistics, 2018</xref>), making the topic one of growing interest and concern. However, there has been almost no research conducted in regard to the predictors of high-school dropout in this community. The large role that the yeshiva plays in the youths’ lives, and the consequences of leaving this framework (as described above), requires a broad perspective that takes into account the familial and communal aspects that typify this community. Based on other studies that have investigated high-school dropout, this study too was based on social capital theory (<xref rid=\"B52\" ref-type=\"bibr\">Perreira et al., 2006</xref>; <xref rid=\"B66\" ref-type=\"bibr\">South et al., 2007</xref>).</p></sec><sec id=\"S1.SS2\"><title>Social Capital Theory</title><p>As stated above, the theory of social capital (<xref rid=\"B15\" ref-type=\"bibr\">Coleman, 1988</xref>) provided the basis for the current study, because it enables a theoretical base to the predictors of dropping out of high school in a closed religious community. <xref rid=\"B15\" ref-type=\"bibr\">Coleman (1988)</xref> was the first to provide evidence of a relationship between social capital and school dropout rates. He argued that students attending private religious schools were less likely to drop out than students attending public high schools due to the relationships of parents with one another, in the schools, and in their community (<xref rid=\"B16\" ref-type=\"bibr\">Coleman and Hoffer, 1987</xref>). Social capital theory focuses on different levels of factors surrounding the individual: human capital and social capital. Human capital refers to individuals’ skills, capabilities, and resources that make them able to act in new ways (<xref rid=\"B15\" ref-type=\"bibr\">Coleman, 1988</xref>). Social capital refers to the value of social structures that act as resources that individuals can use to fulfill their needs. The most common structure is the family, while other conceptualizations of social capital contain various other sources, like school, neighborhood, and community, broader familial forms of social capital (<xref rid=\"B15\" ref-type=\"bibr\">Coleman, 1988</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Long and Perkins, 2007</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Dufur et al., 2008</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Goksen and Cemalcilar, 2010</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Hassan et al., 2016</xref>). Social capital theory emphasizes the importance of the individual’s personal resources, his community resources, and the interaction between the two, as well as the researcher’s ability to use them to predict, among other things, school dropout among youth (<xref rid=\"B52\" ref-type=\"bibr\">Perreira et al., 2006</xref>; <xref rid=\"B66\" ref-type=\"bibr\">South et al., 2007</xref>).</p><p>High levels of solidarity among members and few social connections they have with people outside their communities in closed religious communities may be the factor for the higher levels of social capital that were found in these communities compared to non-religious communities (<xref rid=\"B15\" ref-type=\"bibr\">Coleman, 1988</xref>; <xref rid=\"B54\" ref-type=\"bibr\">Portes, 1998</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Fukuyama, 2001</xref>). It was found that religious participation alters teen social networks, putting teens in greater contact with educational resources and pro-school values from peers, all relating to the lower percentage of drop out compare to non-religious youth (<xref rid=\"B28\" ref-type=\"bibr\">Glanville et al., 2008</xref>). This may result from the ability of congregations to provide a place to meet and develop friendships with non-at-risk youth. Regarding youth from the Ultraorthodox Jewish community, the loss of social capital is considered to be a significant issue in their school dropout process (<xref rid=\"B42\" ref-type=\"bibr\">Kelly, 2014</xref>).</p><p>In a review of 203 published articles, <xref rid=\"B62\" ref-type=\"bibr\">Rumberger and Lim (2008)</xref> found that high-school dropout predictors were divided into two main groups: individual factors including background variables and self-perception, and institutional factors including family and community resources. In the current study, we also investigated background variables, and personal and social resources, as predictors of dropout among Ultraorthodox Jewish youth. We chose to focus on background variables that have been found to be relevant in predicting dropout in the literature in regard to this particular population. Specifically, we investigated the contribution of age, being a member of a newly religious family and/or of a family in which another family member had already dropped out of school. Regarding personal resources, we investigated the contribution of self-esteem and mastery. Regarding social resources, we focused both on the family aspect, via parental conditional regard (PCR) (<xref rid=\"B4\" ref-type=\"bibr\">Assor et al., 2004</xref>), and on the community aspect, via societal conditional regard (SCR) (<xref rid=\"B37\" ref-type=\"bibr\">Itzhaki et al., 2018</xref>).</p></sec><sec id=\"S1.SS3\"><title>Background Variables</title><p>Rumberger and Lim’s meta-analysis (2008) concluded that 31 of the 57 studies that investigated the relationship between age and dropout found that older high school students had a higher chance to drop out and a low probability to graduate, than younger high school students (<xref rid=\"B13\" ref-type=\"bibr\">Chapman et al., 2010</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Landis and Reschly, 2011</xref>). Moreover, dropout could happen in lockstep with the decision of a sibling to drop out (<xref rid=\"B23\" ref-type=\"bibr\">Dupéré et al., 2015</xref>). Consistently, high school students were more likely to drop out if they had a sibling who had done so (<xref rid=\"B63\" ref-type=\"bibr\">Rumberger and Thomas, 2000</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Jacob, 2001</xref>).</p><p>A concept that seems to be somewhat unique to the Ultraorthodox Jewish community is that of the “newly religious family.” The term newly religious family (“hozrim b’tshuvah”) refers to those who “return” (even if they were not brought up as such) to Orthodox Jewish religious observance (<xref rid=\"B17\" ref-type=\"bibr\">Davidman and Greil, 2007</xref>). This group comprises people who for the most part had never observed Jewish religious law until they decided to become part of the Jewish religious community. In most cases, the community chosen was the Ultraorthodox Jewish community. Although the “hazarah b’tshuva” process is generally seen as being praiseworthy by religious Jews, newly religious families rarely succeed in fully integrating into the Ultraorthodox Jewish community, mostly because Ultraorthodox communities are quite closed and wary of assimilation (<xref rid=\"B21\" ref-type=\"bibr\">Doron, 2013</xref>). Perhaps for this reason, the percentage of high-school dropout among newly religious families (a minority group of the Ultraorthodox Jewish community) is higher than that of “established” Ultraorthodox Jewish families (<xref rid=\"B36\" ref-type=\"bibr\">Itzhaki, 2009</xref>; <xref rid=\"B70\" ref-type=\"bibr\">Yogev, 2012</xref>).</p></sec><sec id=\"S1.SS4\"><title>Self-Esteem and Mastery</title><p>In this study, similar to previous studies that investigated youth, the personal resources that were examined are self-esteem and sense of mastery (<xref rid=\"B46\" ref-type=\"bibr\">Lipschitz-Elhawi and Itzhaky, 2005</xref>; <xref rid=\"B53\" ref-type=\"bibr\">Phillips, 2010</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Dwyer et al., 2011</xref>). Unsurprisingly, youth at risk are characterized by low levels of self-esteem and low levels of mastery, among other things (<xref rid=\"B41\" ref-type=\"bibr\">Jessor et al., 1998</xref>; <xref rid=\"B49\" ref-type=\"bibr\">Metz, 2006</xref>), both of which are connected to a failure to adapt into frameworks such as school or the workplace (<xref rid=\"B20\" ref-type=\"bibr\">Dillon, 2004</xref>; <xref rid=\"B2\" ref-type=\"bibr\">Aristilde, 2006</xref>). Both self-esteem and mastery were found to be predictors of high-school dropout (<xref rid=\"B9\" ref-type=\"bibr\">Bong and Skaalvik, 2003</xref>; <xref rid=\"B18\" ref-type=\"bibr\">De Ridder et al., 2013</xref>; <xref rid=\"B55\" ref-type=\"bibr\">Quiroga et al., 2013</xref>).</p></sec><sec id=\"S1.SS5\"><title>The Familial and Communal Aspect</title><p>Both family and community are described in the literature as crucial factors in predicting school dropout among youth. The familial aspect has been investigated via three main components: (1) family structure, (2) family resources, and (3) family practices (<xref rid=\"B62\" ref-type=\"bibr\">Rumberger and Lim, 2008</xref>). The relationship between adolescence and parents was found to have a crucial role in school engagement, while poor parent–teenager relationships were found to be a predictor for high-school dropout (<xref rid=\"B57\" ref-type=\"bibr\">Roberts et al., 2013</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Fortin et al., 2013</xref>; <xref rid=\"B69\" ref-type=\"bibr\">Wang and Fredricks, 2014</xref>). As for the community aspect, it has been investigated via the following: (1) access to institutional resources (e.g., childcare, medical facilities, employment opportunities), (2) parental relationships that can provide access to family and friends, as well as social connections in the neighborhood, and (3) social capital that arises out of mutual trust and shared values and that comes into play in terms of supervising and monitoring the activities of residents, and particularly youth (<xref rid=\"B45\" ref-type=\"bibr\">Leventhal and Brooks-Gunn, 2000</xref>). High level of trust and mutual cooperation among people in the community contributed to reducing high-school dropout (<xref rid=\"B31\" ref-type=\"bibr\">Hassan et al., 2016</xref>). However, negative attitude from community members such as discrimination and prejudice was found to be a predictor of high-school dropout (<xref rid=\"B19\" ref-type=\"bibr\">De Witte et al., 2013</xref>). In the current study, we focused on the examination of the role of family and community via the relationships between the youths and their parents and between the youths and the members of their community. Specifically, these relationships were examined via the construct of conditional regard (CR).</p></sec><sec id=\"S1.SS6\"><title>Parental Conditional Regard and Societal Conditional Regard</title><p>In the current study, we examined the contribution of parental conditional regard (PCR), and societal conditional regard (SCR) (<xref rid=\"B37\" ref-type=\"bibr\">Itzhaki et al., 2018</xref>), to school dropout among youth. Parental conditional regard has its basis in self-determination theory (SDT) (<xref rid=\"B64\" ref-type=\"bibr\">Ryan and Deci, 2000</xref>) and is a socialization strategy wherein parental love/acceptance depends upon the child’s complying with the parents’ expectations. Parental conditional <italic>positive</italic> regard (PCPR) implies a greater degree of parental attention, affection, and esteem as a result of the child meeting parental expectations. Parental conditional <italic>negative</italic> regard (PCNR), conversely, implies a lesser degree of parental affection/warmth as a result of the child <italic>not</italic> meeting parental expectations (<xref rid=\"B4\" ref-type=\"bibr\">Assor et al., 2004</xref>). What the constructs of PCPR and PCNR have in common is that both hinder autonomy and give rise to a number of negative effects (<xref rid=\"B4\" ref-type=\"bibr\">Assor et al., 2004</xref>; <xref rid=\"B60\" ref-type=\"bibr\">Roth et al., 2009</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Assor and Tal, 2012</xref>).</p><p>The authors, in an earlier study (<xref rid=\"B37\" ref-type=\"bibr\">Itzhaki et al., 2018</xref>), argued that in closed traditional societies, CR as practiced by the society could affect the individual in much the same way that CR, as practiced by the parents, would affect him/her. As such, one needs to understand societal conditional regard (SCR) in order to understand individuals’ behaviors in closed religious communities. Societal conditional regard, an adaptation of PCR, refers to the way in which <italic>society</italic> “gives its blessing” to individuals on the basis of their realizing what that particular society expects of them. In a set of studies regarding high-school dropout in the Ultraorthodox Jewish community, the authors found that both PCR and SCR contributed to psychological aspects in the youths’ lives.</p><p>Both PCNR and PCPR were found to contribute to higher levels of loneliness and to lower levels of well-being among high-school dropouts. Societal conditional negative regard (SCNR) made a negative contribution to psychological aspects, i.e., higher levels of SCNR contributed to higher levels of loneliness and lower levels of well-being. However, societal conditional positive regard (SCPR) was found to contribute to higher levels of positive future orientation and well-being (<xref rid=\"B37\" ref-type=\"bibr\">Itzhaki et al., 2018</xref>). Following these novel findings, in the current study, the authors wished to find out whether PCR and SCR might also serve as predictors of high-school dropout among youth from the Ultraorthodox Jewish community, beyond their psychological consequences. According to SDT, less autonomy practices relate to less self-determined motivation among youth, which in turn predicts high-school dropout (<xref rid=\"B68\" ref-type=\"bibr\">Vallerand et al., 1997</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Hardre and Reeve, 2003</xref>). Therefore, the authors examined PCR and SCR in regard to youths who no longer practiced a religious lifestyle that complied with Jewish law (<xref rid=\"B3\" ref-type=\"bibr\">Assor and Friedman, 2005</xref>) and hypothesized that higher levels of conditional regard (CR) would predict their school dropout, as a result of their insecure relationships with both their parents and their community.</p><p>In sum, the goal of this study was to examine the contribution of background variables, personal resources, and social resources to the phenomenon of high-school dropout among Ultraorthodox Jewish youth in Israel. To do so, three groups of students who were at three different stages of the dropout process were examined. Based on social capital theory (<xref rid=\"B15\" ref-type=\"bibr\">Coleman, 1988</xref>), the hypothesis was that older age, belonging to a newly religious family, having a sibling who had dropped out of school previously, having low levels of self-esteem and mastery, and being the recipients of high levels of CR would be predictors of belonging to the dropout group (in comparison to belonging to the other two study groups). By comparing three groups of students representing three distinct levels in the dropout process (i.e., at risk of dropping out; dropping out but participating in alternative educational programs; and dropping out but not participating in any other educational framework), the conditions were set for a deeper inquiry into the contribution of each of the studied predictors to the phenomenon of high-school dropout in a closed religious community.</p></sec></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Participants and Procedure</title><p>Participants comprised 261 Jewish male youths who were born and raised in an Ultraorthodox family and originally were part of the Ultraorthodox community between the ages of 14 and 21. Participants were divided into three research groups representing three different stages in the dropout process. One group consisted of youths in yeshiva high schools who were at risk for dropping out. These youths assigned or received treatment from the social services department designated for yeshiva boys (<italic>n</italic> = 61). A second group consisted of youths who had dropped out of yeshiva and enrolled in a program designed especially for Ultraorthodox high-school dropouts (<italic>n</italic> = 131), and a third group consisted of youths who dropped out of high school and did not enroll in any educational framework (<italic>n</italic> = 69). All participants were members of the same community located in the center of Israel, and they shared the same community background. The average age of the participants was 17 (<italic>SD</italic> = 1.71). Most of the participants (67%) defined themselves, religiously, as Ultraorthodox (or “Haredi”). Some of the participants (19.6%) defined themselves as “not religious,” and the rest of them (13.4%) defined themselves as “religious.”</p><p>Following the approval of data collection and questionnaires by Bar-Ilan University’s institutional review board (IRB), a convenience sample was obtained, and participants were recruited by a variety of youth practitioners who meet the youth in the different services according to the inclusion criteria: male, singles, who originally were part of the Ultraorthodox community, between the ages of 14 and 21. Participants for the yeshiva high school group were recruited by practitioners from the mentoring organizations and from the social services department designated for yeshiva boys. Participants for the program for high-school dropouts were recruited by practitioners who worked in these programs. Participants for the dropout group were recruited by practitioners who worked with dropout Ultraorthodox youth in the street. The practitioners administrate the questionnaires to participants during their meetings. In order to assure anonymity of the respondents, the participants were told not to write their names or provide any identifying information on the forms. Participants’ agreement was voluntary, and participants were told that they were free to stop answering questions whenever they wished to. Of the ∼400 questionnaires that were distributed, 276 of them (or ∼69%) were returned, and 15 of them (about 3.75%) were eliminated due to technical problems, such as partial completion.</p></sec><sec id=\"S2.SS2\"><title>Instruments</title><sec id=\"S2.SS2.SSS1\"><title>Self-Esteem</title><p>This questionnaire was developed by <xref rid=\"B58\" ref-type=\"bibr\">Rosenberg (1965)</xref> and assesses the individual’s sense of self-esteem. It consists of 10 items such as the following: “I feel that I’m a person of worth, at least on an equal plane with others.” The participant in the current study was asked to rate the extent to which he agreed with each item on a 5-point Likert-type scale, ranging from 1 <italic>(strongly disagree)</italic> to 5 <italic>(strongly agree)</italic>. The Cronbach’s alpha reliability of the questionnaire in the present study for this measure was α = 0.77.</p></sec><sec id=\"S2.SS2.SSS2\"><title>Mastery</title><p>This questionnaire was developed by <xref rid=\"B51\" ref-type=\"bibr\">Pearlin and Schooler (1978)</xref> and assesses an individual’s sense of mastery over his surroundings and over the future. It consists of seven items such as “The future and what will happen to me depends mostly on me.” The participant is asked to rate the extent to which he/she agrees with each item on a 5-point Likert-type scale, ranging from <italic>strongly disagree</italic> to <italic>strongly agree</italic>. The Cronbach’s alpha reliability reported by Pearlin and Schooler was α = 0.88, and the reliability of the questionnaire used in the present study was α = 0.78.</p></sec><sec id=\"S2.SS2.SSS3\"><title>Parental Conditional Regard</title><p>This questionnaire was based on the parental conditional regard (PCR) questionnaire originally developed by <xref rid=\"B4\" ref-type=\"bibr\">Assor et al. (2004)</xref>. It assesses the degree of parents’ conditional regard, as experienced by the youth, and also assesses the youth’s perceptions of positive and negative parental conditional regard. In addition, like the research of <xref rid=\"B3\" ref-type=\"bibr\">Assor and Friedman (2005)</xref>, the current research also examined PCR in relation to the individual’s observance of a religious lifestyle and adherence to Jewish law. The questionnaire therefore consisted of 10 items. Results of an exploratory factor analysis yielded the expected two-factor structure with no cross loadings. Five of those items related to parental conditional positive regard (PCPR), such as “When I keep the commandments, I feel that my parents give me more warmth and affection than usual.” The other five items addressed parental conditional negative regard (PCNR), such as “When I do not immerse myself in my studies, my parents give me the feeling that I am not worthy.” The participant was asked to evaluate his agreement on a 5-point Likert-type scale ranging from 1 (<italic>greatly agree)</italic> to 5 (<italic>do not agree at all)</italic>. The Cronbach’s alpha reliability in the present study was.85 for PCPR and.94 for PCNR.</p></sec><sec id=\"S2.SS2.SSS4\"><title>Societal Conditional Regard</title><p>This questionnaire was developed by <xref rid=\"B37\" ref-type=\"bibr\">Itzhaki et al. (2018)</xref> and was based on the parental conditional regard (PCR) questionnaire (<xref rid=\"B4\" ref-type=\"bibr\">Assor et al., 2004</xref>), which has already been described in detail above. While the PCR questionnaire used in this study was adapted with questions regarding observance of a religious lifestyle, the societal conditional regard (SCR) questionnaire was further adapted by replacing the word “parents” with the words “people in the community.” The SCR items were the same as the PCR items, except “my parents” was replaced by “people in the community” (e.g., “If or when I am careful about keeping the commandments, people in the community show me more warmth and affection than usual”). The Cronbach’s alpha reliability of the questionnaire used in the present study was.96 for societal conditional positive regard (SCPR) and.95 for societal conditional negative regard (SCNR).</p></sec><sec id=\"S2.SS2.SSS5\"><title>Sociodemographics</title><p>This questionnaire was used to examine sociodemographic characteristics such as age, place of residence, and educational framework (or lack thereof). In this study, participants were also asked whether their family was newly religious (“<italic>hozrim b’tshuva</italic>”) or whether they had always been Ultraorthodox and whether they had a family member who had previously dropped out of high school.</p></sec></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Correlations and Regressions</title><p>Correlations, means, and standard deviations among the study’s variables are presented in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>. As can be seen, there are significant correlations between some of the variables, without multicollinearity.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Means, standard deviations, and correlations between the study variables.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Measures</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>M</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>SD</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.Age</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17.05</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.71</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.06</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.07</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.03</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2.Newly religious family</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.50</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.20**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.19**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.20**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.13*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.21***</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.11</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3.Dropout family member</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.49</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.09</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.03</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.02</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.02</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4.Self-esteem</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.77</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.72</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.63***</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.44***</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.16**</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.31***</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5.Mastery</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.56</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.89</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–0.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.44***</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.16*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.31***</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">6.PCPR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.96</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.26***</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.31***</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.25***</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7.PCNR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.37</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.38***</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.61***</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8.SCPR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.53</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.59***</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">9.SCNR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.42</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr></tbody></table><table-wrap-foot><attrib><italic>*p < 0.05, **p < 0.01, ***p < 0.001.</italic></attrib></table-wrap-foot></table-wrap><p>In order to examine the effects of the study’s variables as predictors of membership in each study group, we conducted four nominal regression models, where membership in a study group served as the dependent variable in two dummy categories: high-school students vs. dropouts, and youth in a program for dropouts vs. dropouts in no program. In the first model, we examined the contribution of age, newly religious family, and dropout family member. In the second model, we added the personal resources self-esteem and mastery. In the third model, we added the PCPR and the PCNR, and in the last model we added the SCPR and the SCNR. The regression coefficients for high-school students vs. dropouts are displayed in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>. In the first model, having a newly religious family and having a family member who had previously dropped out of school contributed to belonging to the dropout group. In the second model, self-esteem and mastery made no contribution to the group belonging. In the third model, PCR made no contribution to the group belonging. In the final model, entering the SCR, both PCPR and SCNR contributed to belonging to the dropout group. The regression coefficients and correlations for students in a program for dropouts vs. dropouts in no program are displayed in <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>. In the first model, older age contributed to belonging to the dropout group, while having a newly religious family and having a family member who had previously dropped out of school made no significant contribution to group belonging. In the second model, self-esteem and mastery made no contribution to the group belonging. In the third model, PCR made significant contribution to the belonging to the dropout group. In the final model, entering the SCR, PCPR, and SCNR contributed to the belonging to the dropout group. The variables explained 38% of the variance in each of the two models.</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Predictors for high-school dropout among high school students vs. dropouts in the multi-nominal regression models.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Model I<hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Model II<hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Model III<hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Model IV<hr/></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Measures</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B (SE)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Wald (95% CI)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">OR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B (SE)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Wald (95% CI)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">OR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B (SE)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Wald (95% CI)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">OR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B (SE)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Wald (95% CI)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">OR</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Age</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.05 (0.11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.22 (0.85–1.31)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.05</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.04 (0.11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.16 (0.84–1.30)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.04</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.01 (0.12)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.01 (0.81–1.23)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.00 (0.12)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.00 (0.79–1.26)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.00</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Newly religious family</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.75 (0.37)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.02* (0.22–0.98)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.47</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.90 (0.39)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.35* (0.19–0.87)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.00 (0.41)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.77* (0.17–0.84)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.94 (0.42)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.05* (0.17-0.89)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.39</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dropout family member</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.00 (0.038)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.07** (0.18–0.77)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.90 (0.38)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.67* (0.19–0.85)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.88 (0.39)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.03* (0.19–0.89)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.88 (0.40)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.82* (0.19–0.91)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.41</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Self-esteem</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.06 (0.32)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.03 (0.56–2.00)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.06</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.12 (0.36)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.11 (0.44–1.80)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.16 (0.37)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.19 (0.42–1.75)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.85</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mastery</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.4 (0.27)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.40 (0.89–2.54)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.24 (0.29)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.69 (0.72–2.22)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.20 (0.29)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.48 (0.69–2.17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.22</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PCPR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.43 (0.24)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.24 (0.40–1.04)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.65</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.52 (0.26)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.87* (0.35–1.00)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.59</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PCNR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.48 (0.17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.17 (0.44–0.86)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.62</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.33 (0.19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.95 (0.49–1.05)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.72</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCPR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.28 (0.17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.82 (0.95–1.86)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.33</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCNR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.47 (0.19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.89* (0.43–0.91)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.62</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>R</italic><sup>2</sup></td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.21***</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.24***</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.33***</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.38***</td><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic>*p < 0.05, **p < 0.01, ***p < 0.001.</italic></attrib></table-wrap-foot></table-wrap><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>Predictors for high-school dropout stages among youth in a program for dropouts vs. dropouts in the multi-nominal regression model.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Model I<hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Model II<hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Model III<hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Model IV<hr/></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Measures</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B (SE)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Wald (95% CI)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">OR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B (SE)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Wald (95% CI)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">OR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B (SE)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Wald (95% CI)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">OR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B (SE)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Wald (95% CI)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">OR</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Age</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.40 (0.10)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17.33*** (0.55–0.81)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.67</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.40 (0.10)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.85*** (0.55–0.81)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.67</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.43 (0.10)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.48*** (0.53–0.80)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.65</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.46 (0.11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17.75*** (0.51–0.78)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.63</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Newly religious family</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.36 (0.31)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.39 (0.78–2.64)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.44</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.17 (0.32)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.27 (0.63–2.24)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 (0.34)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.06 (0.55–2.14)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.09 (0.36)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.07 (0.54–2.24)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.09</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dropout family member</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.18 (0.30)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.36 (0.46–1.51)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.83</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.06 (0.31)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.04 (0.51–1.73)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.94</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.02 (0.33)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.00 (0.51–1.87)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.98</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.05 (0.34)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.02 (0.49–1.87)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.95</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Self-esteem</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.37 (0.28)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.75 (0.83–2.53)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.45</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.23 (0.32)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.54 (0.68–2.35)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.36</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.19 (0.33)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.33 (0.64–2.29)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.21</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mastery</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.21 (0.23)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.85 (0.79–1.94)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.05 (0.25)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.04 (0.65–1.70)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.05</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.02 (0.26)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.00 (0.61–1.69)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.02</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PCPR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.53 (0.22)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.79* (0.38–0.91)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.59</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.57 (0.24)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.44* (0.35–0.91)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.56</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PCNR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.45 (0.14)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.79** (0.48–0.85)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.18 (0.17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.17 (0.60–1.16)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.83</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCPR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.20 (0.15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.81 (0.91–1.65)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.23</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCNR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.62 (0.18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.45*** (0.38–0.76)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.54</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>R</italic><sup>2</sup></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">0.21***</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">0.24***</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">0.33***</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">0.38***</td><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic>*p < 0.05, **p < 0.01, ***p < 0.001.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS2\"><title>Mediation Model</title><p>A path analysis model was performed using Mplus 8 (<xref rid=\"B50\" ref-type=\"bibr\">Muthén and Muthén, 1998–2017</xref>) to examine the possibility of a mediating model. In the model, the target variables of the model (i.e., high school students vs. dropouts, and students in program for dropouts vs. dropouts) were expected to respond to the study variables. Based on the regression findings, we entered into the model only the variables that made significant contributions to the regression models (i.e., older age, being from a newly religious family, having a family member who had previously dropped out of school, PCR, and SCR). In our path analysis model, we estimated indirect effects from the study’s background variables (i.e., older age, being from a newly religious family, and having a family member who had previously dropped out of school) to outcome variables (i.e., the three study groups) through mediators such as PCPR, PCNR, SCPR, and SCNR. All possible paths from independent variables to dependent variables were estimated.</p><p><xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref> presents the standardized β coefficients and SEs of the direct effects found to be significant in the path analysis modeling, <italic>p</italic> < 0.05 and lower (AIC = 5502, BIC = 5677). <xref rid=\"T4\" ref-type=\"table\">Table 4</xref> presents the β coefficients and SEs of the indirect effects in the path analysis modeling.</p><table-wrap id=\"T4\" position=\"float\"><label>TABLE 4</label><caption><p>Betas and SEs of the indirect effects of the path analysis model.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Independent</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mediator</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dependent</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Independent to mediator</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mediator to dependent</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Direct effect</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Indirect effect</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">95% CI</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Newly religious family</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PCNR</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCPR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.40* (0.17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.34*** (0.06)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.19*** (0.03)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.04* (0.02)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(−0.258, −0.014)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Newly religious parents</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PCNR</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCNR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.40* (0.17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.61*** (0.05)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.03 (0.05)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.09* (0.04)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(−0.451, −0.040)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dropout family member</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PCNR</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCNR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.37 (0.18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.61*** (0.05)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.03 (0.05)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(0.04)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(0.010, 0.440)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PCNR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SCNR</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">High school students vs. Dropouts</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.61*** (0.05)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.48** (0.17)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.35* (0.16)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.30** (0.11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(−0.513, −0.085)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PCNR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SCNR</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Program for dropouts vs. Dropouts</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.61*** (0.05)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.62*** (0.18)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.21 (0.15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.38*** (0.12)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(−0.612, −0.159)</td></tr></tbody></table><table-wrap-foot><attrib><italic>*p < 0.05, **p < 0.01, ***p < 0.001.</italic></attrib></table-wrap-foot></table-wrap><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Standardized results of the path anlysis model of socio-demographic variables, PCR and SCR on stages in the dropout process.</p></caption><graphic xlink:href=\"fpsyg-11-01911-g001\"/></fig><p>Regarding the connection between the study’s variables and “high school students vs. dropouts” group, the findings point to a direct negative correlation between having a newly religious family and having a family member who had previously dropped out of school and between the group. It was found that high school students were less likely to be part of newly religious families (β = −0.38) or to have family members who had previously dropped out of school (β = −0.31) than were dropouts.</p><p>A direct negative correlation was also found between PCPR, PCNR, and SCNR and between the groups. High school students experienced less PCPR (β = −0.38), less PCNR (β = −0.35), and less SCNR (β = −0.55), than did dropouts. Parental conditional negative regard also made an indirect contribution to the group via SCNR (β = −0.30).</p><p>Regarding the connection between the study’s variables and the “students in a program for dropouts vs. dropouts” group, the findings point to a direct negative correlation between age and group. It was found that students in a program for dropouts were older than dropouts who were in no educational framework (β = −0.54).</p><p>A direct negative correlation was also found between PCPR and SCNR and between the groups. Students in a program for dropouts experienced less PCPR (β = −0.36) and less SCNR (β = −0.63) than did dropouts who were in no educational framework. Parental conditional negative regard also made an indirect contribution to the group via SCNR (β = −<italic>0.38)</italic>.</p></sec></sec><sec id=\"S4\"><title>Discussion</title><p>The current study’s findings reveal unique predictive factors of high-school dropout among youth from an Ultraorthodox Jewish community in Israel. Self-esteem, and mastery, which are generally considered to be significant predictors for school dropout among youth, made no significant contribution to school dropout in the current study. Conditional regard (CR), on the other hand, a construct examined for the first time as a predictor of youth dropout, was indeed found to be a significant predictor, both in terms of parental CR and societal CR.</p><p>Consistent with previous studies, both “having a sibling who dropped out of school” and “belonging to a newly religious family” were found to be predictors of high-school dropout (<xref rid=\"B63\" ref-type=\"bibr\">Rumberger and Thomas, 2000</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Itzhaki, 2009</xref>; <xref rid=\"B70\" ref-type=\"bibr\">Yogev, 2012</xref>). A sibling who drops out of yeshiva may serve as an inspiration for a brother who feels he does not “fit in” or who wants to leave the yeshiva as well. A sibling who drops out of yeshiva may also provide his brother with the social support needed to make such a move.</p><p>In terms of the role played by “belonging to a newly religious family,” in the prediction of high-school dropout, poor family socialization theory may shed light on this finding. <xref rid=\"B6\" ref-type=\"bibr\">Battin-Pearson et al. (2000)</xref> used this theory as one of five selected theories to explain predictors of high-school dropout. In their study, they recognized parents’ poor resources as being a predictor of the child’s dropout. In addition, early family socialization is considered to be an influencing factor on school dropout among youth (<xref rid=\"B61\" ref-type=\"bibr\">Rumberger, 1983</xref>). Given that a newly religious family’s original socialization took place in the secular world, they must essentially start over, with no religious resources at hand, or knowledge about Ultraorthodox community’s norms. As such, for these families, “poor family socialization” may indicate their lack of religious belonging and knowledge (i.e., their “outsider-ness” in this very “insider” world), much the way poor family socialization in other contexts indicates factors such as parents’ lack of education, and parents’ divorce (<xref rid=\"B6\" ref-type=\"bibr\">Battin-Pearson et al., 2000</xref>).</p><p>In this study, neither self-esteem nor mastery was found to be a predictor of high-school dropout among Ultraorthodox Jewish youth. This finding is surprising given that a lack of personal resources in general, and lower self-esteem and mastery in particular, has been found to be characteristic of at-risk youth and connected to high-school dropout (<xref rid=\"B20\" ref-type=\"bibr\">Dillon, 2004</xref>; <xref rid=\"B2\" ref-type=\"bibr\">Aristilde, 2006</xref>; <xref rid=\"B49\" ref-type=\"bibr\">Metz, 2006</xref>). These findings may point to the unique method of education in the Ultraorthodox community; that is, to prioritize the community’s needs over the individual’s needs (<xref rid=\"B65\" ref-type=\"bibr\">Shor, 1998</xref>). When the community and family serve as such strong central controlling factors in the youths’ lives, they may become more significant than the youths’ personal resources. The reason that dropping out of high school in the Ultraorthodox Jewish community makes an even bigger statement than it does in other communities is precisely because of the priority ordinarily given by the individual to community and family sentiment, over personal sentiment.</p><p>The role of family and of community as predictors of high-school dropout was investigated via the construct of conditional regard (CR). Parental conditional positive regard was found to be a predictor of high-school dropout both (1) among dropouts vs. high school students and (2) among students in a program for dropouts vs. dropouts. These findings are consistent with previous studies that pointed to a negative contribution of PCR in regard to the well-being and loneliness of Ultraorthodox Jewish high-school dropouts (<xref rid=\"B37\" ref-type=\"bibr\">Itzhaki et al., 2018</xref>) and also with the existing literature, which presents PCPR as a practice that suppresses autonomy and leads to non-optimal self-esteem dynamics, with many negative effects such as compulsive over-investment, avoidance of challenge, and failing to develop the capacity to regulate negative emotions (<xref rid=\"B4\" ref-type=\"bibr\">Assor et al., 2004</xref>; <xref rid=\"B60\" ref-type=\"bibr\">Roth et al., 2009</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Assor and Tal, 2012</xref>). Although <xref rid=\"B59\" ref-type=\"bibr\">Roth and Assor (2010)</xref> PCPR can be considered as an effective method in determining youth behavior in accordance with their parents’ expectations, it seems that for Ultraorthodox Jewish youth it can be a predictor of dropping out of high school – obviously an undesirable behavior.</p><p>Regarding PCNR, comparing high school students with dropouts, this construct made a significant contribution to group belonging both directly and indirectly via SCNR. Given the literature regarding PCNR, describing its many negative effects (<xref rid=\"B4\" ref-type=\"bibr\">Assor et al., 2004</xref>; <xref rid=\"B60\" ref-type=\"bibr\">Roth et al., 2009</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Assor and Tal, 2012</xref>), the contribution of PCNR in this study is not surprising. High levels of PCNR experienced by the Ultraorthodox Jewish youngsters seemed to be a predictor of dropout. As high levels of parental closeness (closeness, satisfaction, warmth, and satisfaction with parental communication) have been found to contribute to lower odds of dropping out (<xref rid=\"B52\" ref-type=\"bibr\">Perreira et al., 2006</xref>), the lack of parental regard/love likely lessens the youth’s feeling of closeness to the parent and thus serves as a predictor of dropout.</p><p>However, comparing students in a program for dropouts vs. dropouts who were not in any type of educational framework, PCNR contributed to the group only indirectly via SCNR. As such, it appears that at some point between enrolling in an alternative educational framework and dropping out of school altogether, the parental aspect shifted to a communal aspect. This finding can perhaps be explained as follows: Yeshiva students who are at risk of dropping out are nevertheless still in the acceptable framework (they have not dropped out yet), and therefore, their “situation” is at this point known only to their parents, not to the community at large. However, when these students take the next step, leaving their sanctioned yeshivas and enrolling in alternative programs, their situation becomes “known,” as do community members’ attitudes. The community attitude, as reflected by SCNR, then becomes significant for the parents’ attitude, as reflected by PCNR.</p><p>In the current study, SCPR was not found to be a predictor of high-school dropout. According to self-determination theory (<xref rid=\"B64\" ref-type=\"bibr\">Ryan and Deci, 2000</xref>), both SCPR and SCNR would be expected to have negative consequences. However, contrary to expectations, SCPR was found in an earlier study to contribute to high levels of well-being, future orientation, and sense of community (<xref rid=\"B37\" ref-type=\"bibr\">Itzhaki et al., 2018</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Itzhaki and Cnaan, 2019</xref>). It may be that participants in the current study reacted to SCPR not as a gratuitous or unwanted judgment of character, but rather as legitimate, i.e., as part and parcel of community membership. As such, it had no significant role in the dropout process. However, SCNR did have a significant role; that is, the youth clearly felt that their acceptance was predicated on giving up negatively perceived behaviors. This tension, as expressed by experiencing SCNR, was the strongest predictor of dropout in both models (i.e., high school students vs. dropouts, and students in a program for dropouts vs. dropouts). This finding is consistent with earlier studies that pointed to the negative psychological consequences of SCNR among high-school dropouts from the Ultraorthodox Jewish community (i.e., higher levels of loneliness and lower levels of well-being). The understanding that beyond the psychological harm SCNR can also serve as a predictor of dropout was a novel aspect of the current study, shedding light on the significant role played by the community in the dropout process of Ultraorthodox Jewish youth.</p><sec id=\"S4.SS1\"><title>Recommendations and Limitations</title><p>The unique relationship between religion and high-school dropout have been investigated before. Religious affiliation and high levels of religious involvement among mainline Protestant, conservative Protestant, Catholic, and Mormons were found to be a significant contribution in preventing high-school dropout (<xref rid=\"B44\" ref-type=\"bibr\">Lehrer, 2006</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Cohen-Zada and Sander, 2008</xref>). However, there is not enough data, regarding risk and protective factors for dropout among these religious communities.</p><p>Although the present study sheds new light on risk and protective factors in explaining high-school dropout among Ultraorthodox Jewish youth, in order to generalize the current study’s finding to other religious closed communities, it would be important to investigate the role of conditional regard in other types of closed religious communities as well. Although every community has its own unique characteristic, some characteristics are common to religious communities as a whole, such as the significance of the community to its members, the importance of observing community and religious norms, and sanctions made by the community leaders and the community members when one is violating these norms (<xref rid=\"B11\" ref-type=\"bibr\">Bromley, 1991</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Cates and Weber, 2013</xref>). We believe that expanding the research in this area would further our understanding of these unique characteristics, especially those related to violation of specific communities’ norms.</p><p>In terms of implementing findings, the present study points to the important role of social capital and SCR in regard to the phenomenon of high-school dropout in closed religious communities. We would recommend professionals who work with youths who are either at risk of dropping out of school or who have already done so, to focus on youths’ social capital and familiarize themselves with using conditional regard by parents and community members, and the consequences of doing so. Program interventions focusing on reducing the use of the CR practice may help in preventing youth dropout.</p><p>The current study had a few limitations. First, there were challenges in getting the agreement of the target population to participate in the research due to the closeness of the Ultraorthodox community. This challenge required our use of self-reports for the independent variables. Although self-reports are acceptable and were found reliable in other studies that have been conducted among high-school dropouts, further studies should add other forms of data, such as parents’ perception and educational success. Second, the aspect of students’ academic success/failure at school has not been examined in the current study, and it could have broadened the understanding of the high-school dropout phenomenon. We recommend that this aspect be investigated in further studies. Moreover, the study was based on cross-sectional design, which is acceptable in social sciences’ studies. However, further studies should investigate the way changes in the independent variables contribute to the dropout process. In addition, longitudinal studies may be relevant for this examination. These examinations would strength the current study’s recommendations.</p></sec></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.</p></sec><sec id=\"S6\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by the Bar-Ilan University’s Institutional Review Board (IRB). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>YI-B conceived of the study, participated in the design, data collection, analysis for the study, and drafted the manuscript. HI and YY conceived of the study, participated in its design, and contributed to drafts of the manuscript. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"methods-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Plant Sci</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Plant Sci</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Plant Sci.</journal-id><journal-title-group><journal-title>Frontiers in Plant Science</journal-title></journal-title-group><issn pub-type=\"epub\">1664-462X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849754</article-id><article-id pub-id-type=\"pmc\">PMC7431704</article-id><article-id pub-id-type=\"doi\">10.3389/fpls.2020.01218</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Plant Science</subject><subj-group><subject>Methods</subject></subj-group></subj-group></article-categories><title-group><article-title>A Leaf-Mimicking Method for Oral Delivery of Bioactive Substances Into Sucking Arthropod Herbivores</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Ghazy</surname><given-names>Noureldin Abuelfadl</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref><xref ref-type=\"author-notes\" rid=\"fn001\">\n<sup>*</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/1034445\"/></contrib><contrib contrib-type=\"author\"><name><surname>Okamura</surname><given-names>Mayo</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/1052149\"/></contrib><contrib contrib-type=\"author\"><name><surname>Sai</surname><given-names>Kanae</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/1052126\"/></contrib><contrib contrib-type=\"author\"><name><surname>Yamakawa</surname><given-names>Sota</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/570358\"/></contrib><contrib contrib-type=\"author\"><name><surname>Hamdi</surname><given-names>Faten Abdelsalam</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/1052162\"/></contrib><contrib contrib-type=\"author\"><name><surname>Grbic</surname><given-names>Vojislava</given-names></name><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff5\">\n<sup>5</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/352841\"/></contrib><contrib contrib-type=\"author\"><name><surname>Suzuki</surname><given-names>Takeshi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff6\">\n<sup>6</sup>\n</xref><xref ref-type=\"author-notes\" rid=\"fn001\">\n<sup>*</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/196722\"/></contrib></contrib-group><aff id=\"aff1\">\n<sup>1</sup>\n<institution>Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology</institution>, <addr-line>Koganei</addr-line>, <country>Japan</country>\n</aff><aff id=\"aff2\">\n<sup>2</sup>\n<institution>Agriculture Zoology Department, Faculty of Agriculture, Mansoura University</institution>, <addr-line>El-Mansoura</addr-line>, <country>Egypt</country>\n</aff><aff id=\"aff3\">\n<sup>3</sup>\n<institution>Japan Society for the Promotion of Science</institution>, <addr-line>Chiyoda</addr-line>, <country>Japan</country>\n</aff><aff id=\"aff4\">\n<sup>4</sup>\n<institution>Department of Biology, The University of Western Ontario</institution>, <addr-line>London, ON</addr-line>, <country>Canada</country>\n</aff><aff id=\"aff5\">\n<sup>5</sup>\n<institution>Instituto de Ciencias de la Vid y el Vino</institution>, <addr-line>Logrono</addr-line>, <country>Spain</country>\n</aff><aff id=\"aff6\">\n<sup>6</sup>\n<institution>Institute of Global Innovation Research, Tokyo University of Agriculture and Technology</institution>, <addr-line>Koganei</addr-line>, <country>Japan</country>\n</aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Carolina Escobar, University of Castilla-La Mancha, Spain</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Wannes Dermauw, Ghent University, Belgium; Juan Manuel Alba, University of Amsterdam, Netherlands</p></fn><corresp id=\"fn001\">*Correspondence: Noureldin Abuelfadl Ghazy, <email xlink:href=\"mailto:noureldinghazy@mans.edu.eg\" xlink:type=\"simple\">noureldinghazy@mans.edu.eg</email>; Takeshi Suzuki, <email xlink:href=\"mailto:tszk@cc.tuat.ac.jp\" xlink:type=\"simple\">tszk@cc.tuat.ac.jp</email>\n</corresp><fn fn-type=\"other\" id=\"fn002\"><p>This article was submitted to Plant Pathogen Interactions, a section of the journal Frontiers in Plant Science</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1218</elocation-id><history><date date-type=\"received\"><day>09</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>27</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Ghazy, Okamura, Sai, Yamakawa, Hamdi, Grbic and Suzuki</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Ghazy, Okamura, Sai, Yamakawa, Hamdi, Grbic and Suzuki</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Spider mites (Acari: Tetranychidae) are pests of a wide range of agricultural crops, vegetables, and ornamental plants. Their ability to rapidly develop resistance to synthetic pesticides has prompted the development of new strategies for their control. Evaluation of synthetic pesticides and bio-pesticides—and more recently the identification of RNA interference (RNAi) target genes—requires an ability to deliver test compounds efficiently. Here we describe a novel method that uses a sheet-like structure mimicking plant leaves and allows for oral delivery of liquid test compounds to a large number of individuals in a limited area simultaneously (~100 mites cm<sup>−2</sup>). The main component is a fine nylon mesh sheet that holds the liquid within each pore, much like a plant cell, and consequently allows for greater distribution of specific surface area even in small amounts (10 µl cm<sup>−2</sup> for 100-µm mesh opening size). The nylon mesh sheet is placed on a solid plane (<italic>e.g.</italic>, the undersurface of a Petri dish), a solution or suspension of test compounds is pipetted into the mesh sheet, and finally a piece of paraffin wax film is gently stretched above the mesh so that the test mites can feed through it. We demonstrate the use of the method for oral delivery of a tracer dye (Brilliant Blue FCF), pesticides (abamectin and bifenazate), dsRNA targeting the <italic>Vacuolar-type H<sup>+</sup>-VATPase</italic> gene, or fluorescent nanoparticles to three species of <italic>Tetranychus</italic> spider mites (Acari: Tetranychidae) and to the cotton aphid, <italic>Aphis gossypii</italic> Glover (Hemiptera: Aphididae). The method is fast, easy, and highly reproducible and can be adapted to facilitate several aspects of bioassays.</p></abstract><kwd-group><kwd>aphids</kwd><kwd>artificial diet</kwd><kwd>membrane feeding assay</kwd><kwd>spider mites</kwd><kwd>RNAi</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Japan Society for the Promotion of Science<named-content content-type=\"fundref-id\">10.13039/501100001691</named-content></funding-source></award-group><award-group><funding-source id=\"cn002\">Japan Science and Technology Agency<named-content content-type=\"fundref-id\">10.13039/501100002241</named-content></funding-source></award-group><award-group><funding-source id=\"cn003\">Japan Society for the Promotion of Science<named-content content-type=\"fundref-id\">10.13039/501100001691</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"5\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"41\"/><page-count count=\"11\"/><word-count count=\"6099\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Effective delivery of xenobiotics to herbivorous arthropods is a prerequisite for evaluating synthetic pesticides and bio-pesticides. The delivery method should be simple and reproducible and allow for precise estimation of the lethal concentration of a candidate agent (<xref rid=\"B19\" ref-type=\"bibr\">Kabir et al., 1993</xref>). Several methodologies are used for evaluating synthetic pesticides against spider mites (Acari: Tetranychidae), including direct spraying, leaf-dip, slide-dip, residual contacted vial, and membrane feeding bioassays (<italic>e.g.</italic>, <xref rid=\"B28\" ref-type=\"bibr\">Potter, 1952</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Dittrich, 1962</xref>; <xref rid=\"B16\" ref-type=\"bibr\">Hanna and Hibbs, 1970</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Kwon et al., 2010</xref>).</p><p>Pesticide spraying—usually using the Potter spray tower that simulates field application (<xref rid=\"B28\" ref-type=\"bibr\">Potter, 1952</xref>)—is probably the most common method of assessing toxicity and resistance in mites. This method, however, requires costly laboratory equipment and a large liquid volume. In the leaf-dip method, an infested or uninfested plant leaf is dipped into a test solution for about 5 s and then air dried (<italic>e.g.</italic>, <xref rid=\"B11\" ref-type=\"bibr\">Dittrich, 1962</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Knight et al., 1990</xref>). A major source of variability is the uneven distributions of the residues and test mites on the dipped leaf in addition to the high probability of mite escape from the treated leaf. In the slide-dip method, mites are affixed on double-sided adhesive tape attached to a glass slide, and then the whole set is dipped into a test solution for several seconds (<xref rid=\"B11\" ref-type=\"bibr\">Dittrich, 1962</xref>). Both the leaf-dip and slide-dip methods share the common drawback of requiring a large volume of test solution. Moreover, according to <xref rid=\"B11\" ref-type=\"bibr\">Dittrich (1962)</xref>, when using the slide-dip method, about 2 h of preparation is needed to affix <italic>ca.</italic> 60 mites onto 10 slides. The residual contacted vial method is used to assay pesticide resistance in field populations (<xref rid=\"B23\" ref-type=\"bibr\">Kwon et al., 2010</xref>); the inner surface of a 5-ml glass vial is coated with 100 µl of an acetone-based test solution and kept for about 1 h under a fume hood until the acetone is completely dried. Although the preparation is simple because plant material is not required, mite handling and mortality scoring after treatment, particularly under non-lethal concentrations, can be problematic. The membrane feeding method was originally developed as a feeding device for the beet leafhopper, <italic>Citculifer tenellus</italic> Baker (Hemiptera: Cicadellidae); artificial diet was placed in a sachet made of fish-skin membrane so that the test insects could feed through it (<xref rid=\"B5\" ref-type=\"bibr\">Carter, 1927</xref>). <xref rid=\"B26\" ref-type=\"bibr\">Mittler and Dadd (1962)</xref> later developed a membrane feeding device using an extensible and waterproof paraffin wax film (<italic>i.e.</italic>, Parafilm) for the green peach aphid, <italic>Myzus persicae</italic> (Sulzer) (Hemiptera: Aphididae). Using the Parafilm membrane feeding method, many nutritional and pharmacological studies have been carried out on aphids, spider mites, planthoppers, thrips, bedbugs, whiteflies, and mosquitoes (<xref rid=\"B9\" ref-type=\"bibr\">Dadd and Mittler, 1966</xref>; <xref rid=\"B40\" ref-type=\"bibr\">Walling et al., 1968</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Mitsuhashi and Koyama, 1969</xref>; <xref rid=\"B16\" ref-type=\"bibr\">Hanna and Hibbs, 1970</xref>; <xref rid=\"B39\" ref-type=\"bibr\">van der Geest et al., 1983</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Montes et al., 2002</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Gotoh et al., 2008</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Upadhyay et al., 2011</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Costa-da-Silva et al., 2013</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Torres-Quintero et al., 2013</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Suzuki et al., 2017a</xref>). Although the membrane feeding method was successfully used, the small specific surface area of the feeding arena that coincides with the liquid under the membrane limits the efficiency of the bioassays. Overall, these methods are not suitable for the delivery of extracts/compounds that are available in small amounts and, with the exception of spraying, are less feasible for testing pesticide toxicity on mite immature stages.</p><p>In addition to the delivery of chemical compounds, there is also a need for efficient delivery of double-stranded RNA (dsRNA) for RNA interference (RNAi)-based functional genomics and pest management. To date, the dsRNA is delivered into mites using three common methods: microinjection, soaking, or orally <italic>via</italic> plant leaf discs. Microinjection is widely used for delivering dsRNA into nematodes and insects (<xref rid=\"B12\" ref-type=\"bibr\">Fire et al., 1998</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Bucher et al., 2002</xref>). Although microinjection was used for dsRNA delivery to the two-spotted spider mite, <italic>Tetranychus urticae</italic> (Acari: Tetranychidae) (<xref rid=\"B21\" ref-type=\"bibr\">Khila and Grbić, 2007</xref>), this method is not feasible for practical application due to the difficulty of injecting mites that are ~0.5 mm in length and the possibility of causing physical damage (<xref rid=\"B29\" ref-type=\"bibr\">Price and Gatehouse, 2008</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Yu et al., 2013</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Suzuki et al., 2017a</xref>). Delivery of dsRNA <italic>via</italic> soaking mites in dsRNA solution was also demonstrated, but the difficulties of recovering mites after soaking and soaking of immature stages reduce the utility of this method for high-throughput RNAi screens (<xref rid=\"B34\" ref-type=\"bibr\">Suzuki et al., 2017a</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Suzuki et al., 2017b</xref>). Ultimately, delivering dsRNA orally <italic>via</italic> feeding is the most attractive method because it is the least invasive (<italic>i.e.</italic>, entails no physical damage to test organisms) and is conducive to dsRNA application as a bio-pesticide.</p><p>The leaf disc-mediated oral delivery of dsRNA (<italic>i.e.</italic>, foliar application) is widely used for triggering RNAi in spider mite species. <xref rid=\"B24\" ref-type=\"bibr\">Kwon et al. (2013)</xref> used leaf discs floating on a dsRNA solution to orally deliver dsRNA to mites. This method requires a large volume of dsRNA solution and consequently a large amount of dsRNA. Leaf disc dehydration is a modified method of foliar application of dsRNA used for the carmine spider mite, <italic>Tetranychus cinnabarinus</italic> (Boisduval), where plant leaf discs are dehydrated at 60°C for 3 min and then soaked in a dsRNA solution for 5 h (<xref rid=\"B33\" ref-type=\"bibr\">Shi et al., 2015</xref>). Thereafter, the surface-dried leaf discs are desiccated and used for the bioassay. Broad application of this method is uncertain, however, because the preparation of leaf discs requires a long time, and different plant species may vary in their tolerance to high-temperature dehydration. More recently, <xref rid=\"B35\" ref-type=\"bibr\">Suzuki et al. (2017b)</xref> have used the leaf coating approach to deliver dsRNA into <italic>T. urticae</italic>. Although this method uses a small volume (~7.6 μL cm<sup>−2</sup> leaf disc) of dsRNA solution compared to the floating leaf discs, it requires the manual spreading of dsRNA in order to cover an entire leaf surface. Alternatively, a surfactant such as Silwet L-77 can be used to promote liquid dispersion, but it has been shown to have a negative impact on some test organisms (<xref rid=\"B8\" ref-type=\"bibr\">Cowles et al., 2000</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Ray and Hoy, 2014</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Abouelmaaty et al., 2019</xref>). Hence, identification of a high-throughput dsRNA delivery method to mites remains a great challenge.</p><p>We recently developed the mesh method for delivering a test solution or suspension to sucking arthropods. This method allows easy manipulation of test arthropods, uses a small liquid volume, requires no plant material, and the overall preparation requires no specialized skills. Here, we demonstrate the use of the mesh method to deliver tracer dye (Brilliant Blue FCF), pesticides (abamectin and bifenazate), dsRNA targeting the <italic>Vacuolar-type H<sup>+</sup>-VATPase</italic> gene, and fluorescent nanoparticles to TSSM. In addition, we also delivered the tracer dye to tomato red mite, <italic>Tetranychus evansi</italic> Baker and Pritchard, Kanzawa spider mite, <italic>Tetranychus kanzawai</italic> Kishida, and cotton aphid, <italic>Aphis gossypii</italic> Glover (Hemiptera: Aphididae), to test the flexibility of our method. We anticipate that this method will enable large-scale, high-throughput screens of active ingredients of synthetic pesticides and bio-pesticides that include environmental RNAi-based pesticides in spider mites and other sucking herbivores.</p></sec><sec sec-type=\"materials|methods\" id=\"s2\"><title>Materials and Methods</title><sec id=\"s2_1\"><title>Mite and Aphid Colonies</title><p>The reference population of <italic>T. urticae</italic> was established in early 2000 and has previously been used for whole-genome sequencing (<xref rid=\"B15\" ref-type=\"bibr\">Grbić et al., 2011</xref>). The <italic>T. urticae</italic> population is maintained in the laboratory on seedlings of kidney beans (<italic>Phaseolus vulgaris</italic> L.) at an air temperature of 25°C, relative humidity of 50%, and light period of 16 h day<sup>−1</sup>. The population of <italic>T. evansi</italic> was collected from black nightshade (<italic>Solanum nigrum</italic> L.) in Tokyo, Japan, in 2006 (<xref rid=\"B14\" ref-type=\"bibr\">Gotoh et al., 2009</xref>). The <italic>T. evansi</italic> population is maintained in the laboratory on detached leaves of eggplant (<italic>Solanum melongena</italic> L. cv. Senryo #2). The <italic>T. kanzawai</italic> population was collected from red clover (<italic>Trifolium pratense</italic> L.) in Sobetsu, Hokkaido, Japan in 2008 and is routinely reared on detached leaves of <italic>P. vulgaris</italic>. The population of <italic>A. gossypii</italic> is maintained in the laboratory on detached leaves of <italic>P. vulgaris</italic>. An air pump-based system (<xref rid=\"B6\" ref-type=\"bibr\">Cazaux et al., 2014</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Suzuki et al., 2017a</xref>) was used to collect mites or aphids used in the following experiments.</p></sec><sec id=\"s2_2\"><title>Preparation of the Feeding Device in the Mesh Method</title><p>In general, the feeding device consists of three main components: a waterproof solid plane (e.g., the undersurface of a Petri dish), a nylon mesh sheet with an opening size of 50, 100, or 500 µm (#62-0866-38, #2-9566-05 or #2-9566-03; As One, Osaka, Japan) and a paraffin wax film (Parafilm M; Bemis, Neenah, WI, USA). First, a piece of the mesh sheet is placed on the undersurface of a polystyrene Petri dish flipped upside down (<xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figure 1A</bold>\n</xref>). Second, an appropriate volume (see below) of test solution or suspension is pipetted into the mesh sheet. Third, the entire mesh sheet is covered with a piece of Parafilm stretched to almost four times its original area to prevent evaporation of the liquid and to allow test arthropods to suck the underlying liquid by piercing the Parafilm membrane (<xref ref-type=\"supplementary-material\" rid=\"SM1\">\n<bold>Supplementary Video S1</bold>\n</xref>). Finally, the liquid-filled area of the mesh sheet is surrounded with a wetted Kimwipe (Nippon Paper Crecia, Tokyo, Japan) or an adhesive (Tangle B; Fuji Pharm, Tokyo, Japan) to form a feeding arena into which mites or aphids, respectively, can be placed. <xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figures 1B, C</bold>\n</xref> represent the assembled feeding devices for mites and aphids, respectively. A food dye (Brilliant Blue FCF; Fujifilm Wako Pure Chemical, Osaka, Japan) was used to visualize the distribution of liquid applied in the mesh sheet and to trace it in the digestive tracts of test arthropods. The structure of the feeding device and its use for oral administration are patent pending (Japanese Patent Application No. 2018-197157).</p><fig id=\"f1\" position=\"float\"><label>Figure 1</label><caption><p>Description of the feeding device in the mesh method. <bold>(A)</bold> Schematic of the procedure for preparation of the feeding device in the mesh method. Prepared feeding device for <bold>(B)</bold> mites and <bold>(C)</bold> aphids, in which a 1 or 2.5% (w/v) blue tracer dye (Brilliant Blue FCF) was added into the nylon mesh sheet (2 × 2 cm) with a 100- or 500-µm opening size, respectively. Scale bar: 1 cm. <bold>(D)</bold> The relationship between the volume of tracer dye solution and the solution-filled area of the nylon mesh sheet (2 × 2 cm) with a 50-, 100-, or 500-µm opening size. Data were collected from three independent experimental runs and are presented with a regression line and a 95% confidence interval band.</p></caption><graphic xlink:href=\"fpls-11-01218-g001\"/></fig></sec><sec id=\"s2_3\"><title>Liquid Volume Required for the Feeding Device</title><p>To determine the volume of liquid required to fill the entire mesh sheet (4 cm<sup>2</sup>) with opening sizes of 50, 100, or 500 µm in the feeding device, we tested 10 to 160 µl of the 1% (w/v) blue dye solution. The test was conducted in three independent experimental runs for each volume and mesh opening size. The feeding devices were then scanned with an image scanner (GTX980; Seiko Epson, Suwa, Japan), and the proportion of the area filled with the blue dye solution was determined using an image processing program (ImageJ 1.52f).</p></sec><sec id=\"s2_4\"><title>Time Required for <italic>T. urticae</italic> Feeding</title><p>Adult emergence of mites was synchronized as described previously (<xref rid=\"B34\" ref-type=\"bibr\">Suzuki et al., 2017a</xref>). To determine the time required for mite feeding in the mesh method, about 150 newly emerged adult <italic>T. urticae</italic> females were placed onto the feeding device using the nylon mesh sheet (2 cm<sup>2</sup>) with a 100-µm opening to which 40 µl of 1% (w/v) blue dye solution was added. The <italic>T. urticae</italic> females were allowed to feed on the solution for 1 to 4 h under standard laboratory conditions. Mite feeding was determined by the change of body color. The test was conducted in three independent experimental runs.</p></sec><sec id=\"s2_5\"><title>dsRNA Synthesis</title><p>Total RNA was extracted from about 800 adult TSSM females frozen in liquid nitrogen with NucleoSpin RNA Plus (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. The quality and quantity of RNA were measured using a spectrophotometer (NanoPhotometer N60; Implen, Munich, Germany). cDNA was synthesized from 3 µg of total RNA using reverse transcriptase (SuperScript II Reverse Transcriptase; Thermo Fisher Scientific, Waltham, MA, USA) and an oligo (dT)<sub>12–18</sub> primer (Thermo Fisher Scientific) according to the manufacturer’s protocol. cDNA was then stored at −30°C until use. Genomic DNA (gDNA) was extracted using a NucleoSpin Tissue extraction kit (Macherey-Nagel) and stored at −30°C. Using cDNA or gDNA as a template, specific primers targeting a 600-bp fragment of the <italic>TuVATPase</italic> gene (<italic>tetur09g04140</italic>) or a 382-fragment of the intergenic region (negative control [NC], genomic coordinates: scaffold 12, position 1690614–1690995) were used, respectively, for PCR amplification using DNA polymerase (Phusion High-Fidelity DNA Polymerase; New England Biolabs, Ipswich, MA, USA). Primers designed to amplify the DNA fragments of <italic>TuVATPase</italic> and NC are shown in <xref rid=\"T1\" ref-type=\"table\">\n<bold>Table 1</bold>\n</xref>. DNA fragments were then purified with NucleoSpin Gel and PCR Clean-Up Kit (Macherey-Nagel). The integrity of the purified DNA fragments was further confirmed with 2% (w/v) agarose gel (Agarose 21; Nippon Gene, Tokyo, Japan) electrophoresis, quantified with the spectrophotometer, and stored at −30°C until use. A template of 0.1 µg of each DNA fragment was used for RNA synthesis with an <italic>in vitro</italic> Transcription T7 Kit (Takara Bio, Kusatsu, Japan) in 1.5-ml centrifuge tubes according to the manufacturer’s protocol. After DNase I (Takara Bio) treatment for 30 min, RNA was denatured at 95°C for 5 min followed by slow cool-down to room temperature to facilitate dsRNA formation (<xref rid=\"B35\" ref-type=\"bibr\">Suzuki et al., 2017b</xref>). The dsRNA fragments (dsRNA-<italic>TuVATPase</italic> and dsRNA-NC) were purified by phenol-chloroform extract and precipitated with ethanol, quantified, and confirmed with the spectrophotometer and 2% (w/v) agarose gel, respectively.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Primers used in this study for dsRNA production and real-time qRT-PCR analysis.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" colspan=\"3\" align=\"left\" rowspan=\"1\">PCR amplification primers</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Primer name</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Oligonucleotide sequence (5′ to 3′)<sup>a</sup>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Size (bp)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tetur-VATP-F</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GTTGCGGTGAGAGAGGTAATG</td><td valign=\"top\" rowspan=\"2\" align=\"left\" colspan=\"1\">600</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tetur-VATP-R</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GAAGAGGTACGAAATCTGGG</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tetur-sc12-F</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GCCCTCTCCTGGTTGTAAACTT</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">382<sup>b</sup>\n</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tetur-sc12-R</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CGACCCCATCAGGCTATTGA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" colspan=\"3\" align=\"left\" rowspan=\"1\">\n<bold>qPCR analysis primers</bold>\n</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Primer name</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Oligonucleotide sequence (5′ to 3′)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Primer efficiency</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RP49 (tetur18g03590) F</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CTTCAAGCGGCATCAGAGC</td><td valign=\"top\" rowspan=\"2\" align=\"left\" colspan=\"1\">100.9%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RP49 (tetur18g03590) R</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CGCATCTGACCCTTGAACTTC</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VATPase qPCR F</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GGGTACCATCACATTCCTCG</td><td valign=\"top\" rowspan=\"2\" align=\"left\" colspan=\"1\">103.3%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VATPase qPCR R</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AATCGGTCTGGTTTGACGAAC</td></tr></tbody></table><table-wrap-foot><fn><p>\n<sup>a</sup>Primers for amplifying the DNA fragments for dsRNAs include the T7 promoter sequence (TAATACGACTCACTATAGGG) at the 5′ end.</p></fn><fn><p>\n<sup>b</sup>Negative control (NC) fragment (<xref rid=\"B35\" ref-type=\"bibr\">Suzuki et al., 2017b</xref>).</p></fn></table-wrap-foot></table-wrap></sec><sec id=\"s2_6\"><title>Oral Delivery of dsRNA to <italic>T. urticae</italic>\n</title><p>Newly emerged adult <italic>T. urticae</italic> females were placed onto the feeding device (~30 mites/device) using 1 cm<sup>2</sup> of nylon mesh sheet (opening size: 100 µm) to which 1 µg µl<sup>−1</sup> of dsRNA-<italic>TuVATPase</italic> or dsRNA-NC and 1% (w/v) of blue dye solution had been added. The <italic>T. urticae</italic> females were allowed to feed for 24 h under standard laboratory conditions. Fed mites were transferred onto 1-cm-diameter kidney bean leaf discs (1 mite/disc), and the survivorship, fecundity, and dark-body phenotype previously reported in oral delivery of dsRNA-<italic>TuVATPase</italic> (<xref rid=\"B35\" ref-type=\"bibr\">Suzuki et al., 2017b</xref>) were observed with a stereomicroscope (SZ40; Olympus, Tokyo, Japan) for 6 days in the laboratory. The RNAi assay was conducted in three or four independent experimental runs.</p></sec><sec id=\"s2_7\"><title>Real-Time Quantitative Reverse Transcription-PCR Analysis</title><p>Approximately 50 adult <italic>T. urticae</italic> females were collected at 2, 3, and 4 days after feeding on dsRNA-<italic>TuVATPase</italic> or dsRNA-NC, kept frozen in liquid nitrogen, and stored at −80°C until use. Total RNA was extracted using NucleoSpin RNA Plus (Macherey-Nagel), and single-stranded cDNA was synthesized by reverse transcription of total RNA using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Real-time quantitative reverse transcription-PCR (qRT-PCR) reactions were performed in three technical replicates with Power SYBR Green Master Mix (Thermo Fisher Scientific) on an ABI StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). A gene encoding a ribosomal protein, <italic>RP49</italic>, was used as a reference gene (<xref rid=\"B35\" ref-type=\"bibr\">Suzuki et al., 2017b</xref>). Primers and amplification efficiencies for the reference gene (E<sub>R</sub>) and target gene (E<sub>T</sub>) are shown in <xref rid=\"T1\" ref-type=\"table\">\n<bold>Table 1</bold>\n</xref>. The threshold cycle (Ct) value was calculated by averaging three technical replicates. The expression value of the target gene (T) was normalized to the reference gene (R), and normalized relative quantity (NRQ) was calculated as follows: NRQ = (1 + E<sub>R</sub>)<sup>CtR</sup>/(1 + E<sub>T</sub>)<sup>CtT</sup>. The real time qRT-PCR analysis was conducted in three independent experimental runs.</p></sec><sec id=\"s2_8\"><title>Oral Delivery of Synthetic Pesticides to <italic>T. urticae</italic>\n</title><p>The efficiency of the mesh method for bioassays was evaluated with two synthetic pesticides: abamectin (Agrimec; 1.8 g L<sup>−1</sup> EC [emulsifiable concentrate], Syngenta Japan, Tokyo, Japan) and bifenazate (Mito-Kohne; 20 g L<sup>−1</sup> FL [flowable], Nissan Chemical, Tokyo, Japan). Ten microliters of water-diluted abamectin (0.018 to 18 mg L<sup>−1</sup> [=ppm]) or bifenazate (0.2 to 200 ppm) was applied to the feeding device using a 1-cm<sup>2</sup> nylon mesh sheet with an opening size of 100 µm. Newly emerged adult <italic>T. urticae</italic> females (<italic>n</italic> = 29–92) were placed onto the feeding device, and the mortality was observed at 24 h and at 48 h for the treatment with abamectin and bifenazate, respectively. The pesticide assay was conducted in three independent experimental runs.</p></sec><sec id=\"s2_9\"><title>Oral Delivery of Nanoparticles to <italic>T. urticae</italic>\n</title><p>To examine whether the mesh method can be used for oral delivery of nanoparticles, newly emerged adult <italic>T. urticae</italic> females were placed onto the feeding device (100 mites/device) using 1 cm<sup>2</sup> of nylon mesh sheet (opening size: 100 µm) to which polystyrene fluorescent microspheres (diameter, 500 nm; Polysciences, Warrington, PA, USA) suspended in 1% (w/v) blue tracer dye solution was added. According to <xref rid=\"B2\" ref-type=\"bibr\">Bensoussan et al. (2018)</xref>, mites can uptake 500-nm-diameter particles with their stylets, and the size cutoff is around 750 nm. After allowing mites to feed for 24 h, fluorescent images were taken with a digital camera (EOS Kiss X7, Canon, Tokyo, Japan) installed on a fluorescence stereomicroscope (M205FA; Leica Microsystems, Wetzlar, Germany) fitted with a GFP filter (395–455 nm excitation, >480 nm emission) with an exposure time of 5 s (ISO: 200). Bright-field images were taken using the same system without filters with an exposure time of 1 s (ISO: 200). The fluorescent microsphere assay was conducted in three independent experimental runs.</p></sec><sec id=\"s2_10\"><title>Oral Delivery Assays in Other Spider Mites and an Insect</title><p>The mesh method was tested for the oral delivery of the blue tracer dye to other spider mite species (<italic>T. evansi</italic> and <italic>T. kanzawai</italic>) and to <italic>A. gossypii</italic> as a representative of sap-sucking insects. The same experimental procedure described for the <italic>T. urticae</italic> was used for <italic>T. evansi</italic> and <italic>T. kanzawai</italic> except that a 24-h fasting pre-treatment of newly emerged adult females prior to the assay was introduced in order to enhance the solution uptake. The starved <italic>T. evansi</italic> and <italic>T. kanzawai</italic> females (<italic>n</italic> = 50) were then introduced onto the feeding device and allowed to feed on 1% (w/v) blue tracer dye solution for 24 h under standard laboratory conditions. After 24 h, the number of mites to which the tracer dye was delivered was counted under a stereomicroscope. For <italic>A. gossypii</italic>, nylon mesh with an opening size of 500 µm was used in the feeding device, applying parameters preliminarily determined. The Parafilm of the feeding device for aphids was stretched less than that for spider mites, resulting in thicker film, in order to prevent them from cutting the Parafilm with their legs. In addition, the feeding arena was isolated with adhesive to prevent aphid escape (<xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figure 1A</bold>\n</xref>). Aphid nymphs and adults of various ages (<italic>n</italic> = 30–50) were placed onto the feeding device and allowed to feed on 2.5% (w/v) blue tracer dye solution for 24 h under the standard laboratory condition. After 24 h, the number of aphids to which the tracer dye was delivered was counted under a stereomicroscope.</p></sec><sec id=\"s2_11\"><title>Data Analysis</title><p>All data analyses were performed with R v3.3.2 or v4.0.0 (<xref rid=\"B30\" ref-type=\"bibr\">R Core Team, 2016</xref>; <xref rid=\"B31\" ref-type=\"bibr\">R Core Team, 2020</xref>). Results for the relative area of distribution of the dye applied in the feeding device and the relative number of dye-fed mites are presented with a regression line and a 95% confidence interval band (R package: <italic>ggplot2</italic>). Survival curves were plotted with the Kaplan–Meier method (R function: survfit, package: <italic>survival</italic>). Differences in the survival curves between the dsRNA treatments were analyzed using the log-rank test (R function: survdiff, package: <italic>survival</italic>). Data normality and equality of variance in fecundity, cumulative proportion of dark phenotype mites, and relative quantity of <italic>TuVATPase</italic> gene expression were analyzed with the Shapiro–Wilk test (R function: shapiro.test) and <italic>F</italic>-test (R function: var.test). Differences in daily mite fecundity between the dsRNA treatments were statistically analyzed using the Wilcoxon–Mann–Whitney test (R function: wilcox.exact, package: <italic>exactRankTests</italic>). Arcsine square-root transformation was applied to normalize the cumulative proportion of mites with dark phenotype. Differences in the normalized proportional data and the relative quantity of <italic>TuVATPase</italic> gene expression between the dsRNA treatments were analyzed with a <italic>t</italic>-test (R function: t.test). Results for the daily fecundity and the cumulative proportion of mites with dark phenotype are presented as overlaid bee-swarm (R function: beeswarm, package: <italic>beeswarm</italic>) and box-and-whisker plots (R function: boxplot). The dose–response curves in the pesticide assay were generated with the two-parameter log-logistic function (R function: drm, R package: <italic>drc</italic>) and are represented with a 95% confidence interval band.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec id=\"s3_1\"><title>Liquid Volume Required for the Feeding Device in the Mesh Method</title><p>The feeding device in which 4 cm<sup>2</sup> of nylon mesh with a pore size of 50, 100, or 500 µm required 30, 40, or 160 µl (7.5, 10, or 40 µL cm<sup>−2</sup>) of liquid, respectively, to saturate the entire area (<xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figure 1D</bold>\n</xref>).</p></sec><sec id=\"s3_2\"><title>Time Required for <italic>T. urticae</italic> Feeding in the Mesh Method</title><p>Adult <italic>T. urticae</italic> females fed on blue tracer dye solution in the feeding device for 24 h showed blue color in the midgut (<xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figure 2A</bold>\n</xref>), which consists of the ventriculus, caeca, and posterior midgut (<xref rid=\"B2\" ref-type=\"bibr\">Bensoussan et al., 2018</xref>). In the fed mites, the blue color was most concentrated in the posterior midgut, consistent with the filtering of small molecules (<1 to 4 kDa) from the ventriculus to the posterior midgut (<xref rid=\"B2\" ref-type=\"bibr\">Bensoussan et al., 2018</xref>). However, blue dye was also visible in the caeca. The proportion of dye-fed mites increased over the feeding time (<xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figure 2B</bold>\n</xref>; <xref ref-type=\"supplementary-material\" rid=\"SM2\">\n<bold>Supplementary Video S2</bold>\n</xref>). The tracer dye was observably delivered to approximately 95% of mites after feeding for 4 h.</p><fig id=\"f2\" position=\"float\"><label>Figure 2</label><caption><p>Efficiency of oral administration to <italic>Tetranychus urticae</italic> using the feeding device. <bold>(A)</bold> Adult females kept on the feeding device in which a 1% (w/v) blue tracer dye (Brilliant Blue FCF) was excluded (left) or included (right) at 25°C for 24 h after molting. C, caeca; v, ventriculus; pm, posterior midgut. Scale bar: 100 µm. <bold>(B)</bold> The relationship between the feeding time and relative number of fed mites. Newly molted adult females were placed onto the feeding device using a nylon mesh sheet (2 × 2 cm) with a 100-µm opening, and ingestion was determined by the change of body color. Data were collected from three independent experimental runs and are represented with a regression line and a 95% confidence interval band. <italic>n</italic> = 127–193.</p></caption><graphic xlink:href=\"fpls-11-01218-g002\"/></fig></sec><sec id=\"s3_3\"><title>Oral Delivery of dsRNA for RNAi of <italic>TuVATPase</italic>\n</title><p>A significantly lower survivorship was observed in mites fed on dsRNA-<italic>TuVATPase</italic> than mites that fed on the control dsRNA (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figure 3A</bold>\n</xref>). The fecundity was significantly lower in mites fed on dsRNA-<italic>TuVATPase</italic> than in the control group at 2 to 6 days after treatment (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figure 3B</bold>\n</xref>). The dark-body phenotype that is associated with <italic>VATPase</italic> gene silencing in <italic>T. urticae</italic> (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figure 3C</bold>\n</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Suzuki et al., 2017b</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Bensoussan et al., 2020</xref>) was observed in around 80 and 90% of mites fed on dsRNA-<italic>TuVATPase</italic> at 2 days and at 3–6 days after treatment, respectively, whereas no mites showed the dark coloration in the control group even at 6 days after treatment (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figure 3D</bold>\n</xref>). The expression level of endogenous <italic>TuVATPase</italic> transcripts was significantly lower in the treatment group than in the control group at 2–4 days after treatment (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figure 3E</bold>\n</xref>).</p><fig id=\"f3\" position=\"float\"><label>Figure 3</label><caption><p>The effect of 1 µg µl<sup>−1</sup> dsRNA-<italic>TuVATPase</italic> or dsRNA-NC (negative control) delivered <italic>via</italic> the mesh method on the survivorship, fecundity, and endogenous <italic>TuVATPase</italic> gene expression in adult <italic>Tetranychus urticae</italic> females at 25°C. <bold>(A)</bold> Survivorship of adult females for 6 days after treatment (DAT) with dsRNA-<italic>TuVATPase</italic> and dsRNA-NC. Survival curves were plotted by using the Kaplan–Meier method and compared by using the log-rank test. <bold>(B)</bold> Daily fecundity of adult females that survived after treatment with dsRNA-<italic>TuVATPase</italic> and dsRNA-NC. Data were represented by bee-swarm and box-and-whisker plots and compared by using Wilcoxon–Mann–Whitney tests (NS, <italic>P</italic> > 0.05; ***, <italic>P</italic> < 0.001). Values in parentheses indicate the number of surviving mites. <bold>(C)</bold> Mite body phenotype associated with dsRNA treatment. Lower photos are of mites soaked in a 50% (v/v) glycerol and 0.1% (v/v) Triton X-100 solution. Scale bar: 100 µm. <bold>(D)</bold> Cumulative frequency of dark-body phenotype observed after treatment with dsRNA-<italic>TuVATPase</italic> and dsRNA-NC. Data were represented by bee-swarm and box-and-whisker plots and compared by using a <italic>t</italic>-test after normalization with arcsine square-root transformation (NS, <italic>P</italic> > 0.05; **, <italic>P</italic> < 0.01; ***, <italic>P</italic> < 0.001). Data were collected from four and three independent experimental runs for treatments with dsRNA-<italic>TuVATPase</italic> (<italic>n</italic> = 4) and dsRNA-NC (<italic>n</italic> = 3), respectively. In each experimental run, 25 to 42 mites were used. <bold>(E)</bold>\n<italic>TuVATPase</italic> gene expression relative to the expression of <italic>RP49</italic> reference gene at 2, 3, and 4 days after treatment with dsRNA-<italic>TuVATPase</italic> and dsRNA-NC. Data were represented as mean ± SE and compared by using a <italic>t</italic>-test (*, <italic>P</italic> < 0.05). <bold>(A, B, E)</bold> Data were collected from three independent experimental runs. <bold>(B, D)</bold> In the box-and-whisker plots, the central line (second quartile, Q2) indicates the median, the distance between the box bottom (first quartile, Q1) and top (third quartile, Q3) indicates the interquartile range (IQR), and the whisker bottom and top indicate the minimum and maximum values, respectively. Outliers that are outside the range between the lower [Q1 − 1.5 × IQR] and upper limits [Q3 + 1.5 × IQR] are plotted outside of the IQR.</p></caption><graphic xlink:href=\"fpls-11-01218-g003\"/></fig></sec><sec id=\"s3_4\"><title>Oral Toxicity of Abamectin and Bifenazate</title><p>More than 90% mortalities were observed in mites placed onto the feeding device and allowed to feed on >1.8-ppm abamectin (<xref ref-type=\"fig\" rid=\"f4\">\n<bold>Figure 4A</bold>\n</xref>) and >20-ppm bifenazate (<xref ref-type=\"fig\" rid=\"f4\">\n<bold>Figure 4B</bold>\n</xref>). The LC<sub>50</sub> values were 0.43 ± 0.05 and 3.41 ± 0.35 ppm for mites ingesting abamectin and bifenazate, respectively. A rapid toxicity was observed in mites fed on 18-ppm abamectin and all mites tested were dead or dying within 2 h after placement on the feeding device (<xref ref-type=\"supplementary-material\" rid=\"SM3\">\n<bold>Supplementary Video S3</bold>\n</xref>).</p><fig id=\"f4\" position=\"float\"><label>Figure 4</label><caption><p>Effects of the acaricides <bold>(A)</bold> abamectin and <bold>(B)</bold> bifenazate delivered <italic>via</italic> the mesh method in adult <italic>Tetranychus urticae</italic> females at 25°C for 24 and 48 h, respectively. Data were collected from three independent experimental runs and presented with a regression curve and a 95% confidence interval band. In each experimental run, 25 to 92 mites were used.</p></caption><graphic xlink:href=\"fpls-11-01218-g004\"/></fig></sec><sec id=\"s3_5\"><title>Oral Delivery of Particle Suspension</title><p>Fluorescent microspheres were delivered to approximately 90% of mites (<italic>n</italic> = 309) that were allowed to ingest 500-nm-diameter fluorescent microsphere suspensions for 24 h, and the fluorescent signal was observed in the ventriculus and caeca in the midgut (<xref ref-type=\"fig\" rid=\"f5\">\n<bold>Figure 5</bold>\n</xref>).</p><fig id=\"f5\" position=\"float\"><label>Figure 5</label><caption><p>Oral delivery of fluorescent nanoparticles (diameter, 500 nm) to adult <italic>Tetranychus urticae</italic> females with the mesh method. Mites were allowed to ingest at 25°C for 24 h. In the control, water or 1% (w/v) blue tracer dye (Brilliant Blue FCF) solution was placed in the feeding device.</p></caption><graphic xlink:href=\"fpls-11-01218-g005\"/></fig></sec><sec id=\"s3_6\"><title>Liquid Delivery to Other Species</title><p>Unlike <italic>T. urticae</italic>, adult female <italic>T. evansi</italic> and <italic>T. kanzawai</italic> required prior starvation for 24 h to enhance their liquid uptake through the feeding device in the mesh method. After this pre-treatment, uptake of 1% (w/v) blue tracer dye solution was observed in more than 90% of <italic>T. evansi</italic> (<italic>n</italic> =105) and <italic>T. kanzawai</italic> females (<italic>n</italic> = 96) after feeding for 3 h (<xref ref-type=\"supplementary-material\" rid=\"SF1\">\n<bold>Supplementary Figure S1A</bold>\n</xref>). In the cotton aphid <italic>A. gossypii</italic>, uptake of 2.5% (w/v) blue tracer dye solution was observed in ~80% of nymphs and adults (<italic>n</italic> = 117) of various ages (<xref ref-type=\"supplementary-material\" rid=\"SF1\">\n<bold>Supplementary Figures S1B, C</bold>\n</xref>, <xref ref-type=\"supplementary-material\" rid=\"SM4\">\n<bold>Supplementary Video S4</bold>\n</xref>).</p></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Resistance to conventional synthetic pesticides in arthropod herbivores imposes severe threats to the productivity of agricultural and horticultural crops. Therefore, it is important to develop new compounds or other strategies for arthropod pest control. However, the development of an effective compound requires numerous candidates to be screened with time-consuming bioassays. Thus, the development of a simple and effective method for the delivery of a wide range of compounds would support high-throughput screening of candidate molecules. Here, we reported a new method for the oral delivery of test compounds into spider mites and aphids. Our experiments demonstrate its applicability in environmental RNAi with exogenously supplied dsRNA and bioassays with synthetic pesticides and nanoparticles as a potential carrier of bio-active compounds in <italic>T. urticae</italic>.</p><p>The mesh method has a high efficiency of oral administration: >90% of mites ingested the tracer dye within 4 h (<xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figure 2B</bold>\n</xref>). Compared to the soaking method, where a 24-h ingestion time is provided (<xref rid=\"B34\" ref-type=\"bibr\">Suzuki et al., 2017a</xref>, b; <xref rid=\"B3\" ref-type=\"bibr\">Bensoussan et al., 2020</xref>), our method is time saving. In addition, the mesh method requires fewer resources, as 10 µl of liquid can be delivered to ~100 mites (<italic>i.e.</italic>, 0.1 µl/mite). This efficiency is quite high compared to previously reported methods for oral administration by soaking (1 µl/mite) (<xref rid=\"B34\" ref-type=\"bibr\">Suzuki et al., 2017a</xref>, <xref rid=\"B35\" ref-type=\"bibr\">b</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Bensoussan et al., 2020</xref>), feeding on artificial diet filled in hemispherical Parafilm bubbles (3.3 µl/mite) (<xref rid=\"B34\" ref-type=\"bibr\">Suzuki et al., 2017a</xref>, b), and feeding on treated leaf discs (2–400 µl/mite) (<xref rid=\"B24\" ref-type=\"bibr\">Kwon et al., 2013</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Suzuki et al., 2017b</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Abouelmaaty et al., 2019</xref>). Furthermore, preparation of the feeding device is simple (<xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figure 1A</bold>\n</xref>). Hemispherical Parafilm bubbles used in the artificial diet method requires a custom-built vacuum device, as described by <xref rid=\"B18\" ref-type=\"bibr\">Jonckheere et al. (2016)</xref> and <xref rid=\"B34\" ref-type=\"bibr\">Suzuki et al. (2017a)</xref>. All materials used in the mesh method are low-cost and general-purpose products that are likely available in most laboratories. Unlike the hemispherical Parafilm bubbles, the feeding device in the mesh method is flat like a leaf disc, making it easy to inoculate and maintain mites and allowing the inoculation of ~100 mites even in a limited area of 1 cm<sup>2</sup>, which enables the execution of the area-efficient bioassays. Moreover, the mesh method does not require post-treatment manipulation of mites, such as the rinsing and drying of soaked mites, which is the bottleneck of the soaking method (<xref rid=\"B35\" ref-type=\"bibr\">Suzuki et al., 2017b</xref>). Thus, mite handling is as easy as that on leaf discs, which enables time-efficient bioassays.</p><p>To evaluate the usefulness of the mesh method to deliver genetic materials, we tested environmental RNAi targeting the <italic>TuVATPase</italic> gene with exogenously supplied dsRNA in mites. The dark-body phenotype (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figure 3C</bold>\n</xref>) associated with RNAi targeting the <italic>TuVATPase</italic> gene was observed in approximately 90% of mites (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figure 3D</bold>\n</xref>), which was 2 to 3 times higher than that reported by <xref rid=\"B35\" ref-type=\"bibr\">Suzuki et al. (2017b)</xref> and comparable to that noted by <xref rid=\"B3\" ref-type=\"bibr\">Bensoussan et al. (2020)</xref>, who tracked dsRNA ingestion using a tracer dye. In addition, we observed reductions in mite survivorship and fecundity by RNAi targeting the <italic>TuVATPase</italic> gene (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figures 3A, B</bold>\n</xref>). According to <xref rid=\"B35\" ref-type=\"bibr\">Suzuki et al. (2017b)</xref> and <xref rid=\"B3\" ref-type=\"bibr\">Bensoussan et al. (2020)</xref>, a marked reduction in mite survivorship was observed at 6 to 10 days after treatment. In the present study, the reduction in mite survivorship was moderate when compared to these reports, partly because the observation period was limited to 6 days after treatment. Thus, an observation period of about 10 days should be used to accurately assess the lethal effect of environmental RNAi in mites. Although the dsRNA concentration used (1 µg µl<sup>−1</sup>) was higher than that used by <xref rid=\"B35\" ref-type=\"bibr\">Suzuki et al. (2017b)</xref> and <xref rid=\"B3\" ref-type=\"bibr\">Bensoussan et al. (2020)</xref> (20 to 320 ng µl<sup>−1</sup>), the reductions in mite survivorship and fecundity were comparable. <xref rid=\"B3\" ref-type=\"bibr\">Bensoussan et al. (2020)</xref> have reported no significant difference in the effects of RNAi targeting the <italic>TuVATPase</italic> and a subunit of coatomer protein complex (<italic>TuCOPB2</italic>) genes at dsRNA concentrations higher than 160 ng µl<sup>−1</sup>, which suggest that the lethal RNAi effect may have already been saturated at 1 µg µl<sup>−1</sup> dsRNA. The <italic>TuVATPase</italic> transcript level reached >75% reduction in mites collected 4 days after treatment (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figure 3E</bold>\n</xref>). Although the reduction of the transcript abundance was 2 to 3 times higher than that reported in previous studies (<xref rid=\"B24\" ref-type=\"bibr\">Kwon et al., 2013</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Suzuki et al., 2017b</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Bensoussan et al., 2020</xref>), this might be due to the high concentration of orally administered dsRNA used in the present study. Optimizing the observation period (~10 days) and dsRNA concentration (>160 ng µl<sup>−1</sup>) in the mesh method should allow for high-throughput screening of candidate genes for RNAi-based <italic>T. urticae</italic> control.</p><p>The mesh method can also be used to evaluate the oral toxicity of synthetic pesticides to sucking arthropod herbivores. The LC<sub>50</sub> values were 0.43 and 3.41 ppm in mites that ingested abamectin and bifenazate, respectively (<xref ref-type=\"fig\" rid=\"f4\">\n<bold>Figure 4</bold>\n</xref>). These LC<sub>50</sub> values are higher than those observed in leaf disc-sprayed bioassays (0.024 and 1.89 ppm, respectively) in which mites are exposed to both contact and oral toxicities (<xref rid=\"B20\" ref-type=\"bibr\">Khajehali et al., 2011</xref>). These results suggest that the mesh method is useful in evaluating the oral toxicity of pesticides and could be particularly applicable for testing the synergic effects between pesticides and RNAi targeting xenobiotic metabolic process genes.</p><p>Delivery of dsRNA <italic>via</italic> nanoparticles is a promising approach for enhancing the efficiency of environmental RNAi not only by increasing the stability of dsRNA but also by increasing the cellular uptake of dsRNA (<xref rid=\"B17\" ref-type=\"bibr\">Joga et al., 2016</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Debnath and Das, 2018</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Taning et al., 2020</xref>). The mesh method supported delivery of 500-nm-diameter particles to approximately 90% of mites (<italic>n</italic> = 309) within 24 h (<xref ref-type=\"fig\" rid=\"f5\">\n<bold>Figure 5</bold>\n</xref>). Thus, the mesh method can be used to evaluate the significance of nanoparticles as dsRNA delivery vehicles.</p><p>The mesh method could be more widely used for delivering experimental solutions to other stylet-feeding arthropods. Although we were able to effectively deliver blue tracer dye to <italic>T. evansi</italic> and <italic>T. kanzawai</italic> by using the mesh method (<xref ref-type=\"supplementary-material\" rid=\"SF1\">\n<bold>Supplementary Figure S1A</bold>\n</xref>), unlike <italic>T. urticae</italic>, <italic>T. evansi</italic> and <italic>T. kanzawai</italic> required 24-h starvation before the experiment to enhance their feeding. It was also necessary to clear the gut of these mite species because their bodies are pigmented red, which obscures the blue color of the tracer dye. Therefore, a starvation period allowed <italic>T. evansi</italic> and <italic>T. kanzawai</italic> to excrete the dark digestive cells in the midgut as feces (<xref rid=\"B2\" ref-type=\"bibr\">Bensoussan et al., 2018</xref>), which helped the visualization of the tracer dye but might influence the outcome of follow-up experiments with dsRNA or acaricides. The impact of 24-h starvation on the follow-up experiments remains to be investigated. We also showed that the mesh method can be used for delivering the blue tracer dye solution to aphids (<xref ref-type=\"supplementary-material\" rid=\"SF1\">\n<bold>Supplementary Figures S1B, C</bold>\n</xref>). Although not tested in the present study, we hypothesize that the mesh method may also be used for delivering test compounds to blood-feeding insects such as bedbugs and mosquitos.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>All datasets presented in this study are included in the article/<xref ref-type=\"supplementary-material\" rid=\"SM1\">\n<bold>Supplementary Material</bold>\n</xref>.</p></sec><sec id=\"s6\"><title>Author Contributions</title><p>NG, VG, and TS planned the study. NG, MO, KS, SY, and FH performed the experiments and collected the data. NG and TS performed data analysis and visualization. NG, VG, and TS wrote the manuscript.</p></sec><sec sec-type=\"funding-information\" id=\"s7\"><title>Funding</title><p>This study was supported by a Japan Society for the Promotion of Science (JSPS) KAKENHI grant (18H02203) and Japan Science and Technology Agency OPERA grant (JPMJOP1833) to TS and was partly supported by the Institute of Global Innovation Research of Tokyo University of Agriculture and Technology to TS and VG. NG was supported by JSPS Invitational Fellowships for Research in Japan (L19542).</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><title>Acknowledgments</title><p>The authors would like to thank Ryutaro Murakami for testing our method for his MSc. work.</p></ack><sec id=\"s9\" sec-type=\"supplementary-material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fpls.2020.01218/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fpls.2020.01218/full#supplementary-material</ext-link>\n</p><supplementary-material content-type=\"local-data\" id=\"SF1\"><label>Figure S1</label><caption><p>Oral delivery of blue tracer dye to other sucking arthropod herbivores with the feeding device in the mesh method. <bold>(A)</bold> Adult females of the tomato red mite, <italic>Tetranychus evansi</italic>, and the Kanzawa spider mite, <italic>Tetranychus kanzawai</italic>, kept for 24 h on the feeding device filled with water (control) or 1% (w/v) blue tracer dye (Brilliant Blue FCF) solution. <bold>(B)</bold> Nymphs and adults of the cotton aphid, <italic>Aphis gossypii</italic>, kept for 24 h on the feeding device filled with water (control) or 2.5% (w/v) blue tracer dye solution. In adult aphids, almost half of individuals kept on the feeding device filled with 2.5% (w/v) blue tracer dye solution turned blue across the whole body, and others turned blue only in the digestive tract. <bold>(C)</bold>\n<italic>Aphis gossypii</italic> adult sucking the dye solution in the feeding device. The white arrowhead points at the stylets of the aphid. Scale bars: <bold>(A)</bold> 100 µm; <bold>(B, C)</bold> 500 µm.</p></caption><media xlink:href=\"Image_1.tif\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM1\"><label>Video S1</label><caption><p>Preparation of the feeding device in the mesh method.</p></caption><media xlink:href=\"Video_1.mp4\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM2\"><label>Video S2</label><caption><p>Adult <italic>Tetranychus urticae</italic> females (<italic>n</italic> ≈ 50) sucking on 1% (w/v) blue tracer dye (Brilliant Blue FCF) solution in the mesh method feeding device for 1.5 h. The video is shown at 64× speed. Feeding arena: 5 × 5 mm.</p></caption><media xlink:href=\"Video_2.mp4\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM3\"><label>Video S3</label><caption><p>Adult <italic>Tetranychus urticae</italic> females (<italic>n</italic> = 50) sucking on 18-ppm abamectin in 1% (w/v) blue tracer dye (Brilliant Blue FCF) solution in the mesh method feeding device for 2.5 h. The video is shown at 128× speed. Feeding arena: 4 × 3 mm.</p></caption><media xlink:href=\"Video_3.mp4\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM4\"><label>Video S4</label><caption><p>\n<italic>Aphis gossypii</italic> nymphs sucking on 2.5% (w/v) blue tracer dye (Brilliant Blue FCF) solution in the mesh method feeding device for 16 min. The video is shown at 32× speed.</p></caption><media xlink:href=\"Video_4.mp4\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\">\n<person-group person-group-type=\"author\"><name><surname>Abouelmaaty</surname><given-names>H. G.</given-names></name><name><surname>Fukushi</surname><given-names>M.</given-names></name><name><surname>Abouelmaaty</surname><given-names>A. G.</given-names></name><name><surname>Ghazy</surname><given-names>N. A.</given-names></name><name><surname>Suzuki</surname><given-names>T.</given-names></name></person-group> (<year>2019</year>). <article-title>Leaf disc-mediated oral delivery of small molecules in the absence of surfactant to the two-spotted spider mite, <italic>Tetranychus urticae</italic>\n</article-title>. <source>Exp. Appl. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Cell Dev. Biol.</journal-id><journal-title-group><journal-title>Frontiers in Cell and Developmental Biology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-634X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850867</article-id><article-id pub-id-type=\"pmc\">PMC7431705</article-id><article-id pub-id-type=\"doi\">10.3389/fcell.2020.00775</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cell and Developmental Biology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Angiogenesis Inhibition by a Short 13 Amino Acid Peptide Sequence of Tetrastatin, the α4(IV) NC1 Domain of Collagen IV</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Vautrin-Glabik</surname><given-names>Alexia</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Devy</surname><given-names>Jérôme</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/921851/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Bour</surname><given-names>Camille</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/335445/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Baud</surname><given-names>Stéphanie</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/983318/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Choulier</surname><given-names>Laurence</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Hoarau</surname><given-names>Anthony</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Dupont-Deshorgue</surname><given-names>Aurélie</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1048291/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Sellier</surname><given-names>Christèle</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Brassart</surname><given-names>Bertrand</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/779446/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Oudart</surname><given-names>Jean-Baptiste</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/681481/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ramont</surname><given-names>Laurent</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/538533/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Monboisse</surname><given-names>Jean Claude</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/296051/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Brassart-Pasco</surname><given-names>Sylvie</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/775269/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Laboratoire de Biochimie, Université de Reims Champagne-Ardenne (URCA)</institution>, <addr-line>Reims</addr-line>, <country>France</country></aff><aff id=\"aff2\"><sup>2</sup><institution>CNRS UMR 7369, Matrice Extracellulaire et Dynamique Cellulaire (MEDyC)</institution>, <addr-line>Reims</addr-line>, <country>France</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Plateau de Modélisation Moléculaire Multi-échelle, URCA</institution>, <addr-line>Reims</addr-line>, <country>France</country></aff><aff id=\"aff4\"><sup>4</sup><institution>CNRS UMR 7021, Laboratoire de Bioimagerie et Pathologies, Université de Strasbourg</institution>, <addr-line>Illkirch</addr-line>, <country>France</country></aff><aff id=\"aff5\"><sup>5</sup><institution>CHU Reims, Service Biochimie-Pharmacologie-Toxicologie</institution>, <addr-line>Reims</addr-line>, <country>France</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Lucas Treps, VIB-KU Leuven Center for Cancer Biology, Belgium</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Stephen Robinson, University of East Anglia, United Kingdom; Parvez Khan, University of Nebraska Medical Center, United States</p></fn><corresp id=\"c001\">*Correspondence: Sylvie Brassart-Pasco sylvie.brassart-, <email>pasco@univ-reims.fr</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>Present address: Anthony Hoarau, Inserm UMR-S 1250, Pathologies Pulmonaires et Plasticité Cellulaire, Reims, France</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Cell and Developmental Biology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>775</elocation-id><history><date date-type=\"received\"><day>19</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>23</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Vautrin-Glabik, Devy, Bour, Baud, Choulier, Hoarau, Dupont-Deshorgue, Sellier, Brassart, Oudart, Ramont, Monboisse and Brassart-Pasco.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Vautrin-Glabik, Devy, Bour, Baud, Choulier, Hoarau, Dupont-Deshorgue, Sellier, Brassart, Oudart, Ramont, Monboisse and Brassart-Pasco</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Angiogenesis is defined as the formation of new capillaries by sprouting from the pre-existing microvasculature. It occurs in physiological and pathological processes particularly in tumor growth and metastasis. α1, α2, α3, and α6 NC1 domains from type IV collagen were reported to inhibit tumor angiogenesis. We previously demonstrated that the α4 NC1 domain from type IV collagen, named Tetrastatin, inhibited tumor growth in a mouse melanoma model. The inhibitory activity was located in a 13 amino acid sequence named QS-13. In the present paper, we demonstrate that QS-13 decreases VEGF-induced-angiogenesis <italic>in vivo</italic> using the Matrigel plug model. Fluorescence molecular tomography allows the measurement of a 65% decrease in Matrigel plug angiogenesis following QS-13 administration. The results are confirmed by CD31 microvessel density analysis on Matrigel plug slices. QS-13 peptide decreases Human Umbilical Vein Endothelial Cells (HUVEC) migration and pseudotube formation <italic>in vitro</italic>. Relevant QS-13 conformations were obtained from molecular dynamics simulations and docking. A putative interaction of QS-13 with α<sub>5</sub>β<sub>1</sub> integrin was investigated. The interaction was confirmed by affinity chromatography, solid phase assay, and surface plasmon resonance. QS-13 binding site on α<sub>5</sub>β<sub>1</sub> integrin is located in close vicinity to the RGD binding site, as demonstrated by competition assays. Collectively, our results suggest that QS-13 exhibits a mighty anti-angiogenic activity that could be used in cancer treatment and other pathologies with excessive angiogenesis such as hemangioma, psoriasis or diabetes.</p></abstract><kwd-group><kwd>angiogenesis</kwd><kwd>matrikine</kwd><kwd>Tetrastatin</kwd><kwd>integrin</kwd><kwd>alpha 5 beta 1 collagen IV</kwd></kwd-group><counts><fig-count count=\"5\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"31\"/><page-count count=\"12\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Angiogenesis is the formation of new blood vessels and capillaries from sprouting of pre-existing blood vessels. It normally occurs during wound healing and embryonic development, but it is also required for tumor growth and metastasis. This process is regulated by growth factors, such as vascular endothelial growth factors (VEGFs), which bind to their receptors on the normal endothelial cell surface, induce transduction pathways and promote proliferation and migration of endothelial cells and vascular tube formation (<xref rid=\"B27\" ref-type=\"bibr\">Teleanu et al., 2019</xref>).</p><p>In recent years, the basement membrane (BM), a specialized extracellular matrix (ECM), has been recognized as a key regulator of cell behavior, and not only as an architectural support. BM is an important structural and functional component of blood vessels (<xref rid=\"B24\" ref-type=\"bibr\">Silva et al., 2018</xref>). Several BM components were reported to largely participate in the regulation of tumor angiogenesis (<xref rid=\"B10\" ref-type=\"bibr\">Kalluri, 2003</xref>).</p><p>Basement membrane are composed of type IV collagen in association with other minor collagens, such as collagens XVIII or XIX, laminins, nidogens, and perlecan. Type IV collagen is composed of three α(IV) chains, out of six possible [α1(IV)-α6(IV)]. Each α(IV) chain comprises a 7S N-terminal domain, a long interrupted triple helical domain and a globular C-terminal non-collagenous (NC1) domain (<xref rid=\"B30\" ref-type=\"bibr\">Wu and Ge, 2019</xref>). The α1(IV) NC1 domain (Arresten), the α2(IV) NC1 domain (Canstatin), the α3(IV) NC1 domain (Tumstatin), and the a6(IV) NC1 domain were reported to inhibit angiogenesis (<xref rid=\"B20\" ref-type=\"bibr\">Petitclerc et al., 2000</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Wietecha et al., 2013</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Monboisse et al., 2014</xref>). The α4(IV) NC1 domain was reported to slightly decrease bFGF-induced angiogenesis while α5(IV) NC1 domain has no effect (<xref rid=\"B20\" ref-type=\"bibr\">Petitclerc et al., 2000</xref>). Karagiannis and Popel reported the inhibitory effects of Pentastatin-1 and -2, two α5(IV) NC1 domain-derived peptides, on Human Umbilical Vein Endothelial Cells (HUVEC) proliferation (<xref rid=\"B11\" ref-type=\"bibr\">Karagiannis and Popel, 2008</xref>). They also pointed out the inhibitory effects of Tetrastatin-2 and Pentastatin-3, two peptides from the α4(IV) and α5(IV) NC1 domains, respectively, on VEGF-induced HUVEC migration We previously demonstrated the potent anti-tumor activity of the α4(IV) NC1 domain, named Tetrastatin (<xref rid=\"B7\" ref-type=\"bibr\">Brassart-Pasco et al., 2012</xref>). We recently identified the minimal active sequence (QKISRCQVCVKYS: QS-13) of Tetrastatin that reproduced whole Tetrastatin anti-tumor properties <italic>in vivo</italic> and <italic>in vitro</italic> on melanoma cell proliferation, migration, and invasion (<xref rid=\"B13\" ref-type=\"bibr\">Lambert et al., 2018</xref>).</p><p>In the present article, we investigated the inhibitory effects of QS-13 on angiogenesis <italic>in vivo</italic> in a Matrigel plug assay and <italic>in vitro</italic> on HUVEC proliferation, migration and pseudotube formation.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Peptide Synthesis</title><p>QS-13 was purchased from Proteogenix<sup>®</sup> (Schiltigheim, France). It was obtained by solid-phase synthesis using a FMOC [N-(9-fluorenyl) methoxy-carbonyl] derivative procedure. It was then purified by reverse phase high performance liquid chromatography using a C18 column, eluted by a gradient of acetonitrile in trifluoroacetic acid and lyophilized. Its purity (>98%) was assessed by HPLC and mass spectroscopy.</p></sec><sec id=\"S2.SS2\"><title>Matrigel Plug Angiogenesis Assay</title><p>Eight-week-old albinos female B6(C)Rj -Tyr c/c mice were purchased from Janvier Laboratories (Saint-Berthevin, France). The animals were fed <italic>ad libitum</italic> with a chlorophyll-free diet 10 days before and during imaging experiments. The study was performed in compliance with “The French Animal Welfare Act” and following “The French Board for Animal Experiments.” Experiments were conducted under approval of the French “Ministère de l’Enseignement Supérieur et de la Recherche” (Ethics Committees Nos. C2EA-56 and C2EA-75) in compliance with the “Directive 2010/63/UE”. Protocol no. 4373_V1 APAFIS (07/09/2016).</p><p>Four hundred μL of Matrigel mix composed of growth factor-reduced Matrigel (Dutscher, Brumath, France) supplemented with 100 ng/mL recombinant mouse VEGF (R&D System-Bio-Techne, Lille, France), 350 ng/mL of recombinant mouse bFGF (R&D System-Bio-Techne, Lille, France) and 25 UI/mL of Heparin (R&D System-Bio-Techne, Lille, France) were injected into the left flank of each mouse. Mice were divided into two groups of 8 mice: positive control (Matrigel mix), QS-13-treated mice (Matrigel mix + 40 μM QS-13). Positive control mice received PBS and QS-13 treated mice received QS-13 (10 mg/kg) intraperitoneally at days 3, 7, and 11.</p></sec><sec id=\"S2.SS3\"><title><italic>In vivo</italic> Fluorescence Imaging</title><p>At day 13, 100 μL of AngioSense680<sup>TM</sup> (PerkinElmer, Inc., United States) were injected into the right orbital plexus of mice. At day 14, mice were anesthetized with 2% isoflurane and images were obtained with a fluorescence molecular tomographic (FMT<sup>®</sup>) imaging system (FMT4000, PerkinElmer, Inc., United States). Then, mice were sacrificed. Matrigel plug were removed and placed into 4% formaldehyde for histological analyses. 3D reconstruction and image analysis were performed using TrueQuant<sup>TM</sup> software (PerkinElmer, Inc., United States).</p></sec><sec id=\"S2.SS4\"><title>CD31 Immunostaining</title><p>CD31 immunostaining were performed on 4 μm thick Matrigel plug sections. After deparaffinization, sections were incubated with a Tris–EDTA buffer, pH 8.4 for 20 min at 97°C, washed with distilled water and then incubated with hydrogen peroxide blocking solution (Abcam, Paris, France) for 10 min at room temperature and washed with PBS. They were incubated with Protein Block (Abcam) for 10 min at room temperature, washed with PBS, and incubated overnight at 4°C with an anti-CD31 rabbit monoclonal antibody diluted 1/1000 (ab 28364 from Abcam) and washed again with PBS. The first antibody was detected using the Rabbit specific HRP/DAB (ABC) Detection IHC Kit (Abcam) according to the manufacturer’s instructions. Sections were counterstained with hematoxylin (Novocastra). To evaluate MicroVessel Density (MVD), three sections per plug were performed at three different depth levels of the plug and three different fields were acquired under an inverted microscope. Each positive endothelial cell cluster of immunoreactivity in contact with the selected field was counted as an individual vessel in addition to the morphologically identifiable vessels with a lumen, according to Weidner’s method (<xref rid=\"B28\" ref-type=\"bibr\">Weidner, 1995</xref>).</p></sec><sec id=\"S2.SS5\"><title>Cell Culture</title><p>Human Umbilical Vein Endothelial Cells were purchased from Promocell (Heidelberg, Germany). Cells were grown in Endothelial Cell Growth Medium (ECGM, Promocell, Heidelberg, Germany) at 37°C in a humid atmosphere with 5% CO<sub>2</sub> in air. At 70–90% confluency, cells were subcultured according to Promocell subcultivation protocol, using the Detach Kit (Hepes BSS, Trypsin/EDTA, Trypsin Neutralization Solution). They were used before passage 5.</p></sec><sec id=\"S2.SS6\"><title>Proliferation Assay</title><p>For cell proliferation measurement, 2,000 HUVECs were seeded in 96-well plates and cultivated in ECGM supplemented with 10 ng/mL of Vascular Endothelial Growth Factor (VEGF-165, Promocell) with or without 40 μM QS-13. After 24, 48, 72, and 96 h, cell proliferation was measured using the WST-1 cell proliferation reagent (Sigma, Saint-Quentin-Fallavier, France), according to the manufacturer’s instructions. Absorbance was read at 450 nm using a Biochrom Asys UVM 340 microplate reader (Biochrom, Yvelines, France).</p></sec><sec id=\"S2.SS7\"><title>Scratch Wound Assay</title><p>Human Umbilical Vein Endothelial Cells were seeded in 24-well plates and cultivated to confluence in ECGM at 37°C in a humid atmosphere (5% CO<sub>2</sub>, 95% air). At confluence, a homogenous wound was created in each well with a sterile 1,000 μL pipet tip. After washing, cells were incubated with fresh ECGM supplemented with 10 ng/mL VEGF with or without 40 μM QS-13 for 24 h. The wound area was measured at the beginning (T0) and end of the experiment (T24h) using the ImageJ analysis program (NIH, Bethesda, MD, United States) and the percentage of wound closure after 24 h was calculated.</p></sec><sec id=\"S2.SS8\"><title>Pseudotube Formation on Matrigel</title><p>The ability of HUVECs to form capillary tube structures was evaluated on Matrigel (Dutscher, Brumath, France). Matrigel (200 μL/well of a 10 mg/mL solution) was allowed to polymerize at 37°C for 30 min. After 30 min, 50,000 cells were suspended in ECGM supplemented with 10 ng/mL of VEGF with or without 40 μM QS-13 and seeded into each well. Plates were incubated at 37°C in a humid atmosphere (5% CO<sub>2</sub>, 95% air) for 6 h. Capillary tube formation was imaged after 6 h under an inverted microscope. Quantitative evaluation of the capillary tubes was performed with ImageJ software using the Angiogenesis Analyzer tool. The number of master junctions, master segments, meshes, and the total mesh area of the capillary tube structures were determined.</p></sec><sec id=\"S2.SS9\"><title>Adhesion Assays</title><p>Cells were detached with 50 mM Hepes, 125 mM NaCl, 5 mM KCl, and 1 mM EDTA, washed three times with ECGM, pre-incubated for 30 min with effectors [mouse anti-human α<sub>5</sub>β<sub>1</sub>, catalog number 555614 from BD Pharmingen (10 μg/mL), irrelevant IgG (10 μg/mL) or RGDS peptide 20 mg/mL (Sigma)]. 10,000 cells were seeded per well of a 96 well-plate previously coated with QS-13 and saturated with 1% BSA. After 60 min, cells were washed three times with HEPES buffered Balanced Salt Solution, fixed with 1.1% glutaraldehyde solution and stained with 1% crystal violet solution. Staining was extracted with 10% acetic acid and absorbance was read at 560 nm.</p></sec><sec id=\"S2.SS10\"><title>Affinity Chromatography</title><p>Human Umbilical Vein Endothelial Cells were cultured in 150 cm<sup>2</sup> culture flasks until 70% confluence and cell layer was scrapped in RIPA buffer (Sigma, St Quentin Fallavier, France) supplemented with a protease inhibitor cocktail (Halt Protease Inhibitor Cocktail, Thermo Fisher Scientific, Illkirch, France). Cell lysate was incubated for 30 min at 4°C and centrifuged at 10,000 <italic>g</italic> for 10 min at 4°C to remove insoluble debris. Concentration of soluble proteins was quantified using Biorad Protein Assay (BioRad, Marnes-La-Coquette, France) according to the manufacturer’s instructions. Chromatography was performed on a HiTrap NHS-activated Sepharose High Performance column (GE Healthcare, Orsay, France) functionalized with QS-13 according to the manufacturer’s instructions. Protein extract was chromatographed at 4°C. Unbound proteins were removed with 30 mL of washing buffer [10 mM Tris, 1 mM CaCl<sub>2</sub>, 1 mM MgCl<sub>2</sub>, pH 7.6 supplemented with PIC (ProteoBlock Protease Inhibitor Cocktail, Fermentas, Illkirch, France; w/v) and 0.1% (w/v) octylglucoside]. Proteins bound to the affinity column were eluted with a buffer containing 10 mM Tris, pH 7.6, 0.1% (w/v) octylglucoside and PIC, supplemented with increasing concentrations of NaCl (0.15, 0.6, and 1 M). SDS sample buffer containing 10 mM DTT was added to eluted proteins; samples were incubated for 30 min at 37°C, denatured for 5 min at 95°C and electrophoresed in a 0.1% SDS, 10% polyacrylamide gel. They were then transferred onto Immobilon-P membranes (Millipore, St Quentin en Yvelines, France). Membranes were blocked with 5% non-fat dry milk, 0.1% Tween 20 in TBS for 2 h at room temperature, incubated overnight at 4°C with a rabbit anti-β<sub>1</sub> integrin polyclonal antibody (AB1952P, Merck Millipore) or a rabbit anti-α<sub>5</sub> integrin polyclonal antibody (#4705, Cell Signaling) diluted 1/1000 in 1% non-fat dry milk, 0.1% Tween 20 in TBS) and then for 1 h at room temperature with a second peroxidase-conjugated anti-IgG antibody. Immune complexes were visualized with the ECL chemiluminescence detection kit (GE Healthcare, Orsay, France).</p></sec><sec id=\"S2.SS11\"><title>Solid Phase Assay for Studying QS-13/Integrin Interaction</title><p>Wells of a 96-well plate were coated with 25 nM α<sub>5</sub>β<sub>1</sub> integrin (3230-A5-050; R&D Systems, Lille, France) in amounts overnight at room temperature. The coating was then blocked with TBS containing 5% dry milk, 1 mM MgCl<sub>2</sub>, and 1 mM CaCl<sub>2</sub> for 2 h at room temperature. After washing three times with washing buffer (0.1% dry milk, 1 mM MgCl<sub>2</sub>, and 1 mM CaCl<sub>2</sub> in TBS), the plate was incubated for 90 min at room temperature with 100 μL per well of ranging from 1.25 to 20.10<sup>–11</sup> moles/well biotinylated-QS-13 diluted in washing buffer. After 3 washes, 100 μL of streptavidin-peroxidase diluted 1/20000 in washing buffer were added to each well and incubated for 15 min at room temperature. After 4 washes, 100 μL per well of tetramethylbenzidine (TMB), a peroxidase substrate, were added and incubated in the dark for 15 min. The enzymatic reaction was stopped by adding 50 μL per well of 0.5 M H<sub>2</sub>SO<sub>4</sub>. The intensity of the yellow coloration was measured at 450 nm with a Biochrom Asys UVM 340 microplate reader.</p><p>For competition experiments, wells of a 96-well plate were coated with 25 nM α<sub>5</sub>β<sub>1</sub> integrin and then blocked as described above. Wells were then incubated for 90 min at room temperature with biotinylated QS-13 with or without unbiotinylated QS-13 (molar ratio ranging from 1/1 to 250/1) diluted in washing buffer. The following steps are as described above.</p></sec><sec id=\"S2.SS12\"><title>Surface Plasmon Resonance Analysis</title><p>All experiments were performed on a Biacore T200 instrument (GE Healthcare) at 25°C. Sensor surfaces and other Biacore consumables were purchased from GE Healthcare. Integrin α<sub>5</sub>β<sub>1</sub> was from R&D Systems. The running buffer, HEPES buffered saline (HBS, composed of 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3.4 mM EDTA) was filtered through a 0.22 μm membrane and supplemented with 0.05% P20. Biotinylated peptides were captured on streptavidin-coated sensor chips. Briefly, CM5 sensor chips (GE Healthcare) were preconditioned by duplicate injections of 10 mM HCl, 50 mM NaOH, each for 10 s, and water for 20 s. Before covalent immobilization of streptavidin, traces of biotinylated products that could remain in the flow system were neutralized by injecting a streptavidin solution (0.1 mg/mL in running buffer) for 5 min through all flow cells (<xref rid=\"B3\" ref-type=\"bibr\">Baltzinger et al., 2013</xref>). Streptavidin was then stably immobilized using standard amine-coupling methods. The flow rate was 10 μL/min. Surfaces were activated by injection of a 1:1 mix of 0.2 M N-ethyl-N’-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and 0.05 M N-hydroxysuccinimide (NHS) for 10 min, followed by a 5 min injection of streptavidin at 200 μg/mL in 10 mM sodium acetate (pH 5) and then deactivated with ethanolamine-HCl (pH 8.5) for 10 min. The surface was then subjected to four pulses (100 μL) of 50 mM NaOH at a flow rate 50 μL/min to wash out all non-covalently bound streptavidin. Biotinylated QS-13 (QKISRCQVCVKYSK-biot) was injected onto streptavidin at 5 μg/mL in running buffer, for 10 s at a flow rate of 100 μL/min. Responses were stabilized by five pulse injections of 50 mM NaOH at a flow rate 50 μL/min. Reference surfaces (Fc1) were treated similarly except that an irrelevant biotinylated peptide was injected. Eleven different concentrations of α<sub>5</sub>β<sub>1</sub> integrin (0.12, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 130 nM) were injected into the flow cells at 30 μL/min for 300 s. Dissociation was followed for 600 s. Binding curves were double-reference substracted from buffer blank and reference flow cell (Fc 1). The equilibrium response (Req) was recorded 5 s before the end of integrin injection. The K<sub>D</sub> was determined by fitting the equilibrium response versus the [integrin] curve to a simple 1:1 interaction model with the Biacore T200 evaluation software (GE Healthcare).</p></sec><sec id=\"S2.SS13\"><title>Docking Experiments</title><p>Docking of QS-13 onto α<sub>5</sub>β<sub>1</sub> integrin (RCSB Protein Data Bank 3VI3) was performed using Autodock software (version 4.2; <xref rid=\"B16\" ref-type=\"bibr\">Morris et al., 2009</xref>). The docking parameters were as previously described (<xref rid=\"B13\" ref-type=\"bibr\">Lambert et al., 2018</xref>). The software was used with a fixed integrin and semi-flexible QS-13 ligand (the backbone was frozen as well as the amide links and guanidinium groups). Because the integrin is a large molecule, we performed several independent dockings targeting different subvolumes of the protein; we considered 125 overlapping boxes with a volume of 47.25 Å × 47.25 Å × 47.25 Å. Each box was divided along the three directions, and the distance between the nodes was equal to 0.375 Å. The Lamarckian genetic algorithm was used, and for each ligand, 150 dockings were performed with the default parameters of Autodock except for the population size (150), number of energy evaluations (5 × 10<sup>6</sup>), and maximum number of generations (30,000; <xref rid=\"B13\" ref-type=\"bibr\">Lambert et al., 2018</xref>). Molecular models were derived from the preliminary study. Molecular models were graphed with VMD software, which is available online.</p></sec><sec id=\"S2.SS14\"><title>Statistical Analyses</title><p>For <italic>in vitro</italic> experiments, results are expressed as the mean ± SD and statistical significance were determined using Student’s <italic>t</italic>-test. For <italic>in vivo</italic> experiments, the non-parametric Mann–Whitney test was performed.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>QS-13 Decreases <italic>in vivo</italic> Matrigel Plug Angiogenesis</title><p>The subcutaneous Matrigel plug assay in mouse is a gold standard <italic>in vivo</italic> assay to screen pro- or anti-angiogenic molecules. Administration of a fluorescent imaging agent (AngioSense680<sup>TM</sup>) and quantitative analyses with a fluorescence molecular tomographic (FMT) imaging system revealed a significant decrease (−57%) of angiogenesis in Matrigel plug of QS-13-treated mice compared to control mice at day 13 (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). The Matrigel plugs were then excised and 4 μm thick sections were performed and stained using an anti-CD31 antibody as endothelial cell marker. The MVD analysis of the different sections confirmed that QS-13 inhibits <italic>in vivo</italic> angiogenesis by 61% (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>QS-13 decreases <italic>in vivo</italic> angiogenesis in a Matrigel plug model. Matrigel (400 μL) was subcutaneously injected into mice. After 13 days of treatment with VEGF alone or with VEGF and QS-13, a fluorescent imaging agent was administrated. <bold>(A)</bold> At day 14, anesthetized mice were imaged using molecular fluorescence tomography and FMT signal intensities were measured. <bold>(B)</bold> Matrigel plugs were then excised, CD31-immunostaining was performed on Matrigel plug sections and microvessel density (MVD) was evaluated. Two independent experiments (<italic>N</italic> = 2) were carried out with 4 mice in each group (<italic>n</italic> = 4). The histograms represent the means of the two experiments ± SD. *<italic>p</italic> < 0.05; **<italic>p</italic> < 0.01.</p></caption><graphic xlink:href=\"fcell-08-00775-g001\"/></fig></sec><sec id=\"S3.SS2\"><title>QS-13 Decreases <italic>in vitro</italic> Endothelial Cell Migration and Pseudotube Formation Without Affecting Cell Proliferation</title><p>In order to decipher the anti-angiogenic effect of QS-13, we performed <italic>in vitro</italic> studies.</p><p>As proliferation of endothelial cells plays an essential role in angiogenesis, HUVEC proliferation under the influence of QS13 was assessed at 24, 48, 72, and 96 h using the WST-1 method. QS-13 does not significantly alter HUVEC proliferation up to 96 h incubation (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>QS-13 decreases <italic>in vitro</italic> endothelial cell migration and pseudotube formation without affecting cell proliferation. To study cell proliferation, HUVECs were incubated for 24, 48, 72, and 96 h with or without 40 μM QS-13. <bold>(A)</bold> Proliferation was measured using the WST-1 reagent and absorbance was read at 450 nm (<italic>n</italic> = 8). Three independent experiments were carried out. The histogram represents the means ± SD of the more representative one. NS, not significant. To study endothelial cell migration, a scratch wound was performed using a pipet tip at confluence. HUVECs were then incubated with or without 40 μM QS-13 for 24 h at 37°C. <bold>(B)</bold> Wounds were microphotographed after 24 h of incubation. Scale bar: 50 nm. <bold>(C)</bold> Wound closure was measured using ImageJ software. Three independent experiments were carried out. The histogram represents the means ± SD of the more representative one. <italic>n</italic> = 8, ***<italic>p</italic> < 0.001. To study pseudotube formation, HUVECs were seeded on Matrigel coated well and incubated with or without 40 μM QS-13. <bold>(D)</bold> Pseudotube formation was observed under an inverted microscope after 6 h and photographed. Scale bar: 200 nm. <bold>(E)</bold> The number of master segments, <bold>(F)</bold> the number of master junction, <bold>(G)</bold> the number of meshes and <bold>(H)</bold> the total mesh area of pseudotube were determined with ImageJ software. Three independent experiments were carried out. The histogram represents the means ± SD of the more representative one. <italic>n</italic> = 8, ***<italic>p</italic> < 0.001.</p></caption><graphic xlink:href=\"fcell-08-00775-g002\"/></fig><p>Human Umbilical Vein Endothelial Cell migration, a key step in angiogenesis, was studied using the scratch wound healing model. Scratch wounds were photographed at 0 and 24 h (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). Compared to control, QS-13 decreased wound closure by 28% (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>).</p><p>Human Umbilical Vein Endothelial Cell pseudotube-formation assay is a well-established <italic>in vitro</italic> angiogenesis assay based on the ability of endothelial cells to form three-dimensional capillary-like tubular structures. We demonstrated that QS-13 strongly altered pseudotube formation (<xref ref-type=\"fig\" rid=\"F2\">Figure 2D</xref>): it decreased the number of master segments by 47% (<xref ref-type=\"fig\" rid=\"F2\">Figure 2E</xref>), the number of master junction by 44% (<xref ref-type=\"fig\" rid=\"F2\">Figure 2F</xref>), the number of meshes by 56% (<xref ref-type=\"fig\" rid=\"F2\">Figure 2G</xref>) and the total mesh area by 87% (<xref ref-type=\"fig\" rid=\"F2\">Figure 2H</xref>).</p></sec><sec id=\"S3.SS3\"><title>Identification of α<sub>5</sub>β<sub>1</sub> Integrin as a QS-13 Receptor on HUVECs</title><sec id=\"S3.SS3.SSS1\"><title><italic>In silico</italic> Studies</title><p>α<sub>5</sub>β<sub>1</sub> integrin was previously reported to be largely expressed in HUVECs. The integrin mediates cell adhesion to ECM (<xref rid=\"B23\" ref-type=\"bibr\">Short et al., 1998</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Zeng et al., 2009</xref>) and cell migration through a VEGFR-dependent mechanism (<xref rid=\"B18\" ref-type=\"bibr\">Orecchia et al., 2003</xref>). Thereby, we investigated a putative interaction of QS-13 with the α<sub>5</sub>β<sub>1</sub> integrin. We previously performed molecular dynamics simulations on isolated QS-13 (<xref rid=\"B13\" ref-type=\"bibr\">Lambert et al., 2018</xref>) and observed that a disulfide bond locked the CQVC sequence in a conformation that exposed the glutamine (Q) side chain. The presence of the disulfide bond was confirmed by MALDI-ToF MS analyses. Molecular docking experiments were carried out to test the hypothesis of a QS-13/α<sub>5</sub>β<sub>1</sub> integrin interaction (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). For the two considered conformations of QS-13 peptide (extracted from molecular dynamics simulations), the best 180 results (from the energy point of view) were used in order to cluster the solutions and to identify the main interaction areas. For each conformation, the most populated cluster (32.2% and 19.4% for conformation 1 and 2, respectively) contained the pose associated to the best free energy of binding (-5.71 kcal/mol and -7.25 kcal/mol for conformation 1 and 2, respectively). The visualization of these poses (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>) demonstrated that both conformations share the same interaction area with the α<sub>5</sub>β<sub>1</sub> integrin at the interface between the two integrin subunits. Despite the fact that the two QS-13 shapes were rather different [one could be considered “folded” (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>) and the second “elongated” (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>)], the contacts they made with the integrin were very similar. Indeed, the key interactions established by QS-13 with the α<sub>5</sub>β<sub>1</sub> integrin mainly involved the following residues: the first lysine (K2) with S134 and D137 of the β<sub>1</sub> integrin subunit; the arginine (R5) with D227 of the α<sub>5</sub> integrin subunit and E320 of the β<sub>1</sub> integrin subunit; the glutamine (Q7) exposed by the disulfide bond with Q189, Q221, and D227 of the β<sub>1</sub> integrin subunit. The number of inter-molecule hydrogen bonds is comparable from one pose to the other, and two pairs of interaction are conserved, namely S134/K2 and D227/Q7.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Docking experiments of QS-13 on α<sub>5</sub>β<sub>1</sub> integrin. The best results of QS-13 docking experiment display the same interaction area with the α<sub>5</sub>β<sub>1</sub> integrin at the interface between the two integrin subunits. <bold>(A)</bold> Integrin subunits and QS-13 are represented using the Quick Surface scheme and colored according to the nature of the chain (cyan for α<sub>5</sub> and magenta for β<sub>1</sub>) or the conformation of QS-13 (first conformation extracted from the MD in red and second conformation extracted from the MD in orange). <bold>(B,C)</bold> Zoomed-in representations of the best energy conformation 1 and 2 of QS-13, respectively. The New Cartoon scheme is used to represent QS-13 and the Quick Surface scheme used for integrin is set to transparent. Residues making contacts between integrin and QS-13 (a contact is defined as a distance between two atoms lower than 3 Å) are represented with the CPK mode. Color labels are adopted in order to distinguish integrin (white) and QS-13 (black) residues. Hydrogen bonds are highlighted with thick black dashed lines.</p></caption><graphic xlink:href=\"fcell-08-00775-g003\"/></fig></sec><sec id=\"S3.SS3.SSS2\"><title><italic>In vitro</italic> Experiments</title><p>To determine whether QS-13 binds to HUVECs through α<sub>5</sub>β<sub>1</sub> integrin, we measured HUVEC adhesion on QS-13 in the presence or absence of an anti-α<sub>5</sub>β<sub>1</sub> integrin blocking antibody (10 μg/mL). The preincubation of HUVECs with the blocking anti-α<sub>5</sub>β<sub>1</sub> antibody significantly inhibited (71%) cell adhesion on QS-13, whereas an irrelevant IgG had no effect (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>), suggesting a putative interaction between QS-13 and α<sub>5</sub>β<sub>1</sub> integrin. To verify this hypothesis, we performed affinity chromatography. HUVEC extracts were loaded onto a QS-13-functionalized affinity column. Proteins bound to the affinity column were eluted with increasing concentrations of NaCl (0.15, 0.6, and 1.0 M; <xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1</xref>). Eluted samples, analyzed by western blot, showed the presence of α<sub>5</sub> and β<sub>1</sub> integrin subunits in the 0.6 M eluted fraction (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>). The existence of a direct interaction between α<sub>5</sub>β<sub>1</sub> integrin and QS-13 was investigated using two different methods: solid phase assay and surface plasmon resonance (SPR). For this purpose, QS-13 was biotinylated at its C-terminus. In the solid phase assay, we demonstrated that biotinylated-QS-13 binds to α<sub>5</sub>β<sub>1</sub> integrin in a ligand concentration dependent manner (<xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>). Biotinylated QS-13 was covalently immobilized on a streptavidin-coated CM5 sensor chip. α<sub>5</sub>β<sub>1</sub> integrin was then injected at eleven concentrations ranging from 0.12 to 130 nM. Moreover, a competitive assay was performed to confirm the specificity of the interaction. Increasing amounts of unbiotinylated QS-13 decreased biotinylated-QS-13 binding to α<sub>5</sub>β<sub>1</sub> integrin (<xref ref-type=\"fig\" rid=\"F4\">Figure 4D</xref>). In SPR experiments, biotinylated-QS-13 was covalently immobilized on a streptavidin-coated CM5 sensor chip. α<sub>5</sub>β<sub>1</sub> integrin was then injected at eleven concentrations ranging from 0.12 to 130 nM. <xref ref-type=\"fig\" rid=\"F4\">Figure 4E</xref> presents the sensorgrams obtained after double referencing (subtraction of reference channel and buffer injection). α<sub>5</sub>β<sub>1</sub> integrin binds to Q-S13 in a dose-dependent manner. The double referenced equilibrium responses recorded 5 s before the end of integrin injections were called Req. <xref ref-type=\"fig\" rid=\"F4\">Figure 4F</xref> shows the plotted responses of Req as a function of α<sub>5</sub>β<sub>1</sub> integrin concentration. The equilibrium affinity parameter (K<sub>D</sub>) was determined by fitting the Req versus the [α<sub>5</sub>β<sub>1</sub> integrin] curve to a simple 1:1 interaction model. We measured a K<sub>D</sub> of 20.3 ± 7.5 nM for the QS-13/α<sub>5</sub>β<sub>1</sub> integrin interaction.</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>QS-13 peptide binds to α<sub>5</sub>β<sub>1</sub> integrin. <bold>(A)</bold> HUVECs were pre-incubated with ECGM alone, ECGM containing an anti-α<sub>5</sub>β<sub>1</sub> blocking antibody or an irrelevant antibody (10 μg/mL). HUVEC adhesion was measured as described in the section “Materials and Methods.” Three independent experiments were carried out. The histogram represents the means ± SD of the more representative one (<italic>n</italic> = 8), **<italic>p</italic> < 0.01. <bold>(B)</bold> HUVEC extracts were submitted to affinity chromatography on a QS-13 -bound column. Lane 1: total cell extracts; lane 2: unbound proteins; lane 3: 0.15 M NaCl eluted fraction; lane 4: 0.6 M NaCl eluted fraction. <bold>(C)</bold> Direct interaction between α<sub>5</sub>β<sub>1</sub> integrin at concentrations ranging from 0.25 to 2.10<sup>– 10</sup> mole/well and QS-13 was studied using solid phase assay as described in the “Materials and Methods” section. Two independent experiments were carried out (<italic>n</italic> = 4), ***<italic>p</italic> < 0.001. <bold>(D)</bold> A competition assay was performed using increasing concentrations of unbiotinylated-QS-13 while biotinylated QS-13 and α<sub>5</sub>β<sub>1</sub> integrin concentrations were kept constant Two independent experiments were carried out (<italic>n</italic> = 4), **<italic>p</italic> < 0.01, ***<italic>p</italic> < 0.001. <bold>(E,F)</bold> The equilibrium affinity of the α<sub>5</sub>β<sub>1</sub> integrin-QS-13 interaction was analyzed by SPR. <bold>(E)</bold> Concentration-dependent responses of integrin α<sub>5</sub>β<sub>1</sub> (0.12 to 130 nM range) to the streptavidin-captured biotinylated QS-13. <bold>(F)</bold> Fit of the equilibrium response (Req) versus [α<sub>5</sub>β<sub>1</sub> integrin] to a 1:1 binding indicates a K<sub>D</sub> of 20.3 ± 7.5 nM.</p></caption><graphic xlink:href=\"fcell-08-00775-g004\"/></fig><p>Taken together, the results confirm that QS-13 directly binds to α<sub>5</sub>β<sub>1</sub> integrin.</p></sec></sec><sec id=\"S3.SS4\"><title>RGDS Peptide Competes With QS-13 for α<sub>5</sub>β<sub>1</sub> Integrin Binding on HUVECs</title><p>As specified in the “Materials and Methods” section, docking experiments were carried out using the structure associated with PDB ID 3VI3: this crystallographic data corresponded to the ligand-free form of α<sub>5</sub>β<sub>1</sub> integrin. The structure associated with PBD ID 3VI4 was related to α<sub>5</sub>β<sub>1</sub> integrin headpiece in complex with RGD peptide: the visualization and comparison of most likely QS-13 conformations with this experimental ligand-associated form (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS2\">Supplementary Figure S2</xref>) evidenced that the QS-13 binding site was in close vicinity to the RGD peptide binding site. In the case of the RGD peptide, the residues implicated in the interactions were the arginine (R) with S134 of the β<sub>1</sub> integrin sub-unit and the aspartic acid (D) with Q189, Q221, and D227 of the α<sub>5</sub> integrin sub-unit. Even though the sizes of the RGD peptide and of QS-13 were very different, the same residues are involved in both interactions with the integrin subunits.</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Comparison of QS-13 and RGD theoretical binding sites on α<sub>5</sub>β<sub>1</sub> integrin and <italic>in vitro</italic> confirmation. <bold>(A)</bold> α<sub>5</sub>β<sub>1</sub> integrin is represented using the Quick Surface scheme and a color code related to the nature of the subunit (cyan for α<sub>5</sub> and magenta for β<sub>1</sub>). The best docking poses of the two QS-13 conformations are superimposed with the conformation of the RDG peptide (from the experimental complex corresponding to the PDB ID 3VI4 solved with X-ray experiments) and depicted with a color coded (first QS-13 conformation extracted from the MD in red, the second in orange and RGD peptide in green) Licorice representation. Residues from the integrin making contacts with QS-13 (as evidenced in <xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>) are highlighted with white surface representation and labeled. <bold>(B)</bold> HUVECs were pre-incubated for 30 min with culture medium alone (control), culture medium supplemented with RGDS peptide (20 μg/mL) or with QS-13 (20 μg/mL) and adhesion was measured. Three independent experiments were carried out. The histogram represents the means ± SD of the more representative one. <italic>n</italic> = 8, ***<italic>p</italic> < 0.001.</p></caption><graphic xlink:href=\"fcell-08-00775-g005\"/></fig><p>To test this hypothesis, competition binding assay were performed. HUVECs were pre-incubated with medium alone or medium supplemented with either QS-13 or RGDS peptide and seeded on QS-13 coating. Cell pre-incubation with RGDS inhibited cell adhesion the same way as QS-13 itself (-60%) (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>).</p></sec></sec><sec id=\"S4\"><title>Discussion</title><p>Angiogenesis plays critical roles in human physiological processes. This is a complex phenomenon regulated in a spatial and temporal manner that depends on the cooperation between angiogenic factors, ECM components, and endothelial cells. Uncontrolled angiogenesis may lead to several angiogenic disorders and to vascular overgrowth (hemangiomas, psoriasis, vascularized tumors…). Numerous pro-angiogenic drivers have been identified such as VEGF. To date, the most common approaches to the inhibition of the VEGF axis include the blockade of VEGF receptors (VEGFRs) or ligands by neutralizing antibodies, as well as the inhibition of receptor tyrosine kinases (RTK). Unfortunately, this type of inhibitors leads to numerous side effects as well as resistance phenomena (<xref rid=\"B9\" ref-type=\"bibr\">Haibe et al., 2020</xref>).</p><p>In the present article, we demonstrate that the Tetrastatin QKISRCQVCVKYS peptide sequence (QS-13) inhibits <italic>in vivo</italic> angiogenesis in the Matrigel plug model. <italic>In vitro</italic>, QS-13 does not affect cell proliferation but decreases cell migration and pseudotube organization on Matrigel. The preincubation of HUVECs with a blocking anti-α<sub>5</sub>β<sub>1</sub> antibody significantly inhibited cell adhesion on QS-13, whereas an irrelevant IgG had no effect, suggesting an interaction between QS-13 and the α<sub>5</sub>β<sub>1</sub> integrin.</p><p>The NC1 domains of the different α(IV) collagen chains were reported to exert anti-tumor or anti-angiogenic effects (<xref rid=\"B17\" ref-type=\"bibr\">Mundel and Kalluri, 2007</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Monboisse et al., 2014</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Brassart-Pasco et al., 2020</xref>). We and others demonstrated that the anti-tumor activities of Tumstatin or Tetrastatin were mediated through binding to α<sub>v</sub>β<sub>3</sub> integrin at the tumor cell surface (<xref rid=\"B19\" ref-type=\"bibr\">Pasco et al., 2000</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Brassart-Pasco et al., 2012</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Lambert et al., 2018</xref>). It was also demonstrated that Arresten, Canstatin or Tumstatin also exerted anti-angiogenic activities mediated through binding to α<sub>1</sub>β<sub>1</sub>, α<sub>1</sub>β<sub>1</sub>/α<sub>v</sub>β<sub>3</sub>/α<sub>v</sub>β<sub>5</sub>, and α<sub>v</sub>β<sub>5</sub>/α<sub>v</sub>β<sub>3</sub> integrin binding, respectively (<xref rid=\"B6\" ref-type=\"bibr\">Brassart-Pasco et al., 2020</xref>). Furthermore, several integrins, including α<sub>5</sub>β<sub>1</sub>, and α<sub>v</sub>β<sub>3</sub>/β<sub>5</sub>, have been described to play an important role in tumor angiogenesis. Their overexpression on tumor neo-vessels suggest new anti-angiogenic therapies (<xref rid=\"B22\" ref-type=\"bibr\">Schaffner et al., 2013</xref>).</p><p>The anti-α<sub>5</sub>β<sub>1</sub> integrin antibody M200/volociximab was reported to inhibit angiogenesis and to suppress tumor growth and metastasis in mice (<xref rid=\"B2\" ref-type=\"bibr\">Almokadem and Belani, 2012</xref>). It showed preliminary evidence of efficacy in advanced NSCLC (<xref rid=\"B4\" ref-type=\"bibr\">Besse et al., 2013</xref>).</p><p>Peptides also emerged as important therapeutic agents in angiogenesis-dependent diseases due to their low toxicity and high specificity (<xref rid=\"B21\" ref-type=\"bibr\">Rosca et al., 2011</xref>). The α<sub>5</sub>β<sub>1</sub>-blocking peptide ATN-161, derived from the synergy region of fibronectin, showed preclinical anti-cancer activities (<xref rid=\"B25\" ref-type=\"bibr\">Stoeltzing et al., 2003</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Khalili et al., 2006</xref>). It entered clinical testing but failed to provide therapeutic benefits (<xref rid=\"B8\" ref-type=\"bibr\">Cianfrocca et al., 2006</xref>).</p><p>Another matrikine, a 20-kDa C-terminal fragment of type XVIII collagen, endostatin, was also reported to inhibit α<sub>5</sub>β<sub>1</sub> integrin (<xref rid=\"B26\" ref-type=\"bibr\">Sudhakar et al., 2003</xref>). It was tested in clinical studies in combination with chemotherapies and radiotherapies but the results were inconsistent, probably due to recombinant protein production (<xref rid=\"B1\" ref-type=\"bibr\">Alday-Parejo et al., 2019</xref>).</p><p>We also demonstrated that the inhibitory effects of Tetrastatin are conformation-dependent with a crucial role of the presence of a disulfide bond in QS-13 (<xref rid=\"B13\" ref-type=\"bibr\">Lambert et al., 2018</xref>). Because of the influence and importance of QS-13 disulfide bond, the present <italic>in silico</italic> investigation of the interaction between α<sub>5</sub>β<sub>1</sub> integrin and QS-13 was designed using peptide conformations displaying the presence of the disulfide bond. The results of the docking experiments demonstrate that QS-13 is able to bind to α<sub>5</sub>β<sub>1</sub> integrin in a stable mode since the evaluated free energy of binding of the best solutions is below the threshold of -3.00 kcal/mol. In addition to the energy aspect, the statistical analysis of the best molecular docking results indicates that the interaction areas with α<sub>5</sub>β<sub>1</sub> integrin are not randomly distributed. Indeed, the clustering of the best poses leads to the identification of a region gathering the highest number of results and corresponds to an interface region between the two integrin subunits. This region overlaps with the binding site of the RGD sequence, a well-known integrin recognition sequence. Despite the size difference between RGD and QS-13, the residues involved in the interaction at the protein level show a strong overlap and are either polar residues or residues with charged side chains prone to hydrogen-bonding. It should also be noted that in the case of QS-13, the glutamine residue exposed by the presence of the disulfide bond (<xref rid=\"B13\" ref-type=\"bibr\">Lambert et al., 2018</xref>), is one of the three residues in contact with the integrin.</p><p>The interaction between QS-13 and α<sub>5</sub>β<sub>1</sub> integrin is confirmed by affinity chromatography, solid phase binding assay and SPR. As the RGD binding site was shown to overlap QS-13 theoretical binding sites determined by molecular docking, a competition experiment was performed <italic>in vitro</italic>. Cell pre-incubation with RGDS inhibited cell adhesion to the QS-13 by about 60%, confirming that QS-13 binding site was in close vicinity to RGD binding site on α<sub>5</sub>β<sub>1</sub> integrin.</p><p>Taken together, our results demonstrate that QS-13 binds to α<sub>5</sub>β<sub>1</sub> integrin and inhibits endothelial cell migration and angiogenesis and is a potent anti-angiogenic agent. Since the disulfide bond forms spontaneously in solution, QS-13 may be protected from protease degradation <italic>in vivo</italic>. It offers new therapeutic strategies in hemangiomas and psoriasis treatment alone or in combination with anti-microbial peptides (<xref rid=\"B15\" ref-type=\"bibr\">Morizane and Gallo, 2012</xref>).</p><p>In tumors, α<sub>5</sub>β<sub>1</sub> integrin is overexpressed and represents an interesting target for the administration of anti-cancer agents <italic>in situ</italic> (<xref rid=\"B22\" ref-type=\"bibr\">Schaffner et al., 2013</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Blandin et al., 2015</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Alday-Parejo et al., 2019</xref>). In addition to its inhibitory effects on endothelial cell migration and angiogenesis, QS-13 also decreases cancer progression by inhibiting tumor cell migration and invasion and <italic>in vivo</italic> tumor growth (<xref rid=\"B13\" ref-type=\"bibr\">Lambert et al., 2018</xref>), It would be of interest to propose new therapeutic strategies based for example on QS-13 grafting on the surface of nanoparticles loaded with cytotoxic agents to a specific targeting and drug delivery to the tumor, allowing a decrease in the drug side effects. The patient will benefit from a targeted delivery of therapeutic agents, as well as the anti-angiogenic and anti-tumor activity of QS-13.</p></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>All datasets presented in this study are included in the article/<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Material</xref>.</p></sec><sec id=\"S6\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by the French “Ministère de l’Enseignement Supérieur et de la Recherche” (Ethics Committees Nos. C2EA-56 and C2EA-75) in compliance with the “Directive 2010/63/UE”. Protocol no. 4373_V1 APAFIS (07/09/2016).</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>AV-G, JD, CB, SB, LC, AH, AD-D, CS, and SB-P carried out the experiment. JD, SB, LC, BB, J-BO, LR, JM, and SB-P contributed to the interpretation of the results. SB-P took the lead in writing the manuscript. All authors provided critical feedback and helped to improve the manuscript.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by the Centre National de la Recherche Scientifique (UMR 7369), the University of Reims Champagne-Ardenne, and the Conférence de Coordination Interrégionale de la Ligue Contre le Cancer du Grand Est (CCIR-GE).</p></fn></fn-group><ack><p>The authors thank the HPC-Regional Center ROMEO, the Multiscale Molecular Modeling Platform (P3M) of the University of Reims Champagne-Ardenne (France) for providing time and support. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Cell Neurosci</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Cell Neurosci</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Cell. Neurosci.</journal-id><journal-title-group><journal-title>Frontiers in Cellular Neuroscience</journal-title></journal-title-group><issn pub-type=\"epub\">1662-5102</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32848623</article-id><article-id pub-id-type=\"pmc\">PMC7431706</article-id><article-id pub-id-type=\"doi\">10.3389/fncel.2020.00232</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cellular Neuroscience</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Altered Sensory Neuron Development in CMT2D Mice Is Site-Specific and Linked to Increased GlyRS Levels</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Sleigh</surname><given-names>James N.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><xref ref-type=\"author-notes\" rid=\"fn001\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/305081/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Mech</surname><given-names>Aleksandra M.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Aktar</surname><given-names>Tahmina</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Yuxin</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn001\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Schiavo</surname><given-names>Giampietro</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"author-notes\" rid=\"fn001\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/3096/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London</institution>, <addr-line>London</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff2\"><sup>2</sup><institution>UK Dementia Research Institute, University College London</institution>, <addr-line>London</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Discoveries Centre for Regenerative and Precision Medicine, University College London Campus</institution>, <addr-line>London</addr-line>, <country>United Kingdom</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: David J. Adams, University of Wollongong, Australia</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Sinead Madeleine Murphy, Tallaght University Hospital, Ireland; Albena Jordanova, University of Antwerp, Belgium</p></fn><corresp id=\"c001\">*Correspondence: James N. Sleigh <email>j.sleigh@ucl.ac.uk</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p><sup>†</sup>ORCID: James N. Sleigh <ext-link ext-link-type=\"uri\" xlink:href=\"https://orcid.org/0000-0002-3782-9045\">orcid.org/0000-0002-3782-9045</ext-link> Yuxin Zhang <ext-link ext-link-type=\"uri\" xlink:href=\"https://orcid.org/0000-0003-0243-2520\">orcid.org/0000-0003-0243-2520</ext-link> Giampietro Schiavo <ext-link ext-link-type=\"uri\" xlink:href=\"https://orcid.org/0000-0002-4319-8745\">orcid.org/0000-0002-4319-8745</ext-link></p></fn><fn fn-type=\"other\" id=\"fn002\"><p><bold>Specialty section:</bold>This article was submitted to Cellular Neuropathology, a section of the journal Frontiers in Cellular Neuroscience</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>14</volume><elocation-id>232</elocation-id><history><date date-type=\"received\"><day>27</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>01</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Sleigh, Mech, Aktar, Zhang and Schiavo.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Sleigh, Mech, Aktar, Zhang and Schiavo</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Dominant, missense mutations in the widely and constitutively expressed <italic>GARS1</italic> gene cause peripheral neuropathy that usually begins in adolescence and principally impacts the upper limbs. Caused by a toxic gain-of-function in the encoded glycyl-tRNA synthetase (GlyRS) enzyme, the neuropathology appears to be independent of the canonical role of GlyRS in aminoacylation. Patients display progressive, life-long weakness and wasting of muscles in hands followed by feet, with frequently associated deficits in sensation. When dysfunction is observed in motor and sensory nerves, there is a diagnosis of Charcot-Marie-Tooth disease type 2D (CMT2D), or distal hereditary motor neuropathy type V if the symptoms are purely motor. The cause of this varied sensory involvement remains unresolved, as are the pathomechanisms underlying the selective neurodegeneration characteristic of the disease. We have previously identified in CMT2D mice that neuropathy-causing <italic>Gars</italic> mutations perturb sensory neuron fate and permit mutant GlyRS to aberrantly interact with neurotrophin receptors (Trks). Here, we extend this work by interrogating further the anatomy and function of the CMT2D sensory nervous system in mutant <italic>Gars</italic> mice, obtaining several key results: (1) sensory pathology is restricted to neurons innervating the hindlimbs; (2) perturbation of sensory development is not common to all mouse models of neuromuscular disease; (3) <italic>in vitro</italic> axonal transport of signaling endosomes is not impaired in afferent neurons of all CMT2D mouse models; and (4) <italic>Gars</italic> expression is selectively elevated in a subset of sensory neurons and linked to sensory developmental defects. These findings highlight the importance of comparative neurological assessment in mouse models of disease and shed light on key proposed neuropathogenic mechanisms in <italic>GARS1</italic>-linked neuropathy.</p></abstract><kwd-group><kwd>aminoacyl-tRNA synthetase (ARS)</kwd><kwd>amyotrophic lateral sclerosis (ALS)</kwd><kwd>axonal transport</kwd><kwd>Charcot-Marie-Tooth disease (CMT)</kwd><kwd>glycyl-tRNA synthetase (GlyRS)</kwd><kwd>neurotrophin receptors (Trks)</kwd><kwd>sensory neuron</kwd><kwd>signaling endosome</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Medical Research Council<named-content content-type=\"fundref-id\">10.13039/501100000265</named-content></funding-source><award-id rid=\"cn001\">MR/S006990/1</award-id></award-group><award-group><funding-source id=\"cn002\">Wellcome Trust<named-content content-type=\"fundref-id\">10.13039/100004440</named-content></funding-source><award-id rid=\"cn002\">103191/Z/13/Z, 107116/Z/15/Z</award-id></award-group><award-group><funding-source id=\"cn003\">Horizon 2020<named-content content-type=\"fundref-id\">10.13039/501100007601</named-content></funding-source><award-id rid=\"cn003\">739572</award-id></award-group></funding-group><counts><fig-count count=\"7\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"78\"/><page-count count=\"14\"/><word-count count=\"10480\"/></counts></article-meta></front><body><sec sec-type=\"introduction\" id=\"s1\"><title>Introduction</title><p>Characterized by distal dysfunction of motor and sensory nerves, Charcot-Marie-Tooth disease (CMT) is a hereditary peripheral neuropathy that usually presents in adolescence and affects 1 in 2,500–5,000 people, which makes it the most common inherited neuromuscular condition (Pipis et al., <xref rid=\"B47\" ref-type=\"bibr\">2019</xref>). Classically, the disease can be categorized as CMT1, typified by demyelination and thus reduced nerve conduction velocity, CMT2 in which there is axon loss but no diminished nerve conduction velocity, and intermediate CMT that shares features of both CMT1 and CMT2 (Reilly et al., <xref rid=\"B50\" ref-type=\"bibr\">2011</xref>). Consistent with length-dependency, patients display slowly progressive, bilateral muscle weakness and sensory deficits predominantly in the extremities, typically starting in the feet. CMT is on a phenotypic spectrum with distal hereditary motor neuropathy (dHMN) and hereditary sensory/autonomic neuropathy (HSN/HSAN), which have mainly motor and sensory/autonomic involvement, respectively, and can be caused by mutations in the same gene (Pisciotta and Shy, <xref rid=\"B48\" ref-type=\"bibr\">2018</xref>).</p><p>To date, mutations in more than 100 different genetic loci have been linked to CMT (Rossor et al., <xref rid=\"B52\" ref-type=\"bibr\">2013</xref>; Pipis et al., <xref rid=\"B47\" ref-type=\"bibr\">2019</xref>). Many causative CMT1 genes are selectively expressed by myelinating Schwann cells or have myelin-specific functions, providing mechanistic justification for pathology. However, CMT2-associated genes are involved in a variety of processes critical to general cell viability (e.g., mitochondrial dynamics, endolysosomal sorting, ubiquitination, heat shock response), and the pathomechanisms underlying neuronal selectivity remain relatively obscure. Fitting with this, the widely and constitutively active aminoacyl-tRNA synthetase (ARS) enzymes, which covalently bind specific amino acids to their partner tRNAs for protein translation (Ibba and Soll, <xref rid=\"B24\" ref-type=\"bibr\">2000</xref>), represent the largest protein family implicated in CMT etiology. To date, dominant mutations in six ARS-encoding genes (<italic>GARS1</italic>, <italic>YARS1</italic>, <italic>AARS1</italic>, <italic>HARS1</italic>, <italic>WARS1</italic>, and <italic>MARS1</italic>) have been linked to CMT with varying degrees of evidence for pathogenicity (Wei et al., <xref rid=\"B76\" ref-type=\"bibr\">2019</xref>).</p><p>Encoding glycyl-tRNA synthetase (GlyRS), which charges glycine, <italic>GARS1</italic> is the first and best-studied ARS gene linked to CMT (designated CMT type 2D, CMT2D, OMIM: 601472; Antonellis et al., <xref rid=\"B3\" ref-type=\"bibr\">2003</xref>). Uncharacteristically and contravening length-dependency, CMT2D patients frequently display upper limb predominance with weakness beginning in dorsal interosseus muscles of the hand and progressing to involve lower limbs in about only half of patients (Antonellis et al., <xref rid=\"B4\" ref-type=\"bibr\">2018</xref>; Sivakumar et al., <xref rid=\"B59\" ref-type=\"bibr\">2005</xref>). Genetic studies across yeast, <italic>Drosophila melanogaster</italic> and mouse models for CMT2D indicate that, although neuropathy-causing mutations can abolish canonical GlyRS function and loss-of-function pathogenesis hypotheses prevail (Meyer-Schuman and Antonellis, <xref rid=\"B31\" ref-type=\"bibr\">2017</xref>), the disease is most likely caused by a toxic gain-of-function (Boczonadi et al., <xref rid=\"B9\" ref-type=\"bibr\">2018</xref>; Wei et al., <xref rid=\"B76\" ref-type=\"bibr\">2019</xref>). Commensurate with mutant protein toxicity, wild-type <italic>GARS1</italic> overexpression in CMT2D mice has no discernible rescue effect on neuromuscular pathologies, while the increased dosage of disease-causing <italic>Gars</italic> alleles causes more severe neuropathy (Motley et al., <xref rid=\"B37\" ref-type=\"bibr\">2011</xref>). Moreover, all assessed GlyRS mutants possess a similar conformational opening that excavates neomorphic surfaces usually buried within the structure of the wild-type enzyme (He et al., <xref rid=\"B22\" ref-type=\"bibr\">2011</xref>, <xref rid=\"B21\" ref-type=\"bibr\">2015</xref>). Given that GlyRS is secreted from several different cell types (Park et al., <xref rid=\"B45\" ref-type=\"bibr\">2012</xref>, <xref rid=\"B46\" ref-type=\"bibr\">2018</xref>; Grice et al., <xref rid=\"B18\" ref-type=\"bibr\">2015</xref>; He et al., <xref rid=\"B21\" ref-type=\"bibr\">2015</xref>), these uncovered protein regions can mediate aberrant deleterious interactions both inside and outside the cell (He et al., <xref rid=\"B21\" ref-type=\"bibr\">2015</xref>; Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>; Mo et al., <xref rid=\"B33\" ref-type=\"bibr\">2018</xref>), likely accounting for non-cell-autonomous aspects of pathology (Grice et al., <xref rid=\"B18\" ref-type=\"bibr\">2015</xref>, <xref rid=\"B17\" ref-type=\"bibr\">2018</xref>). While some of these mis-interactions are with neuronally-enriched proteins, the pathomechanisms underlying neuronal selectivity in CMT2D remain unresolved. Nevertheless, recent studies indicate that impairments in the processes of axonal transport (Benoy et al., <xref rid=\"B8\" ref-type=\"bibr\">2018</xref>; Mo et al., <xref rid=\"B33\" ref-type=\"bibr\">2018</xref>) and protein translation (Niehues et al., <xref rid=\"B41\" ref-type=\"bibr\">2015</xref>) may be playing a causative role.</p><p>Several different mouse models are available for CMT2D (Seburn et al., <xref rid=\"B56\" ref-type=\"bibr\">2006</xref>; Achilli et al., <xref rid=\"B1\" ref-type=\"bibr\">2009</xref>; Morelli et al., <xref rid=\"B36\" ref-type=\"bibr\">2019</xref>), which have mutations in endogenous mouse <italic>Gars</italic>, causing phenotypes akin to human neuropathy. These mice display loss of lower motor neuron connectivity and disturbed neurotransmission at the neuromuscular junction (NMJ), causing muscle weakness and motor function deficits (Sleigh et al., <xref rid=\"B61\" ref-type=\"bibr\">2014a</xref>; Spaulding et al., <xref rid=\"B70\" ref-type=\"bibr\">2016</xref>). Furthermore, there appears to be a pre-natal perturbation of sensory neuron fate in dorsal root ganglia (DRG), such that CMT2D mice have more nociceptive (noxious stimulus-sensing) neurons and fewer mechanosensitive (touch-sensing) and proprioceptive (body position-sensing) neurons (Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>). Perhaps causing this and providing a rationale for neuronal selectivity, mutant GlyRS mis-interacts with the extracellular region of tropomyosin receptor kinase (Trk) receptors. These largely neuron-specific transmembrane proteins mediate the development and survival of sensory neurons by binding with differential affinity to neurotrophins secreted from distal target cells/tissues (e.g., Schwann cells and muscles; Huang and Reichardt, <xref rid=\"B23\" ref-type=\"bibr\">2003</xref>). Activated neurotrophin-Trk receptor complexes are internalized in the periphery, sorted into signaling endosomes, and then retrogradely transported along microtubules to neuronal somas, where they elicit transcriptional events fundamental to nerve survival (Villarroel-Campos et al., <xref rid=\"B74\" ref-type=\"bibr\">2018</xref>).</p><p>The earliest manifestation of CMT2D in many individuals is transient cramping and pain in the hands upon cold exposure (Antonellis et al., <xref rid=\"B4\" ref-type=\"bibr\">2018</xref>). In addition to muscle weakness, this is followed by compromised reflexes and loss of sensation to vibration, touch, temperature, and pin-prick (Sivakumar et al., <xref rid=\"B59\" ref-type=\"bibr\">2005</xref>). Some of these symptoms are reflected in the phenotypes observed in <italic>Gars</italic>-neuropathy mice, highlighting their potential for studying sensory pathomechanisms (Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>).</p><p>However, the motor symptoms of CMT2D patients are the focus of the clinical investigation, given their relative severity. Moreover, <italic>GARS1</italic> neuropathy patients can show little to no sensory involvement and are thus diagnosed with dHMN type V (OMIM 600794; Antonellis et al., <xref rid=\"B3\" ref-type=\"bibr\">2003</xref>). The pathological impact of mutant GlyRS on the sensory nervous system is therefore relatively under-studied and requires further attention if we are to elucidate the cause of its varied involvement in <italic>GARS1</italic>-linked neuropathy. Here, we have thus extended our sensory analyses in CMT2D mice to better understand the importance of anatomical location to pathology and to assess the relevance of some proposed disease mechanisms in afferent nerves.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec id=\"s2-1\"><title>Animals</title><p>All experiments were carried out following the guidelines of the UCL Queen Square Institute of Neurology Genetic Manipulation and Ethics Committees and following the European Community Council Directive of 24 November 1986 (86/609/EEC). <italic>Gars<sup>C201R/+</sup></italic> (<ext-link ext-link-type=\"uri\" xlink:href=\"https://scicrunch.org/resolver/RRID:MGI3849420\">RRID:MGI 3849420</ext-link>) and SOD1<sup>G93A</sup> (<ext-link ext-link-type=\"uri\" xlink:href=\"https://scicrunch.org/resolver/RRID:IMSR_JAX002726\">RRID:IMSR_JAX 002726</ext-link>) mouse handling and experiments were carried out under license from the UK Home Office following the Animals (Scientific Procedures) Act 1986 and were approved by the UCL Queen Square Institute of Neurology Ethical Review Committee. <italic>Gars<sup>Nmf249/+</sup></italic> (<ext-link ext-link-type=\"uri\" xlink:href=\"https://scicrunch.org/resolver/RRID:MGI5308205\">RRID:MGI 5308205</ext-link>) tissue was provided by Drs. Emily Spaulding and Robert Burgess (The Jackson Laboratory, Bar Harbor, ME, USA), as previously described (Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>). <italic>Gars<sup>C201R/+</sup></italic> and <italic>Gars<sup>Nmf249/+</sup></italic> mice were maintained as heterozygote breeding pairs on a C57BL/6J background and genotyped as previously described (Seburn et al., <xref rid=\"B56\" ref-type=\"bibr\">2006</xref>; Achilli et al., <xref rid=\"B1\" ref-type=\"bibr\">2009</xref>). Both males and females were used in the analyses of mutant <italic>Gars</italic> alleles, as no clear sex-specific differences have yet been observed or reported. Genotyped using standard procedures (Gurney et al., <xref rid=\"B20\" ref-type=\"bibr\">1994</xref>), transgenic male mice heterozygous for the mutant human <italic>SOD1</italic> gene (G93A) on a mixed C57BL/6-SJL background [B6SJLTg (SOD1*G93A)1Gur/J] and wild-type male littermate controls were used for the SOD1<sup>G93A</sup> experiments. <italic>Gars<sup>C201R/+</sup></italic> mice sacrificed for 1-month and 3-month timepoints were 29–37 and 89–97 days old, respectively. The <italic>Gars<sup>Nmf249/+</sup></italic> mice used at 1 month were P31–32, while SOD1<sup>G93A</sup> mice were P30–31 and P100–101.</p></sec><sec id=\"s2-2\"><title>Tissue Dissection</title><p>DRG were extracted from either non-perfused or saline-perfused mice as previously described (Sleigh et al., <xref rid=\"B68\" ref-type=\"bibr\">2016</xref>, <xref rid=\"B69\" ref-type=\"bibr\">2020b</xref>). The most caudal pair of floating ribs and the large size of lumbar level 4 (L4) DRG and associated axon bundles were used as markers to consistently and accurately define the spinal level. The forepaws of embryonic day 13.5 (E13.5) embryos were removed between the wrist and elbow joints, as outlined elsewhere (Wickramasinghe et al., <xref rid=\"B77\" ref-type=\"bibr\">2008</xref>).</p></sec><sec id=\"s2-3\"><title>Tissue Immunofluorescence</title><p>Dissected DRG and E13.5 forepaws were processed for immunofluorescence and analyzed as previously described in detail (Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>). The following antibodies were used: rabbit anti-GlyRS (1/200, Abcam, ab42905, <ext-link ext-link-type=\"uri\" xlink:href=\"https://scicrunch.org/resolver/RRID:AB_732519\">RRID:AB_732519</ext-link>), rabbit anti-LysRS (1/200, Abcam, ab129080, <ext-link ext-link-type=\"uri\" xlink:href=\"https://scicrunch.org/resolver/RRID:AB_11155689\">RRID:AB_11155689</ext-link>), mouse anti-neurofilament (1/50, 2H3, developed and deposited by Jessell, T.M./Dodd, J., Developmental Studies Hybridoma Bank, supernatant), mouse anti-NF200 (1/500, Sigma, N0142, <ext-link ext-link-type=\"uri\" xlink:href=\"https://scicrunch.org/resolver/RRID:AB_477257\">RRID:AB_477257</ext-link>) and rabbit anti-peripherin (1/500, Merck Millipore, AB1530, <ext-link ext-link-type=\"uri\" xlink:href=\"https://scicrunch.org/resolver/RRID:AB_90725\">RRID:AB_90725</ext-link>). The analyses of L1-L5 and C4-C8 wild-type DRG were performed at different times, as were the assessments of lumbar DRG dissected from the different genetic strains.</p></sec><sec id=\"s2-4\"><title>Western Blotting of DRG Lysates</title><p>Probing and analysis of DRG lysate western blots were performed as previously described (Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>), using the following antibodies: mouse anti-Gapdh (1/3,000, Merck Millipore, MAB374, <ext-link ext-link-type=\"uri\" xlink:href=\"https://scicrunch.org/resolver/RRID:AB_2107445\">RRID:AB_2107445</ext-link>), anti-GlyRS (1/2,000), anti-LysRS (1/500), anti-NF200 (1/1,000), anti-peripherin (1/1,000), rabbit anti-TrkB (1/1,000, BD Biosciences, 610101, <ext-link ext-link-type=\"uri\" xlink:href=\"https://scicrunch.org/resolver/RRID:AB_397507\">RRID:AB_397507</ext-link>) and rabbit anti-TyrRS (1/500, Abcam, ab150429, <ext-link ext-link-type=\"uri\" xlink:href=\"https://scicrunch.org/resolver/RRID:AB_2744675\">RRID:AB_2744675</ext-link>). Ten microgram of DRG lysate was loaded per lane.</p></sec><sec id=\"s2-5\"><title>Culturing Primary DRG Neurons</title><p>Twenty to twenty-four lumbar to thoracic DRG (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>) were dissociated and cultured on 35 mm glass-bottom dishes (MatTek, P35G-1.5-14-C) in the presence of freshly added 20 ng/ml mouse glial cell line-derived neurotrophic factor (GDNF, PeproTech, 450-44) as detailed elsewhere (Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>). To reduce variability, a wild-type and <italic>Gars<sup>C201R/+</sup></italic> littermate of the same sex were dissected and cultured in parallel for each experimental replicate.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Sensory neuron development is not impaired in <italic>Gars<sup>C201R/+</sup></italic> dorsal root ganglion (DRG) at cervical spinal levels. <bold>(A)</bold> Neurons found in sensory ganglia can be classified into NF200<sup>+</sup> cells, which are mainly medium-large in size and function as either mechanoreceptors or proprioceptors, and peripherin<sup>+</sup> cells that are generally small and nociceptive. <bold>(B)</bold> DRG used in this study were taken from cervical spinal level 4 (C4) to C8 and lumbar level 1 (L1) to L5 for immunofluorescence analysis and from thoracic to lumbar levels for primary cultures. The schematic was created with BioRender (<ext-link ext-link-type=\"uri\" xlink:href=\"https://biorender.com\">https://biorender.com</ext-link>). <bold>(C)</bold> Representative immunofluorescence images of 1-month-old wild-type and <italic>Gars<sup>C201R/+</sup></italic> cervical DRG sections stained for NF200 (cyan) and peripherin (red). Scale bars = 200 μm. <bold>(D)</bold> There was no difference in the proportions of NF200<sup>+</sup> (<italic>P</italic> = 0.812, unpaired <italic>t</italic>-test) or peripherin<sup>+</sup> (<italic>P</italic> = 0.885, unpaired <italic>t</italic>-test) neurons between genotypes in C4-C8 ganglia, <italic>n</italic> = 4. <bold>(E)</bold> Representative western blot of C4-C8 DRG lysates from 1-month-old wild-type and <italic>Gars<sup>C201R/+</sup></italic> mice probed for NF200, peripherin, and the loading control Gapdh. <bold>(F)</bold> Consistent with the immunofluorescence analysis <bold>(D)</bold>, there was no difference between genotypes in levels of NF200 (<italic>P</italic> = 0.835, unpaired <italic>t</italic>-test) or peripherin (<italic>P</italic> = 0.702, unpaired <italic>t</italic>-test) protein, <italic>n</italic> = 5. NS, not significant; WT, wild-type. See also <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figures S1</xref>, <xref ref-type=\"supplementary-material\" rid=\"SM1\">S2</xref>.</p></caption><graphic xlink:href=\"fncel-14-00232-g0001\"/></fig></sec><sec id=\"s2-6\"><title><italic>In vitro</italic> Signaling Endosome Transport Assay</title><p>The atoxic binding fragment of tetanus neurotoxin (H<sub>C</sub>T) was bacterially expressed and labeled with the AlexaFluor647 antibody labeling kit (Life Technologies, A-20186) as previously outlined (Gibbs et al., <xref rid=\"B16\" ref-type=\"bibr\">2016</xref>). Twenty-four hours post-plating of dissociated DRG neurons, H<sub>C</sub>T-647 was added to the neuronal media at a final concentration of approximately 1.5 μg/ml (30 nM), before gentle mixing by rotation and returning to 37°C for 25 min. H<sub>C</sub>T-containing medium was then aspirated, the cells were washed with 2 ml pre-warmed medium, before being slowly flooded with 2 ml standard medium containing all supplements. Within 10–90 min of the media change, endosome transport was imaged on an inverted LSM780 laser scanning microscope (Zeiss) inside an environmental chamber pre-warmed and set throughout the experiment to 37°C. An area containing a single neuronal process retrogradely transporting fluorescent endosomes was imaged using a 63× Plan-Apochromat oil immersion objective (Zeiss). Images were taken at 100× digital zoom (1,024 × 1,024, 1% laser power) every 2.4 s on average. Before selecting a neuronal process for analysis, it was first traced back to the cell body to confirm the directionality of transport and imaged for area measurement (see below). Cultures from wild-type and <italic>Gars<sup>C201R/+</sup></italic> mice were imaged in the same session and, to avoid introducing time-dependent biases, their order was alternated across replicates. Two males and two females of each genotype were analyzed at each timepoint.</p></sec><sec id=\"s2-7\"><title>Endosome Transport Analysis</title><p>Individual endosomes were manually tracked using Tracker (Kinetic Imaging Limited) as described previously (Sleigh et al., <xref rid=\"B66\" ref-type=\"bibr\">2020a</xref>). Briefly, endosomes were included in the analysis if they could be observed for five consecutive frames and did not pause <italic>for</italic> >10 consecutive frames. Endosomes moving solely in the anterograde direction were infrequent and not included in the analysis. Individual frame-to-frame step speeds are included in the frequency histogram (an average of 2, 370 ± 135 frame-to-frame speeds per animal), meaning that an endosome tracked across 21 consecutive frames will generate 20 frame-to-frame speeds in this graph. To determine the endosome speed per animal, individual endosome speeds were calculated, and then the mean of these determined (an average of 95.1 ± 3.5 endosomes per animal). All speed analyses include frames and time during which endosomes may have been paused, i.e., the speed across the entire tracked run length is reported and not the speed solely when motile. An endosome was considered to have paused if it remained in the same position for two or more consecutive frames. The “% time paused” is a calculation of the length of time all tracked endosomes remained stationary, while the “% pausing endosomes” details the proportion of endosomes that displayed at least one pause while being tracked. An average of 26.2 endosomes was tracked per neuron, and at least three individual neurons were assessed per animal replicate.</p></sec><sec id=\"s2-8\"><title>Image Analysis</title><p>Cell body areas of neurons analyzed in endosome transport assays were measured using the freehand tool on ImageJ<xref ref-type=\"fn\" rid=\"fn0001\"><sup>1</sup></xref> to draw around the circumference of the somas. The diameters of neuronal processes imaged for transport were measured in ImageJ using the straight-line tool. The average of five measurements across the width of the process was calculated. To determine in which cells GlyRS levels were highest in <italic>Gars<sup>Nmf249/+</sup></italic> lumbar DRG sections, all cells with increased GlyRS expression were first identified by eye in the single fluorescence channel. Cells positive for NF200 were then independently designated in the second channel. The percentage of GlyRS-elevated cells also positive for N200 was then calculated. Similarly, the percentage of NF200<sup>+</sup> cells without an increase in GlyRS was also determined. All sections used for GlyRS analysis were stained and imaged in parallel with the same confocal settings to permit side-by-side comparison.</p></sec><sec id=\"s2-9\"><title>Statistical Analysis</title><p>Data were assumed to be normally distributed unless evidence to the contrary could be provided by the D’Agostino and Pearson omnibus normality test. GraphPad Prism 8 (version 8.4.0, La Jolla, CA, USA) was used for all statistical analyses. Means ± standard error of the mean are plotted, as well as individual data points in all graphs except for those depicting western blot densitometry. Unpaired <italic>t</italic>-tests and two-way ANOVAs were used throughout the study. Rather than ANOVAs, unpaired <italic>t</italic>-tests were used to analyze the percentages of NF200<sup>+</sup> and peripherin<sup>+</sup> neurons separately, because the two markers are not independently expressed. Similarly, western blot densitometry was also analyzed using unpaired <italic>t</italic>-tests; since expression was calculated relative to wild-type levels for each individual protein, the expression of proteins in wild-type animals are not statistically comparable.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec id=\"s3-1\"><title>Altered Sensory Development Occurs Specifically in Lumbar Segments of CMT2D Mice</title><p>In previous work, we showed that sensory neuron fate is altered during development in the mild <italic>Gars<sup>C201R/+</sup></italic> and more severe <italic>Gars<sup>Nmf249/+</sup></italic> mouse models for CMT2D, the extent of which correlated with overall model severity (Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>). In that study, by co-staining DRG for NF200, a marker of medium-large area mechanosensitive/proprioceptive neurons, and peripherin, which identifies small area nociceptive neurons (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>), we determined that mutant <italic>Gars</italic> DRG had fewer touch- and body position-sensing (NF200<sup>+</sup>) neurons and a concomitant increase in noxious stimulus-sensing (peripherin<sup>+</sup>) neurons. We reported that this phenotype was present at birth and did not change up to 3 months of age, suggesting it is developmental in origin and non-progressive. Ganglia assessed in these original experiments were isolated from lumbar level 1 (L1) to L5, which contains neurons that innervate the lower leg (Mohan et al., <xref rid=\"B34\" ref-type=\"bibr\">2014</xref>); however, CMT2D patients frequently display upper limb predominance (Antonellis et al., <xref rid=\"B4\" ref-type=\"bibr\">2018</xref>).</p><p>To determine whether the phenotype is also observed in forelimb-innervating ganglia (Tosolini et al., <xref rid=\"B72\" ref-type=\"bibr\">2013</xref>), we isolated and immunohistochemically analyzed cervical level 4 (C4) to C8 DRG from 1-month-old wild-type and <italic>Gars<sup>C201R/+</sup></italic> mice (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). Co-labelling DRG for NF200 and peripherin (<xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref>) and calculating the percentages of neurons expressing each marker, we saw no difference between genotypes (<xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>). Corroborating this, western blotting of cervical DRG lysates showed no difference in NF200 or peripherin protein levels (<xref ref-type=\"fig\" rid=\"F1\">Figures 1E,F</xref>). Together, these data indicate that there is no impairment in sensory neuron identity in the C4-C8 ganglia of <italic>Gars<sup>C201R/+</sup></italic> mice.</p><p>To confirm and extend the lumbar phenotype, we assessed levels of the protein TrkB, which binds brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) to ensure the survival of a mechanosensitive sub-population of NF200<sup>+</sup> neurons (Montaño et al., <xref rid=\"B35\" ref-type=\"bibr\">2010</xref>). We found that lumbar ganglia of <italic>Gars<sup>C201R/+</sup></italic> mice have less total TrkB, consistent with there being fewer NF200<sup>+</sup> neurons in the mutant DRG, whereas C4-C8 ganglia showed no difference (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S1</xref>).</p><p>We then statistically compared the proportions of wild-type cervical DRG neurons with previously published data from 1-month-old wild-type L1-L5 DRG (NF200<sup>+</sup> 40.7 ± 1.9%; peripherin<sup>+</sup> 61.5 ± 2.1%; Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>). We found that the ratio of subtypes is more even in cervical ganglia, which possess significantly more NF200<sup>+</sup> and significantly fewer peripherin<sup>+</sup> neurons than lumbar DRG (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S2A</xref>).</p><p>In the past, we also identified a sensory neurodevelopmental phenotype in embryonic <italic>Gars<sup>C201R/+</sup></italic> hindlimbs (Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>). Dissecting hind paws from embryonic day 13.5 (E13.5) mice and staining neurons for neurofilament (2H3), we observed impaired arborization of nociceptive neurons found in the developing dorsal floor plate. To evaluate whether this phenotype is also seen in forelimbs, we analyzed forepaws from E13.5 embryos (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>). Similar to the hind paws, there was no difference in sensory nerve growth between genotypes, assessed by measuring the distance from nerve growth cone to digit tip (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). However, CMT2D forepaws did not display the nociceptive nerve branching defect present in lower limbs (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>). Therefore, similar to the DRG, developing sensory neurons originating at cervical spinal levels do not show the impairments found in lumbar afferent nerves.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Sensory neurodevelopment appears normal in the forelimb of <italic>Gars<sup>C201R/+</sup></italic> embryos. <bold>(A)</bold> A representative single confocal plane, tile scan image of the dorsal aspect of an E13.5 wild-type forepaw stained for neurofilament (2H3, green). The arrow depicts distance from a major nerve branch ending to the tip of a finger, which was measured for B. The nerves to the left of the dashed line were used for branch analysis in <bold>(C)</bold>. Scale <italic>bar</italic> = 250 μm. <bold>(B,C)</bold> There was no difference between wild-type and <italic>Gars<sup>C201R/+</sup></italic> mice in sensory nerve extension into forepaw extremities (<bold>B</bold>, <italic>P</italic> = 0.328, unpaired <italic>t</italic>-test), nor in the amount of branching (<bold>C</bold>, <italic>P</italic> = 0.662, unpaired <italic>t</italic>-test), <italic>n</italic> = 3–8. NS, not significant; WT, wild-type.</p></caption><graphic xlink:href=\"fncel-14-00232-g0002\"/></fig></sec><sec id=\"s3-2\"><title>Sensory Populations Are Unaltered in a Mouse Model of ALS</title><p>We believe that the small, yet physiologically relevant, distortion of sensory populations in lumbar ganglia of CMT2D mice may be associated with aberrant mutant GlyRS-Trk receptor binding during development. To see whether it extends to other mouse models of neuromuscular disease, we analyzed L1-L5 DRG from SOD1<sup>G93A</sup> mice, an established model of <italic>SOD1</italic>-associated amyotrophic lateral sclerosis (ALS), which displays a plethora of defects and dysfunctional pathways in peripheral, albeit mainly motor, nerves (Kim et al., <xref rid=\"B26\" ref-type=\"bibr\">2015</xref>; Nardo et al., <xref rid=\"B39\" ref-type=\"bibr\">2016</xref>). Lumbar DRG were dissected and immunohistochemically processed from SOD1<sup>G93A</sup> and littermate control males at P30–31 (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>) and P100–101 (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>), representing pre-symptomatic and late disease stages, respectively. No distinctions in sensory populations were observed at either age (<xref ref-type=\"fig\" rid=\"F3\">Figures 3B,D</xref>), suggesting that the sensory subtype switch is not observed in all mouse models of neuromuscular diseases.</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>The CMT2D lumbar DRG sensory subtype switch is not observed in SOD1<sup>G93A</sup> mice. <bold>(A,C)</bold> Representative immunofluorescence images of L1-L5 DRG sections from P30–31 <bold>(A)</bold> and P100–101 <bold>(C)</bold> wild-type and SOD1<sup>G93A</sup> mice stained for NF200 (cyan) and peripherin (red). Scale bars = 200 μm. <bold>(B,D)</bold> There was no difference in the percentage of NF200<sup>+</sup> and peripherin<sup>+</sup> neurons between wild-type and SOD1<sup>G93A</sup> mice at P30–31 (NF200 <italic>P</italic> = 0.460, peripherin <italic>P</italic> = 0.391; unpaired <italic>t-</italic>tests) nor P100–101 (NF200 <italic>P</italic> = 0.938, peripherin <italic>P</italic> = 0.782; unpaired <italic>t-</italic>tests), <italic>n</italic> = 5. NS, not significant; WT, wild-type. See also <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S2</xref>.</p></caption><graphic xlink:href=\"fncel-14-00232-g0003\"/></fig><p>SOD1<sup>G93A</sup> and <italic>Gars<sup>C201R/+</sup></italic> mice are maintained on different genetic backgrounds, and it appeared as though there may be a small difference in neuron populations between wild-types of the two strains. We, therefore, compared neuron proportions at 1 and 3 months in lumbar DRG from wild-type mice on a mixed C57BL/6-SJL background (SOD1<sup>G93A</sup> control) vs. a pure C57BL/6J background (<italic>Gars<sup>C201R/+</sup></italic> control). We observed a small, but significant difference between strains in the percentage of NF200<sup>+</sup>, but not peripherin<sup>+</sup>, neurons at 1 month, but not 3 months (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figures S2B,C</xref>).</p></sec><sec id=\"s3-3\"><title>Long-Range Signaling Endosome Transport Is Unaffected in CMT2D Sensory Neurons</title><p>Axonal transport is reliant upon motor proteins traversing microtubule networks to deliver diverse cargoes from one end of an axon to the other (Guedes-Dias and Holzbaur, <xref rid=\"B19\" ref-type=\"bibr\">2019</xref>). Anterograde transport from the cell body to the axonal terminal is key for delivering organelles, proteins, and RNAs towards peripheral synapses. Connecting the axon tip to the cell body, retrograde transport is needed for long-range delivery of autophagosomes and survival-promoting neurotrophic factors. Pre-symptomatic disturbances in axonal trafficking are thought to underlie, or at least contribute to, several neurological diseases (Sleigh et al., <xref rid=\"B65\" ref-type=\"bibr\">2019</xref>). Indeed, primary DRG neurons cultured from 12 day old <italic>Gars<sup>Nmf249/+</sup></italic> mice display reduced retrograde transport speeds of nerve growth factor (NGF)-loaded endosomes (Mo et al., <xref rid=\"B33\" ref-type=\"bibr\">2018</xref>), while reduced mitochondrial motility was also identified in sensory processes of 12-month-old <italic>Gars<sup>C201R/+</sup></italic> mice (Benoy et al., <xref rid=\"B8\" ref-type=\"bibr\">2018</xref>). Disruption of two different cargoes suggests a broad transport impairment (e.g., due to microtubule dysfunction); however, <italic>Gars<sup>C201R/+</sup></italic> neurons were cultured from late symptomatic mice, thus the defective trafficking observed in this model may simply be a secondary consequence of neuropathology.</p><p>To analyze <italic>Gars<sup>C201R/+</sup></italic> transport in early symptomatic sensory neurons, we cultured primary thoracic and lumbar DRG neurons from 1 and 3-month-old mice and assessed retrograde signaling endosome trafficking. These spinal levels were combined to obtain sufficient cell numbers for the assay (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>), and the time points were chosen to allow comparison with several other phenotypes assessed previously in this model (Sleigh et al., <xref rid=\"B64\" ref-type=\"bibr\">2014b</xref>, <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>,<xref rid=\"B63\" ref-type=\"bibr\">b</xref>). Cultures were incubated with fluorescently labeled atoxic binding fragment of tetanus neurotoxin (H<sub>C</sub>T-647), which is taken up by neurons and loaded into signaling endosomes containing Trk receptors and p75 neurotrophin receptor (p75<sup>NTR</sup>), when applied to media (Deinhardt et al., <xref rid=\"B12\" ref-type=\"bibr\">2006</xref>, <xref rid=\"B11\" ref-type=\"bibr\">2007</xref>). Time-lapse confocal microscopy was performed to enable tracking of individual endosomes (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>). Sensory neurons cultured from DRG do not always display a visible axon initial segment and may bear several morphologically indistinguishable axon-like extensions/processes (Nascimento et al., <xref rid=\"B40\" ref-type=\"bibr\">2018</xref>), thus the trafficking analyzed may not always be “axonal” transport <italic>per se</italic>.</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Axonal transport of signaling endosomes is unaffected in large area <italic>Gars<sup>C201R/+</sup></italic> sensory neurons. <bold>(A)</bold> Individual signaling endosomes loaded with fluorescently labeled H<sub>C</sub>T (H<sub>C</sub>T-647, grayscale inverted) were tracked and analyzed from primary sensory neurons. Image series (<italic>i</italic>-<italic>iii</italic>) depicts retrograde (right to left) trafficking of distinct endosomes (arrowheads linked by dashed lines across time series). The imaged neuron was cultured from a P97 wild-type female. Scale bars = 10 μm. <bold>(B)</bold> Frame-to-frame speed distribution curves indicate that there was no clear distinction in endosome kinetics between genotypes or timepoints. <bold>(C–E)</bold> This was confirmed by analyzing average endosome speeds (<bold>C</bold>, genotype <italic>P</italic> = 0.792, age <italic>P</italic> = 0.076, interaction <italic>P</italic> = 0.884; two-way ANOVA), the percentage of time endosomes were paused for (<bold>D</bold>, genotype <italic>P</italic> = 0.123, age <italic>P</italic> = 0.206, interaction <italic>P</italic> = 0.779; two-way ANOVA), and the percentage of pausing endosomes (<bold>E</bold>, genotype <italic>P</italic> = 0.245, age <italic>P</italic> = 0.273, interaction <italic>P</italic> = 0.805; two-way ANOVA), <italic>n</italic> = 4. <italic>1 m</italic>, 1 month; <italic>3 m</italic>, 3 months; NS, not significant; WT, wild-type. See also <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figures S3</xref>, <xref ref-type=\"supplementary-material\" rid=\"SM1\">S4</xref>.</p></caption><graphic xlink:href=\"fncel-14-00232-g0004\"/></fig><p>Contrary to the previous studies, overlapping histograms of endosome frame-to-frame speeds suggest that there is little difference in endosome dynamics between genotypes and timepoints (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>). This was confirmed by analyzing average endosome speeds (<xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>), the percentage of time that endosomes were paused (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>), and the percentage of pausing endosomes (<xref ref-type=\"fig\" rid=\"F4\">Figure 4E</xref>) per animal. There was also no difference in transport parameters between wild-type and <italic>Gars<sup>C201R/+</sup></italic> when axons were used as the experimental unit (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S3</xref>); however, irrespective of the genotype, older cultures did display a general slowing of endosome speeds linked to increased pausing when compared in this manner.</p><p>Transport was assessed in larger area neurons only, because there is a less frequent overlap of moving endosomes in wider processes, likely due to lower microtubule density (Ochs et al., <xref rid=\"B42\" ref-type=\"bibr\">1978</xref>), permitting greater tracking accuracy. Although not confirmed immunohistochemically, analyzed cells were therefore very likely to be medium-large NF200<sup>+</sup> sensory neurons (i.e., mechanosensitive or proprioceptive). Nonetheless, given the distortion in sensory subtypes in CMT2D lumbar DRG (Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>) it is possible that different neuron populations were analysed between genotypes. Thus, we measured cell body areas and process diameters from the neurons in which endosome transport was assessed (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S4</xref>). There were no differences in these morphological properties, indicating that similar neurons were analyzed across genotypes and timepoints.</p></sec><sec id=\"s3-4\"><title>Elevated GlyRS Levels in Select Neurons Are Linked to the Sensory Subtype Switch</title><p>GlyRS protein levels have previously been reported to be elevated in both <italic>Gars<sup>C201R/+</sup></italic> and <italic>Gars<sup>Nmf249/+</sup></italic> brains (Achilli et al., <xref rid=\"B1\" ref-type=\"bibr\">2009</xref>; Stum et al., <xref rid=\"B71\" ref-type=\"bibr\">2011</xref>), perhaps as a compensatory response to impaired protein function. To determine whether GlyRS levels are also altered in sensory neurons, we extracted and performed western blotting on C4-C8 and L1-L5 DRG from 1-month-old <italic>Gars<sup>C201R/+</sup></italic> mice (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>). There was no difference in GlyRS levels in cervical ganglia; however, GlyRS was upregulated more than 2-fold in L1-L5 DRG (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>). This was corroborated by GlyRS immunofluorescence analysis in C4-C8 (<xref ref-type=\"fig\" rid=\"F5\">Figure 5C</xref>) and L1-L5 (<xref ref-type=\"fig\" rid=\"F5\">Figure 5D</xref>) ganglia. We also assessed L1-L5 DRG of 1-month-old <italic>Gars<sup>Nmf249/+</sup></italic> mice and saw the same pattern of enhanced GlyRS fluorescence in a subset of DRG neurons (<xref ref-type=\"fig\" rid=\"F5\">Figure 5E</xref>), thus indicating that the increase of mutant GlyRS levels in L1-L5 DRG is an early event in CMT2D pathogenesis.</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>GlyRS protein levels are elevated in lumbar, but not cervical, DRG of CMT2D mice. <bold>(A)</bold> Representative western blot of C4-C8 (top) and L1-L5 (bottom) DRG lysates from 1-month-old wild-type and <italic>Gars<sup>C201R/+</sup></italic> mice probed for GlyRS and the loading control Gapdh. <bold>(B)</bold> There was no difference between genotypes in GlyRS levels in cervical ganglia (<italic>P</italic> = 0.825; unpaired <italic>t</italic>-test; <italic>n</italic> = 5); however, GlyRS was elevated in mutant L1-L5 DRG (***<italic>P</italic> < 0.001, <italic>NS</italic> not significant; unpaired <italic>t</italic>-test; <italic>n</italic> = 4). <bold>(C,D)</bold> This was confirmed by immunofluorescence analysis of GlyRS in the cervical <bold>(C)</bold> and lumbar <bold>(D)</bold> DRG sections from 1-month-old wild-type and <italic>Gars<sup>C201R/+</sup></italic> mice, <italic>n</italic> = 4. GlyRS levels appear higher in many individual neurons with larger cell bodies in the mutant lumbar DRG (top right image). <bold>(E)</bold> These findings were replicated when comparing sections of L1-L5 ganglia from 1-month-old wild-type and <italic>Gars<sup>Nmf249/+</sup></italic> mice, <italic>n</italic> = 3. Scale bars = 200 μm. WT, wild-type.</p></caption><graphic xlink:href=\"fncel-14-00232-g0005\"/></fig><p>Upon closer inspection, GlyRS immunofluorescence is marginally higher in some of the smaller area neurons of wild-type lumbar DRG; however, in both <italic>Gars</italic> mutants, the upregulation appears to be in larger neurons. To better characterize this, we co-stained <italic>Gars<sup>Nmf249/+</sup></italic> lumbar DRG for GlyRS and NF200 (<xref ref-type=\"fig\" rid=\"F6\">Figure 6A</xref>). We found that ≈87% of neurons with increased GlyRS levels were also NF200<sup>+</sup> (<xref ref-type=\"fig\" rid=\"F6\">Figure 6B</xref>), which is a particularly high proportion considering that these mutant ganglia consist of only ≈22% NF200<sup>+</sup> neurons (Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>). This suggests that there is a preferential increase of GlyRS in mechanosensitive and proprioceptive neurons. We then quantified the percentage of NF200<sup>+</sup> neurons that showed elevated GlyRS and found that ≈32% showed the phenotype (<xref ref-type=\"fig\" rid=\"F6\">Figure 6B</xref>), indicating that GlyRS is differentially upregulated even within this neuronal population, perhaps in a subgroup with a particular function.</p><fig id=\"F6\" position=\"float\"><label>Figure 6</label><caption><p>GlyRS is preferentially increased in some, but not all, NF200<sup>+</sup> sensory neurons of <italic>Gars<sup>Nm249/+</sup></italic> mice. <bold>(A)</bold> Representative immunofluorescence image of an L1-L5 DRG section from a 1-month-old <italic>Gars<sup>Nm249/+</sup></italic> mouse stained for GlyRS (yellow) and NF200 (cyan). Red arrows and magenta arrowheads highlight N200<sup>+</sup> and NF200<sup>−</sup> neurons, respectively, in which GlyRS was increased. Scale bar = 200 μm. <bold>(B)</bold> The majority of neurons in which GlyRS was upregulated, designated “<italic>(+)GlyRS</italic>,” express NF200; however, not all NF200<sup>+</sup> cells have increased GlyRS levels, <italic>n</italic> = 3.</p></caption><graphic xlink:href=\"fncel-14-00232-g0006\"/></fig><p>A global increase in ARS proteins would perhaps suggest dysfunction in a cellular process linked to aminoacylation, for instance, protein translation, which is impaired a gain-of-function manner in CMT2D fly models (Niehues et al., <xref rid=\"B41\" ref-type=\"bibr\">2015</xref>). We, therefore, assessed whether upregulation is GlyRS-specific by analyzing lumbar DRG levels of <italic>Kars</italic>-encoded lysyl-tRNA synthetase (LysRS) and <italic>Yars</italic>-encoded tyrosyl-tRNA synthetase (TyrRS; <xref ref-type=\"fig\" rid=\"F7\">Figure 7A</xref>). LysRS was chosen because, with GlyRS, it is the only other dual-localized synthetase functioning in both cytoplasm and mitochondria, while TyrRS was chosen because there is strong evidence that mutations in its encoding gene, <italic>YARS1</italic>, cause CMT (Wei et al., <xref rid=\"B76\" ref-type=\"bibr\">2019</xref>). <italic>Gars<sup>C201R/+</sup></italic> L1-L5 DRG shows no change in LysRS levels, but a small yet significant rise in TyrRS (<xref ref-type=\"fig\" rid=\"F7\">Figure 7B</xref>). We confirmed the lack of LysRS upregulation by staining lumbar ganglia from <italic>Gars<sup>Nmf249/+</sup></italic> mice (<xref ref-type=\"fig\" rid=\"F7\">Figure 7C</xref>). TyrRS immunohistochemistry was attempted, but the resulting staining pattern was not consistent with a cytoplasmic tRNA synthetase, suggestive of non-specific staining.</p><fig id=\"F7\" position=\"float\"><label>Figure 7</label><caption><p>Not all aminoacyl-tRNA synthetase (ARS) proteins are upregulated in lumbar DRG of CMT2D mice. <bold>(A)</bold> Representative western blot of L1-L5 DRG lysates from 1-month-old wild-type and <italic>Gars<sup>C201R/+</sup></italic> mice probed for aminoacyl-tRNA synthetase enzymes LysRS and TyrRS, and the loading control Gapdh. <bold>(B)</bold> Densitometry analysis indicates that LysRS levels did not differ between genotypes. There was a small, but significant, increase in TyrRS levels in <italic>Gars<sup>C201R/+</sup></italic> lumbar DRG, but the change was much less than that observed for GlyRS (densitometry analysis from <xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref> included for comparison). ***<italic>P</italic> < 0.001, **<italic>P</italic> < 0.01, NS, not significant; unpaired <italic>t</italic>-test, <italic>n</italic> = 4. <bold>(C)</bold> Representative immunofluorescence analysis of LysRS in L1-L5 ganglia sections from 1-month-old wild-type and <italic>Gars<sup>Nmf249/+</sup></italic> mice. Unlike GlyRS (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>), but consistent with the <italic>Gars<sup>C201R/+</sup></italic> LysRS western blot <bold>(A,B)</bold>, individual neurons did not display an increase in LysRS levels, <italic>n</italic> = 3. <italic>N.b</italic>., the positive LysRS signal observed in axons (wild-type image) was also seen in the secondary only control and was therefore likely to be non-specific. Scale bars = 200 μm. WT, wild-type.</p></caption><graphic xlink:href=\"fncel-14-00232-g0007\"/></fig><p>Together, these data indicate that there is a selective increase in <italic>Gars</italic> expression that occurs predominantly in NF200<sup>+</sup> cells and only at spinal levels displaying a developmental perturbation of sensory neuron fate.</p></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><sec id=\"s4-1\"><title>Sensory Phenotypes Are Restricted to the Lower Limbs of CMT2D Mice</title><p>CMT2D mice display developmental phenotypes in sensory neurons innervating the hind paws (Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>). To extend these analyses and determine whether the upper limb predominance of patients is replicated, we assessed afferent nerves from cervical spinal levels in <italic>Gars<sup>C201R/+</sup></italic> mice. The lumbar phenotypes of subtype switching and impaired axon branching were not present in sensory neurons targeting forepaws (<xref ref-type=\"fig\" rid=\"F1\">Figures 1</xref>, <xref ref-type=\"fig\" rid=\"F2\">2</xref>, and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S1</xref>), suggesting that anatomical location has a considerable bearing on neurodevelopmental pathology. Given that these phenotypes are developmental and do not progress in severity from P1 to 3 months, it is unlikely that they occur in cervical DRG later in the disease.</p><p>Although mutations in <italic>GARS1</italic> frequently cause hands to be affected before and more severely than feet, there are examples where the opposite occurs (Sivakumar et al., <xref rid=\"B59\" ref-type=\"bibr\">2005</xref>; Forrester et al., <xref rid=\"B14\" ref-type=\"bibr\">2020</xref>); albeit it is unclear as to whether this also applies to sensory symptoms. Similarly, the <italic>Gars<sup>C201R</sup></italic> mutation could therefore simply preferentially impact lower limbs. However, restriction of weakness to CMT2D patient feet is rare. So, what could be driving the differential pathology between lumbar and cervical ganglia in mice? It may be due to a distinction in the proportions of sensory subtypes. In wild-type mice, NF200 is expressed by ≈41% of neurons in L1-L5 DRG (Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>), whereas ≈56% are N200<sup>+</sup> in cervical ganglia (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S2A</xref>). If mutant GlyRS aberrantly interacts with Trk receptors pre-natally, thus impacting sensory development and skewing the proportions of functional subclasses (Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>), then GlyRS is likely to have less impact on ganglia that have a more equal balance between NF200<sup>+</sup> and peripherin<sup>+</sup> cells, as is observed in C4-C8 DRG. Alternatively, spinal level distinctions may be caused by differences in the amount or kinetics of GlyRS secretion or Trk expression. DRG at different spinal levels develop asynchronously (Lawson and Biscoe, <xref rid=\"B28\" ref-type=\"bibr\">1979</xref>) and possess divergent transcription factor profiles (Lai et al., <xref rid=\"B27\" ref-type=\"bibr\">2016</xref>), which may also contribute to the lower limb predominance observed in CMT2D mice. Indeed, the transcription factors neurogenin 1 and 2, which drive two distinct waves of neurogenesis required for segregation of major classes of Trk-expressing sensory neurons, are differentially required by cervical and more caudal sensory ganglia (Ma et al., <xref rid=\"B29\" ref-type=\"bibr\">1999</xref>).</p><p>Irrespective of the cause, experiments presented here highlight the importance of comparative anatomy in mouse models of neuromuscular disorders to enhance understanding of pathomechanisms. It remains to be seen whether such variations are also observed in the motor nervous system of CMT2D mice, although differential susceptibility of muscles to NMJ denervation has been previously reported (Seburn et al., <xref rid=\"B56\" ref-type=\"bibr\">2006</xref>; Sleigh et al., <xref rid=\"B64\" ref-type=\"bibr\">2014b</xref>, <xref rid=\"B688\" ref-type=\"bibr\">2020</xref>; Spaulding et al., <xref rid=\"B70\" ref-type=\"bibr\">2016</xref>).</p></sec><sec id=\"s4-2\"><title>Developmental Perturbation of Sensory Fate Is Not a Common Phenotype</title><p>A difference in sensory neuron populations has also been reported in lumbar DRG of mice modeling spinal muscular atrophy (SMA; Shorrock et al., <xref rid=\"B58\" ref-type=\"bibr\">2018</xref>). We, therefore, aimed to determine whether this phenotype is a general feature of mouse models of neuromuscular disease, as this would cast doubt on the aberrant binding of mutant GlyRS to Trk receptors as being the cause of impaired sensory development in mutant <italic>Gars</italic> mice. SOD1<sup>G93A</sup> mice modeling <italic>SOD1</italic>-linked ALS show a variety of defects in sensory neurons (Sassone et al., <xref rid=\"B55\" ref-type=\"bibr\">2016</xref>; Vaughan et al., <xref rid=\"B73\" ref-type=\"bibr\">2018</xref>; Seki et al., <xref rid=\"B57\" ref-type=\"bibr\">2019</xref>); however, they do not display a subtype switch in lumbar DRG (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). Moreover, we recently found that mice modeling a developmental form of SMA caused by loss-of-function mutations in <italic>BICD2</italic> also do not show this phenotype (Rossor et al., <xref rid=\"B53\" ref-type=\"bibr\">2020</xref>). Together, these data suggest that perturbed sensory development is not observed in all mouse models of neurodegeneration.</p><p>We did, however, see a small difference in lumbar DRG between wild-type littermates of SOD1<sup>G93A</sup> and <italic>Gars<sup>C201R/+</sup></italic> mice (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figures S2B,C</xref>), which are maintained on different genetic backgrounds, suggesting that genetic background may subtly influence sensory neuron populations, likely contributing to previously reported disparities in sensation between strains (Crawley et al., <xref rid=\"B10\" ref-type=\"bibr\">1997</xref>). This result is not overly surprising considering that mouse genetic background can influence even gross anatomical features such as the number of spinal levels (Rigaud et al., <xref rid=\"B51\" ref-type=\"bibr\">2008</xref>).</p></sec><sec id=\"s4-3\"><title>Long-Range Transport Is Not Universally Impaired in CMT2D Sensory Neurons</title><p>Deficits in axonal transport contribute to many different genetic neuropathies (Prior et al., <xref rid=\"B49\" ref-type=\"bibr\">2017</xref>; Beijer et al., <xref rid=\"B7\" ref-type=\"bibr\">2019</xref>), and its early involvement in disease may be a common driver in peripheral nerve selectivity typical of many forms of CMT2. Indeed, disruption of long-range trafficking has been identified in sensory neurons cultured from CMT2D DRG (Benoy et al., <xref rid=\"B8\" ref-type=\"bibr\">2018</xref>; Mo et al., <xref rid=\"B33\" ref-type=\"bibr\">2018</xref>). Contrastingly, we found no difference in retrograde transport of signaling endosomes between wild-type and <italic>Gars<sup>C201R/+</sup></italic> at 1 and 3 months of age (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S3</xref>). Nonetheless, if thoracic DRG show limited to no pathology, by combining thoracic DRG with L1-L5 ganglia we may have masked a lumbar-specific DRG transport phenotype. Additionally, the medium and associated supplements in which DRG neurons were cultured vary across studies. Neuronal activity can impact the rate and quantity of axonal transport (Sajic et al., <xref rid=\"B54\" ref-type=\"bibr\">2013</xref>; Wang et al., <xref rid=\"B75\" ref-type=\"bibr\">2016</xref>), while proteins such as neurotrophic factors, which are present in primary neuron media, can affect neuronal activity (Dombert et al., <xref rid=\"B13\" ref-type=\"bibr\">2017</xref>). Though unlikely, it is possible therefore that the medium in which our neurons were grown may have selectively enhanced <italic>Gars<sup>C201R/+</sup></italic> transport masking a trafficking deficiency.</p><p>Mo et al. (<xref rid=\"B33\" ref-type=\"bibr\">2018</xref>) identified a slow-down in NGF-containing endosomes tracked in sensory neurons cultured from 12-day old <italic>Gars<sup>Nmf249/+</sup></italic> mice. Firstly, if transport disruption correlates with the overall disease burden, then the more severe mutant allele is more likely to display a defect than the milder <italic>Gars<sup>C201R/+</sup></italic> mutant. Equally as important, the assayed neurons in the two studies were perhaps different. NGF binds to TrkA, which is expressed by nociceptors (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S1A</xref>), thus transport was probably assessed in noxious stimulus-sensing peripherin<sup>+</sup> neurons from <italic>Gars<sup>Nmf249/+</sup></italic> mice. The atoxic binding fragment of tetanus neurotoxin that we used to assess transport is taken up into multiple populations of signaling endosomes (Villarroel-Campos et al., <xref rid=\"B74\" ref-type=\"bibr\">2018</xref>); however, we focused our analyses on large area neurons with wide processes (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S4</xref>), likely to be either TrkB<sup>+</sup> mechanoreceptors or TrkC<sup>+</sup> proprioceptors.</p><p>Benoy et al. (<xref rid=\"B8\" ref-type=\"bibr\">2018</xref>) identified that <italic>in vitro</italic> sensory neurons cultured from 12-month-old <italic>Gars<sup>C201R/+</sup></italic> mice had an almost complete impairment in mitochondrial motility. The disparity with this study could simply reflect the analyzed cargo, i.e., mitochondrial, but not endosomal, transport is altered in this model. Alternatively, the difference may be due to the age at which the neurons were tested (1 and 3 months vs. 12 months). Supporting this idea, we have previously shown that <italic>Gars<sup>C201R/+</sup></italic> sensory neurons cultured from 1-month-old animals show normal neurite/process outgrowth (Sleigh et al., <xref rid=\"B62\" ref-type=\"bibr\">2017a</xref>); however, this was defective in 12-month-old cells (Benoy et al., <xref rid=\"B8\" ref-type=\"bibr\">2018</xref>). Decreased neuronal health may, therefore, be contributing to reduced mitochondrial motility, as may the process of aging. Indeed, we have previously reported that the dynamics of signaling endosome transport <italic>in vivo</italic> remain unaltered in aged wild-type mice (Sleigh and Schiavo, <xref rid=\"B60\" ref-type=\"bibr\">2016</xref>; Sleigh et al., <xref rid=\"B66\" ref-type=\"bibr\">2020a</xref>), whereas mitochondrial transport is known to be altered in old animals (Mattedi and Vagnoni, <xref rid=\"B30\" ref-type=\"bibr\">2019</xref>).</p><p>Our long-range retrograde transport data indicate that cargo trafficking is not globally disrupted in all CMT2D sensory neurons during early disease stages. To unravel the significance of axonal transport impairments to CMT2D etiology, it will be important to assess the trafficking of a variety of different cargoes both in sensory and motor neurons of mutant <italic>Gars</italic> models. Given the complexity of the <italic>in vivo</italic> environment, the kinetics of axonal transport is not always replicated <italic>in vitro</italic> (Sleigh et al., <xref rid=\"B67\" ref-type=\"bibr\">2017c</xref>), hence the analysis of mutant <italic>Gars</italic> mouse transport should also be extended to peripheral nerves <italic>in vivo</italic> (Gibbs et al., <xref rid=\"B16\" ref-type=\"bibr\">2016</xref>, <xref rid=\"B15\" ref-type=\"bibr\">2018</xref>).</p></sec><sec id=\"s4-4\"><title>GlyRS Elevation Is Not a Simple Compensatory Mechanism</title><p>Neuropathy-causing <italic>GARS1</italic> mutations differentially impact the enzymatic activity, with some fully ablating it, whilst others having little effect (Oprescu et al., <xref rid=\"B43\" ref-type=\"bibr\">2017</xref>). The charging function of GlyRS<sup>C201R</sup> and GlyRS<sup>P278KY</sup> in <italic>Gars<sup>C201R/+</sup></italic> and <italic>Gars<sup>Nmf249/+</sup></italic> mice, respectively, were originally reported as unaffected (Seburn et al., <xref rid=\"B56\" ref-type=\"bibr\">2006</xref>; Achilli et al., <xref rid=\"B1\" ref-type=\"bibr\">2009</xref>; Stum et al., <xref rid=\"B71\" ref-type=\"bibr\">2011</xref>). However, a re-evaluation under Michaelis-Menten kinetic conditions suggests that GlyRS<sup>P278KY</sup> has severely decreased kinetics and cannot support yeast viability, commensurate with a loss-of-function (Morelli et al., <xref rid=\"B36\" ref-type=\"bibr\">2019</xref>). Moreover, GlyRS<sup>C201R</sup> aminoacylation was analyzed indirectly in brain lysates that had a 3.8-fold increase in GlyRS (Achilli et al., <xref rid=\"B1\" ref-type=\"bibr\">2009</xref>), which could mask a charging deficiency. Accordingly, brains from severe homozygous <italic>Gars<sup>C201R/C201R</sup></italic> mice showed a 60% decrease in aminoacylation despite an 8.2-fold increase in GlyRS. Further supporting a GlyRS<sup>C201R</sup> loss-of-function, wild-type <italic>GARS1</italic> overexpression in sub-viable homozygous <italic>Gars<sup>C201R/C201R</sup></italic> animals can restore post-natal viability (Motley et al., <xref rid=\"B37\" ref-type=\"bibr\">2011</xref>). Similar to <italic>Gars<sup>C201R/+</sup></italic> brains, GlyRS levels were reported to be higher in <italic>Gars<sup>Nmf249/+</sup></italic> cerebellum, although this was not quantified (Stum et al., <xref rid=\"B71\" ref-type=\"bibr\">2011</xref>). It is, therefore, possible that GlyRS levels are elevated in CMT2D tissues as a compensatory response to diminished aminoacylation.</p><p>To test this hypothesis in sensory tissue, we analyzed the GlyRS protein in CMT2D DRG (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>). Coinciding with the perturbation of sensory neuron fate, we observed enhanced GlyRS levels in lumbar, but not cervical, ganglia of mutant <italic>Gars</italic> mice. Furthermore, the increase was not observed in all lumbar sensory neurons, but preferentially in a portion of NF200<sup>+</sup> neurons (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>). This argues against GlyRS upregulation being a compensatory response to impaired charging, an alteration in a non-canonical function (e.g. Johanson et al., <xref rid=\"B25\" ref-type=\"bibr\">2003</xref>; Park et al., <xref rid=\"B45\" ref-type=\"bibr\">2012</xref>; Mo et al., <xref rid=\"B32\" ref-type=\"bibr\">2016</xref>), or that mutant GlyRS protein stability is altered, because if any of those scenarios were true, then GlyRS increase would also likely occur in cervical DRG and across all sensory neurons equally. That is unless there is a greater requirement for glycine charging in cell bodies of larger sensory neurons with the longest axons (i.e., those innervating lower, but not upper, limbs). Contradictory to this idea, GlyRS levels were enhanced in only about a third of NF200<sup>+</sup> neurons in mutant lumbar DRG, suggesting that a particular subset may be selectively impacted by the disease.</p><p>To further tease apart the basis for increased <italic>Gars</italic> expression, we assessed levels of additional ARS proteins. We found that LysRS remained unchanged, but that there was a small increase in TyrRS in CMT2D DRG (<xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref>). This indicates that there is no global increase in tRNA synthetase in response to GlyRS<sup>C201R</sup> expression. We, therefore, observe a GlyRS-specific upregulation, preferentially occurring in a subdivision of NF200<sup>+</sup> neurons and only in ganglia that display neuropathology. Why might this be the case? Neuropathy-associated <italic>GARS1</italic> mutations have been shown to impair GlyRS localization in neuron-like cell lines (Antonellis et al., <xref rid=\"B5\" ref-type=\"bibr\">2006</xref>; Nangle et al., <xref rid=\"B38\" ref-type=\"bibr\">2007</xref>), which could cause build-up in the soma, although, once again, if this were the cause of increased GlyRS levels then it would probably not be so selectively upregulated. The GlyRS elevation is only present in DRG that display a developmental perturbation in sensory neuron fate, suggesting that the two phenotypes may be linked. Perhaps the NF200<sup>+</sup> neurons resident in lumbar ganglia are under stresses not experienced by neighboring subtypes. The integrated stress response (ISR), which is linked to amino acid deprivation (Pakos-Zebrucka et al., <xref rid=\"B44\" ref-type=\"bibr\">2016</xref>), maybe especially activated in these cells. Consistent with impaired protein translation reported in CMT2D fly models (Niehues et al., <xref rid=\"B41\" ref-type=\"bibr\">2015</xref>), the ISR causes a global downregulation of cap-dependent translation of mRNAs, except for a select few that possess upstream open reading frames (uORFs) in their 5′-UTRs, which under non-stressed conditions usually restrict translation initiation of the main downstream ORF (Barbosa et al., <xref rid=\"B6\" ref-type=\"bibr\">2013</xref>). Although not classically thought of as an ISR-associated gene, human and mouse <italic>GARS1</italic> express two mRNA isoforms, one of which possesses an uORF that may, under conditions of stress, play a role in the observed GlyRS increase (Alexandrova et al., <xref rid=\"B2\" ref-type=\"bibr\">2015</xref>). However, some <italic>KARS1</italic> variants also possess an uORF (AceView, NCBI) and LysRS levels remained unchanged. Alternatively, the increase may be an active, compensatory response by a subset of NF200<sup>+</sup> cells to combat degeneration. Indeed, the NF200<sup>+</sup> class of neurons includes vibration-sensing mechanoreceptors, which are most impacted in CMT2D patients (Sivakumar et al., <xref rid=\"B59\" ref-type=\"bibr\">2005</xref>).</p></sec></sec><sec sec-type=\"conclusion\" id=\"s5\"><title>Conclusion</title><p>Sensory dysfunction of <italic>GARS1</italic>-neuropathy patients and mouse models of CMT2D is chronically understudied. This is unsurprising given the relative severity of motor symptoms; however, by studying pathology in both types of peripheral nerve and performing comparative anatomical studies on mouse motor and sensory nervous systems, we are much more likely to determine key pathomechanisms causing the selective pathology characteristic of CMT. Here, we have made four key discoveries: (1) sensory pathology is not equal across all CMT2D ganglia, thus anatomical location dictates disease involvement; (2) perturbed sensory neuron fate is not a general feature of different neuromuscular disease models, supporting its specificity to <italic>GARS1</italic> neuropathy; (3) signaling endosome trafficking in a sub-population of <italic>Gars<sup>C201R/+</sup></italic> sensory neurons remains unaffected, indicating that a widespread CMT2D defect in axonal transport is unlikely; and (4) <italic>Gars</italic> expression is selectively enhanced in NF200<sup>+</sup> lumbar DRG neurons and is thus linked to the subtype switch, perhaps in response to active degeneration.</p></sec><sec sec-type=\"data-availability\" id=\"s6\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p></sec><sec id=\"s7\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by the UCL Queen Square Institute of Neurology Genetic Manipulation and Ethics Committees and performed in accordance with the European Community Council Directive of 24 November 1986 (86/609/EEC). <italic>Gars</italic><sup>C201R/+</sup> (<ext-link ext-link-type=\"uri\" xlink:href=\"https://scicrunch.org/resolver/RRID:MGI3849420\">RRID:MGI 3849420</ext-link>) and SOD1<sup>G93A</sup> (<ext-link ext-link-type=\"uri\" xlink:href=\"https://scicrunch.org/resolver/RRID:IMSR_JAX002726\">RRID:IMSR_JAX 002726</ext-link>) mouse handling and experiments were carried out under license from the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986 and were approved by the UCL Queen Square Institute of Neurology Ethical Review Committee.</p></sec><sec id=\"s8\"><title>Author Contributions</title><p>JS conceived the experiments, analyzed the data and wrote the manuscript. JS, AM, TA, and YZ performed the research. GS provided expertise and discussion. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s9\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>We would like to thank Drs. Emily Spaulding and Robert Burgess (The Jackson Laboratory, Bar Harbor, ME, USA) for providing <italic>Gars<sup>Nmf249/+</sup></italic> tissue and commenting on the manuscript, James Dick (University College London) for genotyping the P100-101 SOD<sup>G93A</sup> and wild-type mice, and Drs. Alexander M. Rossor and David Villarroel-Campos (University College London) for critical comments on the manuscript.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by the Medical Research Council Career Development Award (MR/S006990/1; JS), the Wellcome Trust Sir Henry Wellcome Postdoctoral Fellowship (103191/Z/13/Z; JS), the Wellcome Trust Senior Investigator Award (107116/Z/15/Z; GS), the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement 739572 (GS), and the UK Dementia Research Institute Foundation award (UKDRI-1005; GS).</p></fn></fn-group><fn-group><fn id=\"fn0001\"><p><sup>1</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://imagej.nih.gov/ij/\">https://imagej.nih.gov/ij/</ext-link></p></fn></fn-group><sec id=\"s10\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fncel.2020.00232/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fncel.2020.00232/full#supplementary-material</ext-link>.</p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Achilli</surname><given-names>F.</given-names></name><name><surname>Bros-Facer</surname><given-names>V.</given-names></name><name><surname>Williams</surname><given-names>H. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Oncol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Oncol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Oncol.</journal-id><journal-title-group><journal-title>Frontiers in Oncology</journal-title></journal-title-group><issn pub-type=\"epub\">2234-943X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850466</article-id><article-id pub-id-type=\"pmc\">PMC7431707</article-id><article-id pub-id-type=\"doi\">10.3389/fonc.2020.01614</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Oncology</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Methods of Skull Base Repair Following Endoscopic Endonasal Tumor Resection: A Review</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Hannan</surname><given-names>Cathal John</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/987982/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Kelleher</surname><given-names>Eoin</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Javadpour</surname><given-names>Mohsen</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Neurosurgery, Manchester Centre for Clinical Neurosciences</institution>, <addr-line>Manchester</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Nuffield Department of Population Health, University of Oxford</institution>, <addr-line>Oxford</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff3\"><sup>3</sup><institution>National Neurosurgical Centre, Beaumont Hospital</institution>, <addr-line>Dublin</addr-line>, <country>Ireland</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Royal College of Surgeons in Ireland</institution>, <addr-line>Dublin</addr-line>, <country>Ireland</country></aff><aff id=\"aff5\"><sup>5</sup><institution>School of Medicine, Trinity College Dublin</institution>, <addr-line>Dublin</addr-line>, <country>Ireland</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Marc Andrew Cohen, Cornell University, United States</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Ricardo Luis Carrau, The Ohio State University, United States; Martin Hanson, The University of Queensland, Australia</p></fn><corresp id=\"c001\">*Correspondence: Mohsen Javadpour, <email>mjavadpour@rcsi.ie</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Head and Neck Cancer, a section of the journal Frontiers in Oncology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>1614</elocation-id><history><date date-type=\"received\"><day>26</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>24</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Hannan, Kelleher and Javadpour.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Hannan, Kelleher and Javadpour</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Following the introduction of fully endoscopic techniques for the resection of pituitary tumors, there was a rapid expansion of the indications for endonasal endoscopic surgery to include extrasellar tumors of the skull base. These techniques offer significant advantages over traditional open surgical approaches to the skull base, including improved tumor resection, and better post-operative neurological outcomes. Following their introduction, however, the initial rate of post-operative CSF leak was unacceptably high. Post-operative CSF leak following skull base surgery is a major source of morbidity, and can lead to the development of life-threatening intracranial infection. The use of vascularized naso-septal flaps transformed the management of these patients, significantly reducing the rate of post-operative CSF leak and increasing the number of patients that could benefit from this less invasive treatment modality. Adequate repair of iatrogenic defects in the skull base is of crucial importance for patients with skull base tumors, as the development of a post-operative CSF leak, and the associated complications can significantly delay the administration of the adjunctive oncological therapies these patients require. In this review, we provide an overview of the latest evidence regarding skull base reconstruction following endoscopic skull base surgery, and describe the skull base repair technique in use at our institution.</p></abstract><kwd-group><kwd>skull base</kwd><kwd>endoscopic</kwd><kwd>CSF leak</kwd><kwd>nasoseptal flap</kwd><kwd>lumbar drain</kwd><kwd>pituitary</kwd><kwd>meningioma</kwd><kwd>chordoma</kwd></kwd-group><counts><fig-count count=\"7\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"71\"/><page-count count=\"10\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>The endoscopic endonasal approach to the skull base was initially introduced as an adjunct to the microscope in the resection of pituitary tumors in 1979, with fully endoscopic approaches described in the early 1990s (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>–<xref rid=\"B3\" ref-type=\"bibr\">3</xref>). The endoscope has since come to supersede the operative microscope in pituitary surgery, due to the improved visualization offered as a result of a wider field of vision, better illumination of the operative field and the ability to inspect anatomical areas using angled endoscopes that are impossible to see using the microscope (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Following the adoption of this technique for the resection of pituitary tumors, it was adapted for resection of extrasellar skull base lesions (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>–<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). Fully endoscopic approaches are now in widespread use in the management of malignancies of the ventral skull base, including esthesioneuroblastoma, chordoma, and chondrosarcoma, as well as aggressive, locally invasive pathologies such as meningiomas, and craniopharyngiomas (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>–<xref rid=\"B14\" ref-type=\"bibr\">14</xref>).</p><p>The advantages of these extended endonasal approaches (EEA) to skull base tumors are that they provide a direct trajectory to lesions of the ventral skull base, avoiding the parenchymal retraction and the traversal of cranial nerves inherent to transcranial approaches for these tumors. This less invasive approach is associated with better neurological outcomes and a shorter length of stay than more traditional open approaches (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>). When used in the management of chordomas and esthesioneuroblastomas, the endoscopic approaches offer higher rates of gross total resection than their transcranial alternatives (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>, <xref rid=\"B17\" ref-type=\"bibr\">17</xref>). However, early series utilizing these approaches were complicated by post-operative CSF leak rates as high as 40%, and this shortcoming was regarded as a major obstacle in their widespread adoption (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). Post-operative CSF leak is the major source of morbidity following endoscopic skull base surgery, and can lead to the development of meningitis and/or hydrocephalus (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B20\" ref-type=\"bibr\">20</xref>). Moreover, CSF leak leads to longer length of stay and increases the chances of unplanned readmission to hospital following surgery, both of which have the potential to delay, or interrupt adjunctive therapy in patients with skull base malignancies (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>, <xref rid=\"B22\" ref-type=\"bibr\">22</xref>).</p><p>The resection of pituitary adenomas is often an extra-arachnoidal procedure, with a small dural defect created to access the pathology and is therefore associated with a low rate of CSF leak that was not observed to increase following the introduction of the endoscopic technique (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). EEA to skull base malignancies, however, necessitate larger bony and dural defects, causing high flow intra-operative CSF leaks which are demonstrably associated with higher rates of post-operative CSF leak (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). Therefore, the reconstruction of the skull base following extended EEA to the skull base is of paramount importance in avoiding post-operative CSF leak. Advances in skull base reconstruction, particularly the use of vascularized local flaps, have greatly reduced the incidence of this complication and have been instrumental in the expansion of these approaches for the management of skull base malignancy (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). The importance of using vascularized flaps as part of a multi-layer, rather than a monolayer closure of skull base defects to prevent post-operative CSF leak has also been highlighted in a recent study by Simal-Julián et al. (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). In this review, we will provide an overview of the latest methods used to reconstruct large skull base defects leading to high flow CSF leaks following tumor resection, as well as describing our preferred method for the repair of these defects.</p></sec><sec id=\"S2\"><title>Discussion</title><sec id=\"S2.SS1\"><title>Skull Base Reconstruction Methods</title><sec id=\"S2.SS1.SSS1\"><title>Pedicled Nasoseptal Flap</title><p>The development of the naso-septal flap (NSF) in by Hadad et al. in 2006 revolutionized the field of endoscopic endonasal skull base surgery and has facilitated the expansion of this treatment modality (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). Prior to its development, skull base repair was undertaken using multilayered techniques employing autologous fat grafts and synthetic dural substitutes as inlay and onlay grafts secured with fibrin glue, which could be supported by the intranasal placement of Foley catheters (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). As mentioned above, this repair technique was associated with an unacceptably high rate of post-operative CSF leak and the requirement for an alternative technique was clear. The NSF consists of a vascularized mucoperichondrial/periosteal flap harvested from the midline nasal septum and pedicled on the posterior septal branch of the sphenopalatine artery. This allows for the creation of a large flap, capable of covering skull base defects extending from the frontal sinus to the sella antero-posteriorly, and spanning the width of the distance between both orbits. This vascularized flap was used in combination with an inlay synthetic collagen graft and an autologous fat graft, secured using fibrin glue. In a series of 44 patients undergoing endoscopic skull base surgery involving large dural defects and high flow intra-operative CSF leaks, the authors reported a post-operative CSF leak rate of 4.5% (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). In the setting of very large skull base defects, involving the anterior and posterior cranial fossa, bilateral NSF have been harvested to effectively prevent CSF leak (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>).</p><p>This technique was widely adopted soon after its introduction, and Kassam et al. published their experience of NSF utilization in 75 patients following EEA to a variety of skull base tumors. A large dural defect with high flow intra-operative CSF leak was noted in 55 patients. In similar fashion to that reported by Hadad et al. the authors combined the nasoseptal flap with the use of an inlay synthetic dural graft, secured using a biological glue or Foley catheter. In the first 1/3 of the series, the authors noted a post-operative CSF leak rate of 33% in cases with a high-flow intra-operative CSF leak rate, which dropped to 5.4% in the latter 2/3 of the series (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). With craniopharyngiomas in particular, the authors noted in a separate publication that the use of a NSF dramatically decreased the rate of post-operative CSF leak from 58 to 5% (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). As a testament to the versatility of this technique, it has also been successfully utilized following EEA to skull base lesions in pediatric cohorts, in spite of initial concerns regarding the small size of the nasal septum in children (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B33\" ref-type=\"bibr\">33</xref>). Certain skull base tumors, such as craniopharyngiomas, chordomas and chondrosarcomas have a propensity for local recurrence, necessitating revisional surgery for further tumor resection. NSF can be successfully re-used in this setting, by dissecting it from the initial defect site and re-applying it in the standard fashion, with no increase in the rate of post-operative CSF leak (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). Traditional open approaches to skull base tumors are often closed with local vascularized pericranial flaps, and the options for skull base defect repair in the setting of a post-operative CSF leak can be limited. The use of an endoscopically harvested NSF to successfully control CSF leak following open skull base surgery has been reported, expanding the repertoire of this technique even further (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>).</p><p>Although the development of the NSF was a significant advance in skull base surgery, the technique itself is subject to some limitations. Although it is a rare occurrence, the flaps are subject to necrosis due to compromise of the vascularized pedicle: this is reported to occur in <1% of cases, but these patients will often require revisional surgery for alternative skull base reconstruction (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>, <xref rid=\"B37\" ref-type=\"bibr\">37</xref>). The removal of the mucosa from the nasal septum leaves a large defect, that heals by secondary intention over an extended period. This process can lead to significant nasal crusting and a perception of nasal obstruction in the ipsilateral nostril (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>). More significant structural abnormalities of the nose can also occur, such as septal perforations and collapse of the nasal dorsum, with the rates of these complications varying from 1 to 14% in the published literature (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>, <xref rid=\"B39\" ref-type=\"bibr\">39</xref>, <xref rid=\"B40\" ref-type=\"bibr\">40</xref>). Overall, the use of a NSF for skull base reconstruction can lead to additional morbidity due to the sinonasal complications associated with this technique. A recent review of over 700 patients who underwent endoscopic skull base surgery found that the use of a NSF conferred additional sino-nasal morbidity post-operatively, and had a negative impact on the sino-nasal quality of life outcomes of patients (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>).</p><p>The NSF is an effective, versatile technique that has gone on to form the basis of skull base reconstruction protocols in a number of high-volume skull base centers the world over, with some modifications which will be explored in the sections that follow. <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> summarizes the results of the use of the NSF within skull base reconstruction protocols following EEA and resection of skull base tumors.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Table summarizing the results of studies using a vascularized nasoseptal flap following EEA and intra-operative CSF leak.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Author, Year</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Technique</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Number of Cases</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Post-operative leaks (%)</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hadad et al., 2006 (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Collagen inlay graft ± fat graft + NSF + fibrin glue + nasal packing/Foley catheter</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">44</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (4.5)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Kassam et al., 2008 (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Collagen inlay graft ± fat graft + NSF + dural sealant + nasal packing/Foley catheter</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8 (14.5)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Zanation et al., 2009 (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Collagen inlay graft + NSF ± fat graft + DuraSeal<sup>®</sup> + Gelfoam<sup>®</sup> nasal packing/Foley catheter</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (5.7)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Luginbuhl et al., 2010 (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dual layer “button” fascia lata graft + NSF + dural sealant + Nasopore<sup>®</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (6.3)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Liu et al., 2012 (<xref rid=\"B61\" ref-type=\"bibr\">61</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fascia lata graft inlay/overlay graft ± fat graft + Surgicel<sup>®</sup> ± fascia lata + NSF + Merocel<sup>®</sup> tampon</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">93</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3 (3.2)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Garcia-Navarro et al., 2013 (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fat graft + onlay fascia lata + MEDPOR/Bone + NSF + DuraSeal<sup>®</sup> ± Lumbar Drain</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (4.7)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Thorp et al., 2014 (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NSF ± middle turbinate graft (no further details provided)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">144</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3 (2.1)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cavallo et al., 2019 (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fat graft + NSF + Merocel<sup>®</sup> tampon</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (4)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Conger et al., 2019 (<xref rid=\"B67\" ref-type=\"bibr\">67</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fat graft + collagen sponge + MEDPOR/Bone + NSF + Fat graft + collagen sponge + dural sealant + Merocel<sup>®</sup> tampon</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">83</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (4.3)</td></tr></tbody></table></table-wrap></sec><sec id=\"S2.SS1.SSS2\"><title>Gasket Seal Technique</title><p>Long-term outcomes from a series of 46 patients who underwent EEA and skull base reconstruction using the gasket seal technique were published by Garcia-Navarro et al. in 2013 (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>). This technique involves the placement of an autologous fat graft to eliminate intracranial dead space, covered by an autologous fascia lata graft over the bony skull base defect, with the fascial graft sized such that it extends 1 cm beyond the defect circumferentially. Following the placement of this graft, an autologous bone graft, or synthetic polyethylene implant is laid over the fascial graft, sized to fit snugly inside the bony defect. In the latter stages of their series, the authors then placed a NSF over this solid buttress, secured with DuraSeal (Confluent Surgical, United States). In 67% of cases, this repair technique was combined with a 24–48 h period of prophylactic lumbar drainage. In the cases where the gasket seal technique was combined with a NSF, the authors reported a post-operative CSF leak rate of 4.7%. The authors commented that as the solid buttress they use is not curved, this technique is suboptimal for the closure of large skull base defects that cross two geometric planes (e.g., anterior skull base and clivus).</p></sec><sec id=\"S2.SS1.SSS3\"><title>Bilayer Button Technique</title><p>This technique, originally described by Luginbuhl et al. in 2010 utilizes a bilayer fascia lata graft, in conjunction with a NSF. In this method, the authors suture an onlay fascia lata graft slightly larger than the bony defect onto a much larger piece of fascia lata that goes on to act as an inlay graft. The inlay graft is directly opposed to the dura, with the appropriately sized onlay graft acting to prevent graft migration from the dural defect. This fascial construct was then covered in 16/20 cases by a NSF, secured with a fibrin glue. Using this technique, the authors noted a decrease in the rate of post-operative CSF leak in patients with large dural defects from 45 to 10% (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>). Although the authors introduced the sutured fascia lata construct at the same time as the NSF, given the results from other series, it is highly likely the greatest contributor to the decreased rate of post-operative CSF leak was the NSF.</p></sec><sec id=\"S2.SS1.SSS4\"><title>The 3F Technique</title><p>Cavallo et al. recently published a modification to their skull base reconstruction technique following EEA, having previously employed a modification of the gasket seal technique combined with a NSF (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). In this modification, which the authors call the 3F technique, the first F (fat) is the placement of an autologous fat graft into the dead space created by tumor resection, which acts to span the entirety of the osteodural defect, secured with fibrin glue. The second F (flap) is the placement of the NSF, bolstered with cellulose sponges, and secured with nasal tamponades for 72 h. The authors mobilize the patient to a sitting position as soon as possible after surgery and they are encouraged to walk and stand as much as possible, the third F (flash). Using this skull base reconstruction protocol, the authors reported a post-operative CSF leak rate of 4% in 25 patients who had large osteodural defects following EEA (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>). Post-operative lumbar drainage was not used.</p></sec></sec><sec id=\"S2.SS2\"><title>Alternative Options</title><p>In situations where the pedicled NSF is not available, for example when sinonasal malignancies invade the nasal septum or pterygopalatine fossa, or when the patient has undergone previous reconstruction with a NSF, alternative vascularized regional flaps are available. The lateral nasal wall flap is harvested from the opposite side of the nasal cavity to the NSF, and is based on the lateral nasal wall artery, a branch of the sphenopalatine artery. In a series of 24 patients with high flow intra-operative CSF leaks, Lavigne et al. reported a post-operative CSF leak rate of 25% (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>). Although at first glance this figure appears to be high, it should be borne in mind that this reconstructive method was used as a salvage method, after necrosis of an existing NSF or when the a NSF was not available, having been used in previous surgery. The authors comment that the lateral nasal wall flap cannot cover as great a surface area as the NSF, and due to the fact it is harvested from the conchal surfaces of the lateral nose, it has a greater “memory” and may migrate from its intended position more often. When local vascularized reconstruction options are not available, due to extensive tumor invasion/previous radiotherapy, or where the expertise in vascularized flap reconstruction does not exist, avascular free grafts are an option. In this technique, layered reconstruction of the skull base defect created following EEA is undertaken using a variety of autologous and non-autologous dural substitutes. In a large series of EEA to skull base tumors, Roxbury et al. reported a post-operative CSF leak rate of 6.85%, using a multi-layer closure consisting of an underlay layer of synthetic dural substitute or fat graft, an overlay layer of dural substitute and a further overlay layer of Alloderm<sup>®</sup> (Lifecell, United States) acellular matrix in combination with a free mucosal flap (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>). However, the authors noted that on multi-variate analysis that a high-flow CSF leak, as is often generated in EEA to skull base tumors, was associated with a higher rate of post-operative CSF leak and the majority of the cases in their series were pituitary adenomas, which are known to be associated with a lower rate of post-operative CSF leak (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). More convincing evidence of the potential efficacy of free graft reconstruction techniques is provided by a recent study published by Matavelli et al., wherein the authors describe the results following the resection of 186 sinonasal malignancies, resulting in large anterior cranial fossa defects. Using autologous iliotibial tract and fat tissue in a three-layer reconstruction protocol, the authors reported a post-operative CSF leak rate of 5.8% (<xref rid=\"B47\" ref-type=\"bibr\">47</xref>). Although these studies do suggest that acceptable results can be obtained with the use of free graft techniques, in the absence of a trial comparing both techniques, the weight of the evidence suggests that lower rates of CSF leak are obtained with the use of local vascularized flaps, and this view is supported by the results of a systematic review comparing the efficacy of skull base reconstruction methods following EEA (<xref rid=\"B48\" ref-type=\"bibr\">48</xref>). A further reconstruction option in the context of unavailability or unsuitability of the NSF is the endoscopic pericranial flap. This technique, originally described by Zanation et al., involves the minimally invasive, endoscopic harvesting of a pericranial flap through a small scalp incison. This flap is then brought through into the nasal cavity via a bony defect drilled in the nasion (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>). Following this, it can be used to cover osteodural defects in an identical manner to the NSF and it has been successfully utilized in the reconstruction of anterior and posterior fossa skull base defects (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>, <xref rid=\"B51\" ref-type=\"bibr\">51</xref>).</p><p>In the setting of previous radiotherapy to the skull base, resulting in delayed CSF leak, transposition of a temporo-parietal fascial flap pedicled on the superficial temporal artery has been utilized (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>, <xref rid=\"B53\" ref-type=\"bibr\">53</xref>). This involves harvesting of the flap through an external skin incision overlying the temporal fossa, which is then transposed through the infratemporal fossa into the nasal cavity via an endoscopic <italic>trans</italic>-maxillary sinus or <italic>trans</italic>-pterygoid approach. Although there are reports of its success, the requirement for an external skin incision, as well as the risk of injury to the frontal branch of the facial nerve mean this approach is uncommonly used, and reserved for when local flap options are unsuitable.</p><p>In the setting of locoregional flap failure, the use of free myo-cutaneous flaps, facilitated by microvessel anastomosis to reconstruct skull base defects following EEA has been described. Kang et al. have described the successful use of a vastus lateralis flap, pedicled on the descending branch of the lateral femoral circumflex artery in four patients with anterior skull base defects following EEA. In all four cases, initial locoregional flap methods failed to adequately reconstruct the skull base and the vastus lateralis flap was employed as a salvage procedure, whereby the facial artery was used as a recipient vessel and the flap was tunneled through a maxillotomy to cover the skull base defect (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>). These techniques have also been utilized in the repair of posterior fossa defects following EEA; the radial forearm free flap has been employed effectively to reconstruct a cranio-cervical junction defect following EEA for a clival chordoma. Similar to the four cases above, the patient had undergone previous attempts to reconstruct the skull base using a NSF (<xref rid=\"B55\" ref-type=\"bibr\">55</xref>). The use of free flaps for the reconstruction of the skull base following EEA is a significant undertaking, requiring complex mutli-discplinary input and in our view should only be considered when loco-regional reconstruction methods have failed.</p></sec><sec id=\"S2.SS3\"><title>Adjuncts to Skull Base Repair</title><sec id=\"S2.SS3.SSS1\"><title>Lumbar Drainage</title><p>The value of post-operative lumbar drainage of CSF following EEA to the skull base has been the source of debate since the introduction and widespread adoption of these approaches. The initial high rate of post-operative CSF leak with EEA prompted some centers to adopt lumbar drainage as a matter of course following EEA, providing observational evidence for their efficacy (<xref rid=\"B56\" ref-type=\"bibr\">56</xref>). Others called into question the necessity of lumbar drainage when a NSF is used, and suggested they may in fact be harmful, citing the risk of meningitis, CSF over-drainage and spinal headache and longer hospital stay with their use (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>–<xref rid=\"B59\" ref-type=\"bibr\">59</xref>). In reality, the heterogeneity of the skull base repair methods in these studies, as well as their observational nature leaving them highly susceptible to selection bias, limited the conclusions that could be drawn from them.</p><p>The requirement for a randomized controlled trial, with clearly defined inclusion criteria and controls in place for selection bias was clear, and the results from such a trial were published in 2018. In this trial, published by Zwagerman et al. all patients undergoing EEA resulting in a dural defect >1 cm<sup>2</sup> along with extensive arachnoid dissection and/or entry into a ventricle were eligible for recruitment (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>). Patients were randomized to drain or no drain after the completion of skull base reconstruction, with the lumbar drain placed in the operating room and left in place for 72 h, draining 10 ml/h. All patients had skull base reconstruction with a local vascularized flap. The trial was terminated early having recruited 170 patients, due to evidence of benefit in the lumbar drain arm of the trial. 18/85 (21.2%) of patients with no drain suffered a post-operative CSF leak compared with 7/85 (8%) of patients who had a lumbar drain placed. There were no instances of meningitis associated with lumbar drain use, and only two patients developed spinal headache requiring a blood patch. There was also no significant increase in the risk of venous thromboembolism in the patients who had a lumbar drain placed. In a subgroup analysis based on lesion location, the authors concluded that there was a significant decrease in the incidence of post-operative CSF leak with use of a lumbar drain in patients with pathology located in the anterior and posterior cranial fossa, but that patients with tumors in the suprasellar area did not benefit from lumbar drain insertion. The authors suggested this may have been because the vascularized local flaps used are most effective in the suprasellar region, but they may not provide enough coverage to cover larger defects anteriorly and posteriorly. The results from this trial are striking, but should be interpreted with caution given that this a single center study where one skull base reconstruction protocol is used; the applicability of these results to centers utilizing different methods of skull base repair are uncertain. Moreover, the rates of post-operative CSF leak in both groups were higher than those previously reported in defects closed with vascularized local flaps, and the authors did not provide any data on length of stay in the two cohorts (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B30\" ref-type=\"bibr\">30</xref>, <xref rid=\"B42\" ref-type=\"bibr\">42</xref>). Despite the shortcomings of this trial, it is likely that there are a subset of patients at particularly high risk of CSF leak that stand to benefit from “prophylactic” lumbar drain insertion.</p></sec><sec id=\"S2.SS3.SSS2\"><title>Direct Support of Repair</title><p>Following the positioning of the materials used in the skull base reconstruction, the majority of authors would advocate some form of physical support for the reconstruction, to allow time for epithelisation of the defect and for the mucosa of the NSF to integrate with the mucosa adjacent to the operative site. A number of series have utilized the placement of a Foley catheter with the balloon inflated to provide an upward pressure on the skull base repair, whereas others use nasal tampons or inflatable Merocel<sup>®</sup> (Medtronic, United States) sponges (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>, <xref rid=\"B61\" ref-type=\"bibr\">61</xref>, <xref rid=\"B62\" ref-type=\"bibr\">62</xref>). Prior to the insertion of any buttressing material, the use of tissue sealants to secure the NSF to the skull base is commonplace, although Liu et al. argue that this is not required and merely contributes to unnecessary surgical costs (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>, <xref rid=\"B42\" ref-type=\"bibr\">42</xref>, <xref rid=\"B62\" ref-type=\"bibr\">62</xref>, <xref rid=\"B63\" ref-type=\"bibr\">63</xref>).</p><p>A further technique to provide support for skull base reconstructions following EEA that has been suggested is the suturing of an onlay fascia lata graft to the edges of the dural defect, following the placement of an inlay synthetic dural substitute in the subdural space and combined with a NSF. Xue et al. found that the rates of post-operative CSF leak decreased following their implementation of this practice, although this was confounded by the fact that there was a significantly higher rate of intra-operative lumbar drain insertion in the group with dural suturing (<xref rid=\"B64\" ref-type=\"bibr\">64</xref>). The requirement for dural suturing has also been reported in endoscopic re-intervention for post-operative CSF leak, but at present there is no evidence to support its routine use in all EEA for skull base tumors or for its superiority over non-suture techniques (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>, <xref rid=\"B65\" ref-type=\"bibr\">65</xref>).</p></sec></sec><sec id=\"S2.SS4\"><title>Skull Base Repair: The Dublin Technique</title><p>In our center, we employ a standardized method of skull base reconstruction for all EEA as well as endoscopic <italic>trans</italic>-sphenoidal approaches to pituitary tumors, even in the absence of an intra-operative CSF leak. Following the establishment of this protocol, we have reported a post-operative CSF leak rate of 1%, although this was higher in the early part of the senior author’s experience prior to the introduction of this standardized technique, in keeping with the experience of other surgeons (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). In the latter third of a series of 270 patients (operated between July 2006 and June 2016) undergoing endoscopic surgery for resection of pituitary and skull base tumors, 1/90 (1%) patients experienced a post-operative CSF leak. When only EEA with high flow intra-operative CSF leaks were repaired using the following technique, 1/28 (4%) of patients experienced a post-operative CSF leak (<xref rid=\"B66\" ref-type=\"bibr\">66</xref>).</p><p>A NSF flap is harvested at the beginning of the procedure, and stored in the posterior nasopharynx/maxillary sinus until completion of the tumor resection. <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref> is a diagrammatic representation of the harvesting of a NSF at the beginning of the procedure. Following tumor removal, an inlay graft of autologous fascia lata is inserted in the subdural space, and is sized to be larger than the osteodural defect in all dimensions. The only fascia lata donor site complication in our series was one case of wound hematoma requiring evacuation in a patient with Cushings disease (1/28, 4%). <xref ref-type=\"fig\" rid=\"F2\">Figures 2</xref>, <xref ref-type=\"fig\" rid=\"F3\">3</xref> are intra-operative photographs demonstrating the variety of skull base defects that can be closed using this technique. Placement of the fasica lata as an inlay larger than the dural opening ensures that the graft does not migrate out of the defect, and that it is opposed to the dura mater with each CSF pulsation. This intradural fascial layer is not secured using sutures/clips and contrary to concerns raised by some authors, we have not noted any issues regarding migration of the graft material (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>). We then place the vascularized NSF directly over the dural and bony defects, with no intervening exogenous material. We avoid placing any intervening material between the dura and the NSF because in our view, natural tissues with good blood supply are more likely to adhere to each other and any intervening material may hinder this. The NSF is then covered with a further layer of fascia lata, and the entire construct is secured with dural sealant. Our preferred dural sealant is Bioglue<sup>®</sup> (CryoLife, Inc., United States). <xref ref-type=\"fig\" rid=\"F4\">Figures 4</xref>–<xref ref-type=\"fig\" rid=\"F6\">6</xref> demonstrate the major components of our skull base repair technique. We do not insert further bolstering materials (Foley catheters, nasal tampons) and we do not use any prophylactic lumbar drains. Prior to the adoption of this technique in 2013, we utilized a fat graft, covered with an onlay graft of dural substitute/fascia lata secured with dural sealant. In the setting of a high flow intra-operative CSF leak, a post-operative CSF leak was noted in 7 of 20 cases (35%) (<xref rid=\"B66\" ref-type=\"bibr\">66</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p><bold>(A)</bold> Diagrammatic representation of the endoscopic view during harvesting of the naso-septal flap via the right nostril. An incision is made 1–2 cm below the cribriform plate along the mucosa of the nasal septum. A further incision is made along the medial aspect of the floor of the nasal cavity, which can be extended further medially (dotted line) if a large naso-septal flap is required. Both incisions are then connected by an anterior vertical incision. The flap is then dissected from the nasal septum in retrograde fashion and stored in the nasopharynx or maxillary sinus, to be used for skull base reconstruction at the end of the case. <bold>(B)</bold> Sagittal view of the boundaries of the nasoseptal flap, with the dotted line indicating an optional extension of the incision if a large flap is required.</p></caption><graphic xlink:href=\"fonc-10-01614-g001\"/></fig><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Intra-operative endoscopic view of the skull base defect following a <italic>trans</italic>-tubercular approach to a planum sphenoidale meningioma. C: Optic Chiasm, F: Frontal Lobe, LA2: A2 segment of left anterior cerebral artery, LICA: Left Internal Carotid Artery, LON: Left Optic Nerve, P: Pituitary Gland, RA2: A2 segment of right anterior cerebral artery, RICA: Right Internal Carotid Artery, and RON: Right Optic Nerve.</p></caption><graphic xlink:href=\"fonc-10-01614-g002\"/></fig><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Intra-operative endoscopic view of the skull base defect following the resection of a clival chordoma. B: Basilar Artery, RP: P1 segment of right posterior cerebral artery, and RS: Right superior cerebellar artery.</p></caption><graphic xlink:href=\"fonc-10-01614-g003\"/></fig><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Intra-operative endoscopic view demonstrating the placement of the inlay fascia lata graft. FL: Fascia Lata, P: Pituitary Fossa Dura.</p></caption><graphic xlink:href=\"fonc-10-01614-g004\"/></fig><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Intra-operative endoscopic view demonstrating the naso-septal flap placed to cover the osteo-dural defect in its entirety. NSF: Nasoseptal flap, Ped: Vascular Pedicle.</p></caption><graphic xlink:href=\"fonc-10-01614-g005\"/></fig><fig id=\"F6\" position=\"float\"><label>FIGURE 6</label><caption><p>Intra-operative endoscopic view of the naso-septal flap secured with dural sealant.</p></caption><graphic xlink:href=\"fonc-10-01614-g006\"/></fig><p>Our technique is different from the Gasket-seal technique, insofar as an inlay rather than an onlay fascia lata graft is used, and is more similar to the bilayer button technique and indeed that originally described by Hadad et al. in that respect (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B42\" ref-type=\"bibr\">42</xref>, <xref rid=\"B43\" ref-type=\"bibr\">43</xref>). <xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref> allows for a comparison of the two major alternatives to our technique. We also diverge from the protocol of Conger et al. who argue that a solid buttress is required for the closure of high flow intra-operative CSF leaks (<xref rid=\"B67\" ref-type=\"bibr\">67</xref>). The other published series that most closely resembles our method is that of Eloy et al., describe the use of a NSF to cover an initial layer of autologous fat, fascia lata or dural substitute, secured with dural sealant, and a Merocel tampon. In concordance with our preferred method, the authors do not routinely use a lumbar drain and they reported a post-operative CSF leak rate of 0% in 59 patients with a high flow intraoperative CSF leak.</p><fig id=\"F7\" position=\"float\"><label>FIGURE 7</label><caption><p>Comparative sagittal view of the Gasket seal technique <bold>(A)</bold>, the Bilayer button technique <bold>(B)</bold>, and the Dublin technique <bold>(C)</bold> for skull base reconstruction following endoscopic endonasal resection of skull base tumors. <bold>(A)</bold> The Gasket seal closure technique consists of the placement of an autologous fat graft to eliminate dead space (this is not used when the 3rd ventricle has been opened), with a layer of fascia lata larger than the dural defect wedged in place with a solid buttress of bone or MEDPOR. This construct is then covered with a nasoseptal flap and secured with DuraSeal<sup>®</sup>. <bold>(B)</bold> In the bilayer button technique, two pieces of fascia lata are sutured together, with one much larger than the other. The larger piece of fascia lata is placed intradurally, and the smaller piece placed on the outside of the dural defect. This is then covered with a nasoseptal flap and secured with NasoPore and dural sealant. <bold>(C)</bold> In the Dublin technique, a fascia lata graft larger than the dural defect is placed intradurally as an inlay graft. This fascia lata graft is covered directly by a NSF, which may be secured with a further layer of fascia lata. Bioglue<sup>®</sup> is used to complete the skull base repair. Panel <bold>(A)</bold> adapted with permission from figure in Garcia-Navarro et al. (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>). Panel <bold>(B)</bold> adapted with permission from figure in Luginbuhl et al. (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>).</p></caption><graphic xlink:href=\"fonc-10-01614-g007\"/></fig></sec><sec id=\"S2.SS5\"><title>Post-operative CSF Leak: Risk Factors</title><p>Identification of patients at higher risk of post-operative CSF leak following EEA allows the surgeon to ensure particularly meticulous skull base reconstruction following tumor resection, as well as considering the pre-emptive insertion of a lumbar drain. A number of studies have been performed to identify these risk factors, and BMI ≥ 30 has frequently been identified as being associated with an increased risk of post-operative CSF leak (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>, <xref rid=\"B68\" ref-type=\"bibr\">68</xref>, <xref rid=\"B69\" ref-type=\"bibr\">69</xref>). The presence of an intra-operative CSF leak is strongly associated with a greater risk of post-operative CSF leak, as highlighted by the much higher rates of this complication in patients undergoing EEA compared to those having endoscopic <italic>trans</italic>-sphenoidal approaches to pituitary tumors (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>).</p><p>There is also evidence to suggest that posterior fossa defects have a higher proclivity for post-operative CSF leak, which may not be surprising given their dependent location and the requirement for any vascularized nasoseptal flap to be transposed to a greater extent than if they were being used for an anterior fossa or sellar defect (<xref rid=\"B69\" ref-type=\"bibr\">69</xref>, <xref rid=\"B70\" ref-type=\"bibr\">70</xref>). The rate of post-operative CSF leak has been shown to decrease as the experience of the operating surgeon increases, with data from our series of 270 endoscopic cases identifying a CSF leak rate of 21% in the first 90 cases, as compared to 1% in the last 90 cases (<xref rid=\"B66\" ref-type=\"bibr\">66</xref>).</p></sec></sec><sec id=\"S3\"><title>Conclusion</title><p>Effective closure of the large osteodural defects created by EEA to skull base tumors is of vital importance in the prevention of post-operative CSF leak and meningitis. The addition of the NSF to multi-layered closure has been transformative in this regard, and as demonstrated in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, has brought the risk of post-operative CSF leak below 5%. The role of routine, pre-emptive lumbar drain insertion requires further clarification but one randomized controlled trial has shown benefit in selected cases.</p></sec><sec id=\"S4\"><title>Author Contributions</title><p>CH and MJ drafted and reviewed the manuscript. EK created the medical illustrations. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Halves</surname><given-names>E</given-names></name><name><surname>Bushe</surname><given-names>KA.</given-names></name></person-group>\n<article-title>Transsphenoidal operation on craniopharyngiomas with extrasellar extensions. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864062</article-id><article-id pub-id-type=\"pmc\">PMC7431708</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7450</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Intraocular Pressure Changes after Water Drinking Test in Surgically Treated Primary Congenital Glaucoma</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Razeghinejad</surname><given-names>Reza</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Tajbakhsh</surname><given-names>Zahra</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Masoumpour</surname><given-names>Masoumeh Beigom</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Nowroozzadeh</surname><given-names>M. Hossein</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Glaucoma Service, Wills Eye Hospital, Philadelphia, PA, USA</aff><aff id=\"I2\">\n<sup>2</sup>Poostchi Ophthalmology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>M. Hossein Nowroozzadeh, MD. Poostchi\nOphthalmology Research Center, Poostchi Clinic,\nZand St., Shiraz 713499, Iran.\nEmail: nowroozzadeh@hotmail.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>318</fpage><lpage>325</lpage><history><date date-type=\"received\"><day>14</day><month>4</month><year>2019</year></date><date date-type=\"accepted\"><day>03</day><month>4</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Razeghinejad et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><sec><title>Purpose</title><p>To assess intraocular pressure (IOP) changes after the water drinking test (WDT) in patients with primary congenital glaucoma (PCG).</p></sec><sec><title>Methods</title><p>In this prospective interventional study, 20 eyes of 20 patients with PCG were included. All patients had undergone trabeculotomy. Six out of twenty eyes had received a glaucoma drainage device (GDD) implantation. IOP was measured using an air-puff tonometer at baseline, and 15, 30, 45, and 60 min after WDT. The repeated-measures analysis of variance test was used to compare the mean IOPs at different time points.</p></sec><sec><title>Results</title><p> The mean (<inline-formula><mml:math id=\"M1\"><mml:mo>±</mml:mo></mml:math></inline-formula> standard deviation) of participants' age was 9.9 <inline-formula><mml:math id=\"M2\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.7 years (range, 6 to 16 years), and 8 (40%) participants were male. The mean IOPs at baseline and 15, 30, 45, and 60 minutes after the WDT were 15.8 <inline-formula><mml:math id=\"M3\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.7, 18.6 <inline-formula><mml:math id=\"M4\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.4, 19.0 <inline-formula><mml:math id=\"M5\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.8, 17.9 <inline-formula><mml:math id=\"M6\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.8, and 16.9 <inline-formula><mml:math id=\"M7\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.5 mmHg, respectively (<italic>P</italic>\n<inline-formula><mml:math id=\"M8\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001). Pairwise comparisons revealed that the mean IOPs after 15 and 30 min were significantly greater than the baseline IOP (<italic>P</italic>\n<inline-formula><mml:math id=\"M9\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001 and <italic>P</italic> = 0.002, respectively); however, the difference in mean IOPs after 45 and 60 min were not statistically significant from the baseline IOP. The averages of IOP peak and IOP fluctuation after the WDT were 20.0 <inline-formula><mml:math id=\"M10\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.5 and 4.2 <inline-formula><mml:math id=\"M11\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.9 mmHg, respectively. IOP fluctuation in those who underwent trabeculotomy alone was twice that of those with GDDs, but the difference was not statistically significant (5.0 vs 2.5 mmHg; <italic>P</italic> = 0.08).</p></sec><sec><title>Conclusion</title><p> In patients with PCG, WDT induced significant IOP elevation 15 and 30 min after the test, which returned to pre-test values after 45 min.</p></sec></abstract><kwd-group><kwd>Glaucoma Drainage Device</kwd><kwd> Intraocular Pressure</kwd><kwd> Primary Congenital Glaucoma</kwd><kwd> Trabeculotomy</kwd><kwd> Water Drinking Test</kwd></kwd-group><counts><fig-count count=\"2\"/><table-count count=\"3\"/><ref-count count=\"28\"/><page-count count=\"8\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> INTRODUCTION</title><p>Primary congenital glaucoma (PCG) is the most common hereditary type of glaucoma in childhood.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> Several mechanisms have been suggested for the development of PCG, which result in angle dysgenesis and compromise outflow through the trabecular meshwork. Goniotomy and trabeculotomy have been recommended as the initial procedures to improve outflow by removing the abnormal trabecular tissue and making a direct connection between the anterior chamber and the Schlemm's canal. Trabeculectomy and glaucoma drainage device (GDD) implantation are employed if the intraocular pressure (IOP) cannot be controlled with the aforementioned procedures or glaucoma medications.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> The goal of glaucoma medical and surgical interventions is to keep the IOP at a specific level in order to halt or slow down glaucoma progression.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup>\n</p><p>IOP is a dynamic parameter with an individual circadian rhythm. Currently, management of glaucoma include IOP measurements during clinic hours performed a few times a year. A diurnal curve may be used to evaluate glaucoma progression in a patient when the office IOP is within an acceptable range. The most common methods for assessing the diurnal curve in glaucoma patients are IOP readings at different time points during clinic hours and hospitalization in a sleep laboratory; both are cumbersome and costly. The Water Drinking Test (WDT) has been suggested as a practical and easy-to-perform test to estimate the diurnal IOP profile in a more feasible and controlled fashion.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup>\n</p><p>IOP changes after WDT have been evaluated in adult patients with various types of glaucoma,<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>,<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup> but not in children with PCG. Previous studies also evaluated the WDT-IOP profile of adult glaucoma patients who were taking glaucoma medications or had undergone trabeculectomy, deep sclerectomy, and GDD implantation.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>,<xref rid=\"B9\" ref-type=\"bibr\">9</xref>,<xref rid=\"B10\" ref-type=\"bibr\">10</xref>,<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> However, there is no study in patients with PCG with prior trabeculotomy or GDD implantation.</p><p>The main objective of the present study was to evaluate the IOP changes after WDT in patients with PCG and to compare the IOP changes in those with the history of trabeculotomy and those who underwent trabeculotomy followed by GDD surgery.</p></sec><sec sec-type=\"section\"><title> METHODS</title><p>This prospective interventional study was conducted in a tertiary eye care hospital after getting approval from the local Ethics Committee. The study followed the principles of the Declaration of Helsinki, and informed consent was obtained from the parents of all participants. All enrolled patients underwent a complete ophthalmological examination, which included checking visual acuity, IOP measurement, and a dilated stereoscopic fundus examination to assess the amount of optic nerve head damage using Disc Damage Likelihood Scale.<sup>[<xref rid=\"B12\" ref-type=\"bibr\">12</xref>]</sup> Subsequently, those who met the eligibility criteria were included. The average thickness of the peripapillary retinal nerve fiber layer (using optical coherent tomography) and the central corneal thickness were also recorded.</p><p>At our center, all congenital glaucoma patients undergo trabeculotomy at the superonasal and inferotemporal sites in one session, and if the IOP cannot be controlled using medications, Ahmed Glaucoma Valve (FP7, New World Medical, Rancho Cucamonga, LA, USA) is implanted in the superotemporal quadrant. We do not perform trabeculectomy because of the high chance of failure. Therefore, all patients in the current study had the history of trabeculotomy procedure as the first line treatment. The inclusion criterion was having a controlled PCG with office IOP equal to or under 22 mmHg with or without medication. The exclusion criteria were the presence of ocular infection, corneal opacity, or scar preventing reliable IOP measurements; active heart or renal diseases; and refusal of parents to enroll their children in the study.</p><sec sec-type=\"subsection\"><title>Water drinking test</title><p>Subjects were instructed to refrain from food and fluid intake 3 hours preceding the WDT. After checking the baseline IOP, patients drank 15 mL/kg of bottled water in five minutes. Subsequently, IOP was measured every 15 min for 1 hour. The IOP was measured five times (baseline, 15, 30, 45, and 60 min after drinking water). One examiner measured the IOP using a non-contact tonometer (CT80; Topcon Co., Tokyo, Japan). The average of three measurements was recorded and the measurements were repeated if the differences between the three measurements were greater than 3 mmHg. The following parameters were assessed: IOP trough (lowest IOP after drinking water), IOP peak (highest IOP after drinking water), IOP mean (the mean of the four IOPs after drinking water), IOP fluctuation (difference between peak IOP and baseline), IOP range (difference between peak IOP and lowest IOP reading after drinking water), and end-pressure difference (IOP at 60 min versus baseline).</p></sec><sec sec-type=\"subsection\"><title>Statistical analysis</title><p>The IOP of both eyes was measured, but one eye was randomly selected (using a randomization chart generated by a randomization software) for inclusion in the study. All data were analyzed using IBM SPSS Statistics software version 21 (SPSS Inc., Chicago, IL) and MedCalc version 12.2.1 (<italic>MedCalc</italic> Software, Mariakerke, Belgium). Descriptive results were presented as the mean <inline-formula><mml:math id=\"M12\"><mml:mo>±</mml:mo></mml:math></inline-formula> standard deviation (SD). A <italic>P</italic>-value <inline-formula><mml:math id=\"M13\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05 was considered to be statistically significant.</p></sec></sec><sec sec-type=\"section\"><title> RESULTS</title><p>Patients' baseline characteristics are presented in Table 1. Of the 20 studied patients, 17 (85%) had no associated systemic disease. Cardiac disease (repaired ventricular septal defect), phenylketonuria, and glucose-6-phosphate dehydrogenase deficiency were each found in one patient. However, no subject was on systemic medications, and no patient was prohibited from undergoing the WDT by the pediatrician.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Baseline characteristics of patients with primary congenital glaucoma</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"2\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Characteristic</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Value</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Age, year(s)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9.9 <inline-formula><mml:math id=\"M14\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.7 (6 to 16)<inline-formula><mml:math id=\"M15\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Gender, (Male/Female)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8/12</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Eye, (Right/Left)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12/8</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Weight, kg</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">30.3 <inline-formula><mml:math id=\"M16\"><mml:mo>±</mml:mo></mml:math></inline-formula> 12.2 (14 to 53)<inline-formula><mml:math id=\"M17\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Height, cm</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">134 <inline-formula><mml:math id=\"M18\"><mml:mo>±</mml:mo></mml:math></inline-formula> 17 (104 to 169)<inline-formula><mml:math id=\"M19\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Body Mass Index, kg/m<inline-formula><mml:math id=\"M20\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16.1 <inline-formula><mml:math id=\"M21\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.4 (12.9 to 23.0)<inline-formula><mml:math id=\"M22\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Spherical Equivalent Refraction, Diopter(s)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–2.7 <inline-formula><mml:math id=\"M23\"><mml:mo>±</mml:mo></mml:math></inline-formula> 4.7 (–17.5 to +1.3)<inline-formula><mml:math id=\"M24\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Astigmatism, Diopter(s)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–1.1 <inline-formula><mml:math id=\"M25\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.1 (–4.5 to 0.0)<inline-formula><mml:math id=\"M26\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Central Corneal Thickness, µm</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">582 <inline-formula><mml:math id=\"M27\"><mml:mo>±</mml:mo></mml:math></inline-formula> 47 (488 to 653)<inline-formula><mml:math id=\"M28\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Cup-to-Disc ratio, %</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">48 <inline-formula><mml:math id=\"M29\"><mml:mo>±</mml:mo></mml:math></inline-formula> 24 (10 to 80)<inline-formula><mml:math id=\"M30\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Average Retinal Nerve Fiber Layer Thickness, µm</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">92 <inline-formula><mml:math id=\"M31\"><mml:mo>±</mml:mo></mml:math></inline-formula> 23 (51 to 140)<inline-formula><mml:math id=\"M32\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Number of Topical Medications</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.4 <inline-formula><mml:math id=\"M33\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.1 (0 to 3)<inline-formula><mml:math id=\"M34\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Lens status, </bold>\n<italic><bold>n</bold></italic>\n<bold> (%)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phakic: 20 (100)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Operation, </bold>\n<italic><bold>n</bold></italic>\n<bold> (%)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Trabeculotomy only: 14 (70) GDD 6 (30)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Baseline Intraocular Pressure, mm Hg</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15.8 <inline-formula><mml:math id=\"M35\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.7 (8.5 to 21.0)<inline-formula><mml:math id=\"M36\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula>\n</td></tr><tr><td align=\"left\" colspan=\"2\" rowspan=\"1\">\n<inline-formula><mml:math id=\"M37\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula>Scalar data are presented as mean <inline-formula><mml:math id=\"M38\"><mml:mo>±</mml:mo></mml:math></inline-formula> standard deviation (range)</td></tr></tbody></table></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Comparison of WDT-IOP parameters between the GDD (<italic>n</italic> = 6) and the trabeculotomy (<italic>n</italic> = 14) group</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"4\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Parameter (mmHg)</bold>\n</td><td align=\"center\" colspan=\"2\" rowspan=\"1\"><bold>Operation</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold/>\n<italic><bold>P</bold></italic>\n<bold>-value<inline-formula><mml:math id=\"M39\"><mml:msup><mml:mrow/><mml:mi>b</mml:mi></mml:msup></mml:math></inline-formula></bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Trabeculotomy<inline-formula><mml:math id=\"M40\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>GDD<inline-formula><mml:math id=\"M41\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>IOP Trough</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16.0 <inline-formula><mml:math id=\"M42\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16.7 <inline-formula><mml:math id=\"M43\"><mml:mo>±</mml:mo></mml:math></inline-formula> 4.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.66</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>IOP Peak</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20.1 <inline-formula><mml:math id=\"M44\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">19.6 <inline-formula><mml:math id=\"M45\"><mml:mo>±</mml:mo></mml:math></inline-formula> 4.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.75</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>IOP Mean</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.0 <inline-formula><mml:math id=\"M46\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.3 <inline-formula><mml:math id=\"M47\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.86</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>IOP Fluctuation</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.0 <inline-formula><mml:math id=\"M48\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.5 <inline-formula><mml:math id=\"M49\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.08</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>IOP Range</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.2 <inline-formula><mml:math id=\"M50\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.9 <inline-formula><mml:math id=\"M51\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.14</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>End Pressure Difference</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.5 <inline-formula><mml:math id=\"M52\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3 <inline-formula><mml:math id=\"M53\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.41</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">\n<inline-formula><mml:math id=\"M54\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula>All data are presented as mean <inline-formula><mml:math id=\"M55\"><mml:mo>±</mml:mo></mml:math></inline-formula> standard deviation; <inline-formula><mml:math id=\"M56\"><mml:msup><mml:mrow/><mml:mi>b</mml:mi></mml:msup></mml:math></inline-formula>Calculated with Independent Samples <italic>T</italic>-test; all measurements passed the Shapiro–Wilk test of normality\nIOP, intraocular pressure; GDD, glaucoma drainage device; WDT, water drinking test</td></tr></tbody></table></table-wrap><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><p>The results of the water drinking test in normal subjects, primary open angle glaucoma, ocular hypertension, and pseudoexfoliation syndrome</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"7\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Authors</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Diagnosis</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Management of glaucoma</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Average age of patients (years)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>IOP baseline (mmHg)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>IOP peak (mmHg)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>IOP fluctuation (mmHg)</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Ozyol et al</bold>\n<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">XFS (34)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No treatment</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.8</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">XFG(30)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Medical therapy</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">65.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.2</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>De Moraes et al</bold>\n<sup>[<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">POAG (22)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Medical therapy</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">54.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20.00</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.6</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Danesh-Meyers et al</bold>\n<inline-formula><mml:math id=\"M57\"><mml:msup><mml:mrow/><mml:mrow><mml:mo>[</mml:mo><mml:mn>29</mml:mn><mml:mo>]</mml:mo></mml:mrow></mml:msup></mml:math></inline-formula>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">POAG</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Trabeculectomy</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.3</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Medical therapy</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">68</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.9</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Mansouri et al</bold>\n<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">POAG</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Trabeculectomy</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">67.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.4</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Deep Sclerectomy</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">72.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13.8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.8</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Latanoprost</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">71.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5.2</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Mansouri et al</bold>\n<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Normal subjects (25)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No treatment</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">35.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.9</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Guedes RA et al</bold>\n<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Normal subjects (20)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No treatment</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">58.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.9</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Glaucoma subjects (21)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No treatment</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.4</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Glaucoma subjects (21)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Dorzolamide-timolol</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.3</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Glaucoma subjects (15)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Deep sclerectomy</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.7</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Glaucoma subjects (21)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Trabeculectomy</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.6</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Kocabeyoglu et al</bold>\n<inline-formula><mml:math id=\"M58\"><mml:msup><mml:mrow/><mml:mrow><mml:mo>[</mml:mo><mml:mn>30</mml:mn><mml:mo>]</mml:mo></mml:mrow></mml:msup></mml:math></inline-formula>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Normal subjects (20)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No treatment</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">64.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.5</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">XFS (20)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No treatment</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">66.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.2</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Kerr et al</bold>\n<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">POAG and OHTN</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Before SLT</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">73</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.1</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">After SLT</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.3</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Martinez et al</bold>\n<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">POAG</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Trabeculectomy (20)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">67.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16.25</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.95</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">GDD (20)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">66.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16.15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.6</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">XFS, pseudoexfoliation syndrome; XFG, pseudoexfoliative glaucoma; POAG, primary open angle glaucoma; OHTN, ocular\nhypertension; SLT, selective laser trabeculoplasty</td></tr></tbody></table></table-wrap><p>The mean IOPs at baseline, and 15, 30, 45, and 60 min after WDT were 15.8 <inline-formula><mml:math id=\"M59\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.7, 18.6 <inline-formula><mml:math id=\"M60\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.4, 19.0 <inline-formula><mml:math id=\"M61\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.8, 17.9 <inline-formula><mml:math id=\"M62\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.8, and 16.9 <inline-formula><mml:math id=\"M63\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.5 mm Hg, respectively (<italic>P</italic>\n<inline-formula><mml:math id=\"M64\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001, repeated-measures analysis of variance (ANOVA); Figure 1). Pairwise comparisons using Bonferroni correction revealed that the mean IOPs 15 and 30 min after WDT were significantly greater than the baseline IOP (<italic>P</italic>\n<inline-formula><mml:math id=\"M65\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001 and <italic>P</italic> = 0.002, respectively), however, the mean IOPs after 45 and 60 min were not (<italic>P</italic> = 0.062 and <italic>P</italic> = 1, respectively). The IOP after 60 min was significantly lower than the IOP after 30 min (<italic>P</italic> = 0.03).</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>The average intraocular pressure at baseline and at each time-point after the water drinking test in patients with congenital glaucoma.</p></caption><graphic xlink:href=\"jovr-15-318-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>(A) The scatter diagram and regression line showing direct association between IOP baseline and IOP peak. (B) The scatter diagram and regression line showing reverse association between IOP baseline and IOP fluctuation. (C) The IOP profile after the water drinking test in the trabeculotomy and glaucoma drainage device implantation groups. (D) The IOP profile after the water drinking test in eyes that underwent trabeculotomy with and without adjunctive topical antiglaucoma medications.</p></caption><graphic xlink:href=\"jovr-15-318-g002\"/></fig><p>The values of different WDT-IOP parameters were as following: IOP trough, 16.2 <inline-formula><mml:math id=\"M66\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.2 (range, 10.0 to 22.0) mm Hg; IOP peak, 20.0 <inline-formula><mml:math id=\"M67\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.5 (13.0 to 25.0); IOP mean, 18.1 <inline-formula><mml:math id=\"M68\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.3 (12.0 to 23.3); IOP fluctuation, 4.2 <inline-formula><mml:math id=\"M69\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.9 (0.0 to 11.0); IOP range, 3.8 <inline-formula><mml:math id=\"M70\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.8 (1.0 to 7.0); and end-pressure difference, 1.1 <inline-formula><mml:math id=\"M71\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.1 (–4.0 to 7.0). The first time-point to show an IOP peak was 15 min in nine patients (45%), 30 min in six (30%) patients, 45 min in three (15%) patients, and 60 min in two (10%) patients.</p><p>Linear regression analysis revealed the IOP baseline to be the only statistically significant determinant of the IOP peak (R<inline-formula><mml:math id=\"M72\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula> = 0.463; <italic>P</italic> = 0.001; Figure 2A). The use of a higher number of topical medications was also associated with a trend toward higher IOP peak values (R<inline-formula><mml:math id=\"M73\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula> = 0.170; <italic>P</italic> = 0.071). IOP fluctuation was significantly associated with the IOP baseline (R<inline-formula><mml:math id=\"M74\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula> = 0.216; <italic>P </italic>= 0.039; Figure 2B); and it was lower in the GDD group compared with the trabeculotomy group (R<inline-formula><mml:math id=\"M75\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula> = 0.158; <italic>P</italic> = 0.082).</p><p>Figure 2C and Table 2 summarize the results of WDT in the GDD group and trabeculotomy alone group. The repeated measures analysis of covariance (assuming age, gender, body mass index (BMI), and number of topical medications as possible covariates) revealed no significant difference in WDT-IOP changes between the two surgical groups (<italic>P</italic> = 0.46; Figure 2C). Similarly, with the exception of the IOP fluctuation, which was marginally greater in the trabeculotomy alone group than in the GDD group (5.0 vs 2.5 mm Hg; <italic>P</italic> = 0.08; Table 2), the WDT-IOP parameters were not significantly different. However, because of the small sample size of the groups, the possibility of a type 2 error should be considered while interpreting the insignificant <italic>P</italic>-values.</p><p>Figure 2D shows the WDT-IOP changes in eyes that underwent trabeculotomy with and without adjunctive topical antiglaucoma medications.</p></sec><sec sec-type=\"section\"><title> DISCUSSION</title><p>Previous studies evaluated the WDT response in medically treated glaucoma and in adults who underwent trabeculectomy or GDD implantation.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>,<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> In our study involving PCG patients, the mean IOPs 15 and 30 min, but not 45 and 60 min, after WDT were significantly greater than the baseline IOP. The highest mean IOP was observed after 30 min. In the study by Martinez et al,<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> comparing the results of the WDT in 40 eyes of 34 primary open angle glaucoma (POAG) patients who underwent trabeculectomy or GDD implantation, the highest mean IOP in both groups was observed 30 min after WDT. Similarly, 20 eyes from 20 POAG or ocular hypertension patients had the highest mean IOP after WDT 30 min following selective laser trabeculoplasty; however, before the laser procedure the highest mean IOP was observed after 45 min.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>]</sup> In the study by Mansouri et al<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup> involving normal subjects, the highest mean IOP was detected 15 min after the WDT. The ability of the outflow pathway to handle the load after WDT may have affected the time at which the highest mean IOP was detected. In normal adults with normal outflow facility, the highest IOP was observed after 15 min; however, in adult patients who underwent trabeculectomy or GDD implantation and in our patients, the highest IOP was observed after 30 min.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>,<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup>\n</p><p>In our study the IOP fluctuation in all patients (GDD plus trabeculectomy), and in each of the trabeculectomy, and GDD groups were 4.2, 5.0, and 2.5 mmHg, respectively. The reported IOP fluctuation in adult glaucoma patients managed medically ranged from 4.3 to 8.4 mmHg.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>,<xref rid=\"B9\" ref-type=\"bibr\">9</xref>,<xref rid=\"B10\" ref-type=\"bibr\">10</xref>,<xref rid=\"B15\" ref-type=\"bibr\">15</xref>,<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup> The reported IOP fluctuations in eyes that underwent trabeculectomy ranged from 1.6 to 3.95 (Table 3).<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>,<xref rid=\"B10\" ref-type=\"bibr\">10</xref>,<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup>\n</p><p>A study on GDDs reported an IOP fluctuation of 3.6 mmHg.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> The IOP fluctuation in our trabeculotomy group (4.2 mmHg) was greater than the values reported in trabeculectomy (3.95 mmHg) and GDD (3.6 mmHg) groups in previous studies. However, the IOP fluctuation in our GDD group (2.5 mmHg) was lower than the value in POAG patients with GDD (3.6 mmHg).<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup>\n</p><p>Several studies have suggested that IOP fluctuation is an important contributor to the risk of glaucoma progression.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>,<xref rid=\"B17\" ref-type=\"bibr\">17</xref>]</sup> The Early Manifest Glaucoma Trial showed that even a 1 mmHg increase in IOP was associated with an 11% increase in the hazard ratio for glaucoma progression.<sup>[<xref rid=\"B18\" ref-type=\"bibr\">18</xref>]</sup> The Advanced Glaucoma Intervention Study Group suggested that IOP peaks should be below 18 mmHg to prevent visual-field deterioration in patients with moderate- or advanced-stage glaucoma.<sup>[<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup> As glaucoma progression is correlated with IOP peaks and fluctuations,<sup>[<xref rid=\"B20\" ref-type=\"bibr\">20</xref>]</sup> accurate identification of at-risk patients has become imperative as the first step in preventing further irreversible glaucomatous damage.<sup>[<xref rid=\"B21\" ref-type=\"bibr\">21</xref>]</sup> It has been shown that, in two-thirds of glaucoma patients, the highest IOP values occur outside regular clinic hours, frequently during the nocturnal/sleep period.<sup>[<xref rid=\"B22\" ref-type=\"bibr\">22</xref>]</sup> Therefore, significant IOP fluctuation may be missed if relying only on clinic IOP measurements. Twenty-four hour IOP monitoring and provocative tests such as WDT were suggested as viable options for identifying a greater number of patients with poorly controlled glaucoma. A group of normal tension glaucoma patients underwent several clinical tests for predicting the progression of visual field loss, and the WDT was the most useful clinical predictor for visual field progression.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> The IOP peak occurred during home tonometry in approximately 30% of patients with progressive visual field loss while it occurred during home tonometry in 5% of patients with stable visual fields.<sup>[<xref rid=\"B23\" ref-type=\"bibr\">23</xref>]</sup> After drinking water, the ability of the outflow system to modulate the stress of an IOP rise is the only mechanism that can control the IOP. Interventions that improve outflow facility can be expected to induce fewer changes in the IOP after WDT.</p><p>The smoother WDT-IOP profile in our GDD group may have a protective effect on the damaged optic nerve. It is plausible that trabeculotomy increases aqueous outflow, but not as effective as GDD surgery, which bypasses the congenitally abnormal aqueous drainage pathway in PCG. The IOP fluctuation in the trabeculotomy group was two times greater than that in the GDD group (5.0 vs 2.5 mmHg; <italic>P</italic> = 0.08).</p><p>The IOP profile in the trabeculotomy group on glaucoma medications was greater, though not statistically significant, compared to the trabeculotomy group who were not on medication (Figure 2D). A trend toward a greater IOP peak was observed as the number of topical medications increased (R<inline-formula><mml:math id=\"M76\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula> = 0.170; <italic>P</italic> = 0.071). The use of glaucoma medications following the surgical procedure indicates insufficient IOP control and suggests the existence of increased resistance to the outflow. In other words, the higher number of medications may be an indirect measure of the increased resistance in the outflow pathway.</p><p>The baseline IOP was the only significant determinant of the IOP peak (R<inline-formula><mml:math id=\"M77\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula> = 0.463; <italic>P</italic> = 0.001). This is in line with the findings of previous studies in adult patients demonstrating that a higher IOP at baseline is associated with greater 24-hour IOP changes when measured in the seated position.<sup>[<xref rid=\"B24\" ref-type=\"bibr\">24</xref>]</sup> The rate of aqueous production is steady and the outflow facility is the only determinant factor of IOP. When the baseline IOP is low, the possibility of IOP fluctuation might be reduced because the outflow pathway can handle the load and vice versa.</p><p>IOP variation over time may be divided into diurnal, short-term, and long-term fluctuations. It is often difficult to get a true picture of a patient's IOP profile when it is measured only several times a year. The current method of IOP measurements is simply a snapshot of the real IOP over time and does not represent the actual IOP profile. The WDT is utilized as a provocative test to evaluate outflow capacitance and the effect of medical or surgical glaucoma treatments on the IOP peak and fluctuation.<sup>[<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup> Studies have shown that the WDT-IOP peak strongly correlates with the peak of shortened diurnal curves and the long-term IOP profile.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>,<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup> The exact mechanism that underlies IOP elevation after water ingestion remains unclear. The proposed mechanisms include choroidal expansion, plasma hypo-osmolality-enhanced aqueous ultrafiltration, autonomic nervous system stimulation, and increased episcleral venous pressure.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup> Compared to the 24-hour IOP curve measurement that requires the patient to stay in the hospital and involves the measurement of IOPs at night, the WDT could be an inexpensive and feasible alternative.</p><p>This study has several limitations including the small number of patients, especially in the GDD group, and the fact that the IOP was measured using an air-puff tonometer. In most studies that involved performing WDT in adult glaucoma patients, the number of participants was around 20–30 patients, and in some studies both eyes were included (Table 3). In this study, we included one eye from each patient. The global prevalence of glaucoma for the population aged 40–80 years is 3.54%, which is much greater than that for PCG (0.01–0.001%).<sup>[<xref rid=\"B25\" ref-type=\"bibr\">25</xref>,<xref rid=\"B26\" ref-type=\"bibr\">26</xref>]</sup> The rarity of this disease makes recruiting PCG patients challenging. With respect to cooperation for IOP measurement, non-Goldmann tonometer are usually used for IOP measurement in pediatric patients. It has been shown that, in PCG patients, IOP values obtained using an air-puff tonometer are similar to those obtained using a Goldmann tonometer.<sup>[<xref rid=\"B27\" ref-type=\"bibr\">27</xref>]</sup> Additionally, in a recent meta-analysis that compared all available tonometers with the Goldmann applanation tonometer, air-puff tonometers yielded the least amount of variability in IOP values (mean difference of 0.2 mm Hg).<sup>[<xref rid=\"B28\" ref-type=\"bibr\">28</xref>]</sup>\n</p><p>In conclusion, the WDT induced significant IOP elevation 15 and 30 min after the test in patients with PCG. This increased IOP returned to pre-test values after 45 min. In eyes previously treated with trabeculotomy, the IOP fluctuation was greater, though not statistically significant.</p></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>Nil.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Taylor RH, Ainsworth JR, Evans AR, Levin AV. The epidemiology of pediatric glaucoma: the Toronto experience. <italic>Journal of AAPOS </italic>1999;3<bold>:</bold>308–315.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">Ko F, Papadopoulos M, Khaw PT. Primary congenital glaucoma. <italic>Prog Brain Res </italic>2015;221<bold>:</bold>177–189.</mixed-citation></ref><ref id=\"B3\"><label>3</label><mixed-citation publication-type=\"other\">Collaborative Normal-Tension Glaucoma Study Group. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"editorial\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864076</article-id><article-id pub-id-type=\"pmc\">PMC7431709</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7464</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Letter to Editor</subject></subj-group></article-categories><title-group><article-title>Suicide and Laser Refractive Surgery</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Salimi</surname><given-names>Ali</given-names></name><degrees>MD, MS</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ing</surname><given-names>Edsel</given-names></name><degrees>MD, FRCSC, MPH, MIAD</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Nianiaris</surname><given-names>Nicholas</given-names></name><degrees>MD, FRCSC</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Department of Ophthalmology, McGill University, Montreal, Canada</aff><aff id=\"I2\">\n<sup>2</sup>Department of Ophthalmology, University of Toronto, Toronto, Canada</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Edsel Ing, MD, FRCSC, MPH, MIAD. Michael Garron\nHospital, 650 Sammon Ave., K306, Toronto, ON. M4C\n5M5, Canada.\nE-mail: edingLidStrab@gmail.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>432</fpage><lpage>434</lpage><history><date date-type=\"received\"><day>10</day><month>11</month><year>2019</year></date><date date-type=\"accepted\"><day>21</day><month>3</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Salimi et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><kwd-group><kwd>Depression</kwd><kwd> Laser Refractive Surgery</kwd><kwd> LASIK</kwd><kwd> Suicide</kwd></kwd-group><counts><table-count count=\"1\"/><ref-count count=\"14\"/><page-count count=\"3\"/></counts></article-meta></front><body><p>Dear Editor,</p><p>Laser refractive surgery (LRS) is one of the most frequently performed and successful operations in medicine with 96% postoperative patient satisfaction.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> The possible sequelae of LRS include dry eye syndrome, blurred vision, glare, and night vision disturbance that are usually transient, but sometimes persist.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> Psychiatric complications such as psychosis, depression, suicidal ideation, attempted suicide or completed suicide (PDS) following LRS are rare,<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> but generate marked media attention.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup> Given the tragedy of suicide after LRS, we reviewed the PubMed, Embase, PsycINFO, and Google Scholar databases from inception to October 2019 using keywords and MeSH terms “laser refractive surgery” and “suicide”.</p><p>We found the details of six patients, mainly young men, who completed suicide after LRS (Table 1).<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>,<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup> The patient-support website lasikcomplications.com<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> lists approximately 34 patients with PDS following LRS. From 2007 to 2018, approximately 8,230,000 LASIK procedures were performed in the United States.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> Given that, the incidence rate of completed suicide and PDS in the US is estimated to be 7 per 100,000,000 individuals and 4 per 10,000,000 individuals undergoing LRS per annum, respectively. In the US, the age-adjusted suicide rate has increased by 33% over the last two decades, with 13.9 suicides per 100,000 individuals reported in 2018.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>]</sup> The proportion of patients with either completed suicide or PDS after LRS is markedly lower than the proportion of suicide in the general population (<italic>P</italic>\n<inline-formula><mml:math id=\"M1\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001).</p><p>A thorough informed consent before LRS may help to exclude inappropriate surgical candidates. Although it is impossible to list every possible outcome after LRS, and postoperative suicide is extremely rare, under a patient-centered standard of informed consent, the mandate to disclose the possibility of PDS after LRS merits consideration. In addition, impaired vision and chronic pain were two of the five most common adverse outcomes resulting in legal disputes over duties to disclose treatment risks in a 2012 study from Australia.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup>\n</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Patients with completed suicide after LRS</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"8\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Author, Year</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Age</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Sex</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Post-op eye pain</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Blurred vision</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Procedure</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Latency between LRS and suicide</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Clinical factors</bold>\n</td></tr><tr><td colspan=\"8\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Favaro, 2018<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">54</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">PRK</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20 years</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Reindl, 2018<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">F</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">SMILE</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8 weeks</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">FDA, 2016</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">PRK enhancement</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1 year</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Veteran. Post-traumatic stress disorder and depression</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">van Setten, 2015<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Subjective</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">LASIK</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8 weeks</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Pre-existing psychologic instability. Saw psychiatrist numerous times.</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">LASIKComplications.com, 2011<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">54</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">LASIK</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1 year</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Puglionesi, 2007<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">LASIK</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.5 years</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Pre-operative dry eyes, mydriasis and depressive symptoms.</td></tr><tr><td colspan=\"8\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" colspan=\"8\" rowspan=\"1\"> M, male; F, female; LASIK, laser-assisted in situ keratomileusis; LRS, laser refractive surgery; SMILE, small incision lenticule extraction; PRK, photorefractive keratectomy; FDA, U.S. Food & Drug Administration. MAUDE Adverse Event Report: LASIK 2016 <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfmaude/detail.cfm?mdrfoi__id=5434049\">https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfmaude/detail.cfm?mdrfoi__id=5434049</ext-link>\nEye pain, postoperative dry eye pain.</td></tr></tbody></table></table-wrap><p>Dry eye syndrome was associated with suicidal ideation at an odds ratio of 1.24 and LRS can exacerbate dry eye. Psychologic and pharmacologic predispositions to post-LRS dissatisfaction include preoperative depression, the use of retinoic acid and antidepressants, antipsychotics or hypnotics with anticholinergic activity that may compound dry eye symptoms in patients with LRS.<sup>[<xref rid=\"B12\" ref-type=\"bibr\">12</xref>,<xref rid=\"B13\" ref-type=\"bibr\">13</xref>]</sup> Patients suffering from refractory pain after LASIK can be referred to clinics specializing in dry eye syndrome, scleral contact lenses, or chronic pain. Emergency psychiatric resources in addition to the hospital emergency room include psychiatry and suicide prevention hotlines.</p><p>In conclusion, suicide following LRS is exceedingly rare. Suicide is a complex mental health issue with a myriad of contributing factors, and to ascribe blame to LRS is a single cause fallacy. Various publications have reported that: (i) patients with compensated pre-existing psychiatric disorders showed no increased incidence of PDS postoperatively, (ii) mental health-related quality of life has been shown not to decrease after LRS, and (iii) LRS can improve psychological well-being.<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup>\n</p><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>Nil.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.\n</p></sec></body><back><ack><title> Acknowledgements</title><p>None</p></ack><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Eydelman M, Hilmantel G, Tarver M, Hofmeister E, May J, Hammel K, et al. Symptoms and satisfaction of patients in the patient-reported outcomes with laser in situ keratomileusis (PROWL) studies.\n<italic>JAMA Ophthalmol</italic> 2017;135:13–22.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">van Setten G. Suicide after excimer laser refractive surgery: on the importance of matching expectations.\n<italic>Open J Ophthalmol </italic>2015;5:145–148.</mixed-citation></ref><ref id=\"B3\"><label>3</label><mixed-citation publication-type=\"other\">W5. Canadian Ophthalmological Society and Canadian Cornea, External Disease and Refactive Surgery Society statement to CTV News and W5. CTV News [Internet]. 2019 April 5 [cited 2019 Oct 14]. 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Available from:\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ctvnews.ca/health/painful-side-effects-from-laser-eye-surgery-linked-to-man-s-suicide-family-1.4196890\">https://www.ctvnews.ca/health/painful-side-effects-from-laser-eye-surgery-linked-to-man-s-suicide-family-1.4196890</ext-link>.</mixed-citation></ref><ref id=\"B8\"><label>8</label><mixed-citation publication-type=\"other\">LASIKComplications.com. LASIK complications. Are you considering LASIK, ReLEx SMILE? [Internet]. 2019 [cited 2019 Oct 14]. Available from: <ext-link ext-link-type=\"uri\" xlink:href=\"https://lasikcomplications.com/\">https://lasikcomplications.com/</ext-link>.</mixed-citation></ref><ref id=\"B9\"><label>9</label><mixed-citation publication-type=\"other\">Statista. Number of LASIK surgeries in the United States from 1996 to 2019 (in 1,000). [Internet]. 2016 July 18 [cited 2019 Oct 15]. 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] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"editorial\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864057</article-id><article-id pub-id-type=\"pmc\">PMC7431710</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7445</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Editorial</subject></subj-group></article-categories><title-group><article-title>The role of Guidance and Planning on Safety of Ophthalmic Practice during the COVID-19 Pandemic</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Shahraz</surname><given-names>Saeid</given-names></name><degrees>MD, PhD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mohammadi</surname><given-names>Seyed Farzad</given-names></name><degrees>MD, MPH</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Safi</surname><given-names>Sare</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Institute for Clinical Research and Health Policy Studies, Tufts Medical Center, Boston, Massachusetts, USA</aff><aff id=\"I2\">\n<sup>2</sup>Translational Ophthalmology Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran</aff><aff id=\"I3\">\n<sup>3</sup>Ophthalmic Epidemiology Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran</aff><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>275</fpage><lpage>278</lpage><permissions><copyright-statement>Copyright © 2020 Shahraz.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><counts><fig-count count=\"1\"/><ref-count count=\"24\"/><page-count count=\"4\"/></counts></article-meta></front><body><p>Coronavirus Disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported for the first time in China in December 2019.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> It has been spreading rapidly worldwide and has emerged as the most massive health crisis since World War II.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup> The World Health Organization (WHO) declared the SARS-CoV-2 outbreak as a pandemic on March 12, 2019.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup> The COVID-19 pandemic is not only a health crisis but also has a significant impact on societies, economies, and progress rates toward the United Nations' sustainable development goals.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> The Eastern Mediterranean Region (EMR) ranks third in the world in terms of the total number of confirmed cases among the six WHO regions.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> Iran, as one of the EMR countries, has reported 217,724 confirmed cases and 10,239 deaths from February 19 to June 26, 2020 (Figure 1).<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup>\n</p><p>A recently published meta-analysis reported that the risk of transmission is reduced by more than 75% through implementing three strategies by both healthcare workers and communities: at least a 1-m social distancing, use of a face masks (surgical or similar masks (12–16-layer cotton or gauze masks), N95 respirators or similar), and eye protection.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup> Recently, the WHO has released interim guidance on the use of masks for avoiding transmission of COVID-19.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup>\n</p><p>Infected droplets can find their way into the ocular surfaces where the virus can replicate.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> An overall pooled prevalence of ocular manifestations in patients with COVID-19 was 5.5% in one report.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>]</sup> There is limited published evidence on the prevalence of ocular manifestations of COVID-19 in Iran.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> In a case series including 43 patients in Iran, reverse transcription-polymerase chain reaction (RT-PCR) on nasopharyngeal and tear samples indicated presence of viral material in 30% and 7% of cases, respectively. The nasopharyngeal RT-PCR results were indicative of the presence of the virus in all patients with positive tear RT-PCR results, which comprised a case with clinical conjunctivitis.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> Although the rate of ocular manifestations is low, protecting against viral transmission is vital in ophthalmology practice due to the proximity between the examiner and patients, the lengthy period of exposure during examinations, and direct contact with patient's eye secretions.<sup>[<xref rid=\"B12\" ref-type=\"bibr\">12</xref>]</sup> Many non-urgent ophthalmic services were ceased at the ophthalmology centers in Iran, similar to other countries, during the early months of the pandemic to tackle the spread of COVID-19.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Cumulative number of confirmed cases of COVID-19 and death from February 2020 to June 2020 in Iran.</p></caption><graphic xlink:href=\"jovr-15-275-g001\"/></fig><p>Government agencies, ophthalmology societies, and eye health centers have developed guidance and policy statements to reduce the risk of transmission on one hand and maintain continuity of eye care on the other.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>,<xref rid=\"B14\" ref-type=\"bibr\">14</xref>,<xref rid=\"B15\" ref-type=\"bibr\">15</xref>,<xref rid=\"B16\" ref-type=\"bibr\">16</xref>,<xref rid=\"B17\" ref-type=\"bibr\">17</xref>,<xref rid=\"B18\" ref-type=\"bibr\">18</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup> These blueprints have included recommendations for modifying the clinical pathway and disinfecting protocols, initial screening, and necessary protective equipment for eye care providers, including ophthalmologists, optometrists, clinic/hospital managers, and staff. Furthermore, triage for ophthalmic disorders and cessation of elective examinations, diagnostic procedures, and surgeries were addressed in the guidance.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>,<xref rid=\"B14\" ref-type=\"bibr\">14</xref>,<xref rid=\"B15\" ref-type=\"bibr\">15</xref>,<xref rid=\"B16\" ref-type=\"bibr\">16</xref>,<xref rid=\"B17\" ref-type=\"bibr\">17</xref>,<xref rid=\"B18\" ref-type=\"bibr\">18</xref>]</sup> In response to the COVID-19 pandemic in Iran, the Knowledge Management Unit, Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, in collaboration with the Iranian Society of Ophthalmology published a joint guidance in March 2020, which appears in this issue of the journal.<sup>[<xref rid=\"B20\" ref-type=\"bibr\">20</xref>]</sup> The ad hoc committee urged ophthalmologists to restrict non-emergent services and provide care for conditions such as retinal detachment, trauma, chemical burns, dangerously elevated intraocular pressure, and severe ocular infections. It recommended postponing elective surgeries and counsel patients using remote approaches. The use of face masks, goggles, gloves, and slit-lamp shields were emphasized for visiting urgent cases. Infection control strategies, including handwashing, disinfecting ophthalmic examination equipment with 70% alcohol, and surfaces with bleach-based disinfectants were also recommended.<sup>[<xref rid=\"B20\" ref-type=\"bibr\">20</xref>]</sup>\n</p><p>After leaving behind the first peak of the COVID-19 infection, ophthalmology centers were reopened, and eye care services resumed to prevent sight-threatening eye disorders and to manage the backlog of postponed appointments.<sup>[<xref rid=\"B21\" ref-type=\"bibr\">21</xref>]</sup> New guidance was developed for the post-peak era, and existing recommendations were updated. Naveed et al prepared guidance for modifying the ophthalmic workplace for the post-peak period.<sup>[<xref rid=\"B22\" ref-type=\"bibr\">22</xref>]</sup> The post-peak recommendation addressed different strategies, including engineering and administrative controls and protecting workers with personal protective equipment. The authors suggested tele-triage by a senior physician for urgent cases, minimizing face-to-face time in routine clinics, and limiting general anesthesia to absolutely necessary surgical cases.<sup>[<xref rid=\"B22\" ref-type=\"bibr\">22</xref>]</sup> The joint guidance was also updated to provide recommendations for protecting ophthalmic health workers and ophthalmology centers during the post-peak era. It now mandates physical distancing as well as requirements for protecting ophthalmologists and staff during surgeries. Cataract surgery was considered as a semi-urgent operation that could be performed in cases prone to falls and subjects with significantly impaired vision.<sup>[<xref rid=\"B20\" ref-type=\"bibr\">20</xref>]</sup> Eye Health and Prevention of Blindness Office of the Center for Non-communicable Diseases Control of the Ministry of Health and Medical Education issued an official point of view in late June. It offered detailed guidance for the public and health workers, in addition to eye health professionals during the COVID-19 pandemic. It addresses ocular involvement by SARS-CoV-2 as well.</p><p>The WHO Regional Director for the EMR notified a risk of an accelerated trend in the number of confirmed cases in EMR countries as a consequence of easing restrictions.<sup>[<xref rid=\"B23\" ref-type=\"bibr\">23</xref>]</sup> Estimations by the Institute for Health Metrics and Evaluation (IHME) also confirmed this trend.<sup>[<xref rid=\"B24\" ref-type=\"bibr\">24</xref>]</sup> Therefore, to continue providing care for ophthalmic patients, it is crucial that ophthalmic care providers and staff follow the guidance strictly to reduce the risk of infection transmission from patients to medical care and vice versa.</p><p>In summary, it seems that initial screening at the time of admission, mandating social distancing, washing hands, wearing face masks by healthcare providers and patients, use of slit lamp shields, and disinfecting the instruments are reasonable protective strategies recommended by various guidance. We have to consciously monitor the pandemic status, public health developments, and the evidence which emerges to tailor and update our control measures and eye care strategies. And we all know that the COVID-19 pandemic, directly and indirectly, has affected medical care and will continue to transform it further in the coming decade.</p></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. <italic>Lancet</italic> 2020;395:497–506.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">Worldometer. COVID-19 Coronavirus Pandemic. 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] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864070</article-id><article-id pub-id-type=\"pmc\">PMC7431711</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7458</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Perspective</subject></subj-group></article-categories><title-group><article-title>Ophthalmic Workplace Modifications for the Post-COVID Era</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Naveed</surname><given-names>Hasan</given-names></name><degrees>MBBS, BS (Hons)</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Leung</surname><given-names>Victor</given-names></name><degrees>MSc, CIH, ROH, CRSP</degrees><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zarei-Ghanavati</surname><given-names>Mehran</given-names></name><degrees>MD, FICO;</degrees><xref ref-type=\"aff\" rid=\"I4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Leak</surname><given-names>Christopher</given-names></name><degrees>BS, BA, BMBS, MCOptom, MSc, MPhil, FRCOphth</degrees><xref ref-type=\"aff\" rid=\"I5\">\n<sup>5</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Liu</surname><given-names>Christopher</given-names></name><degrees>OBE, FRCOphth, FRCSEd, FRCP, CertLRS</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref><xref ref-type=\"aff\" rid=\"I5\">\n<sup>5</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Sussex Eye Hospital, Brighton and Sussex University Hospitals NHS Trust, UK</aff><aff id=\"I2\">\n<sup>2</sup>Brighton and Sussex Medical School, UK</aff><aff id=\"I3\">\n<sup>3</sup>Core Extension Health & Safety, Canada</aff><aff id=\"I4\">\n<sup>4</sup>Eye Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran</aff><aff id=\"I5\">\n<sup>5</sup>Moorfields Eye Hospital, Croydon, UK</aff><aff id=\"I6\">\n<sup>6</sup>Tongdean Eye Clinic, UK</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Christopher Liu, OBE, FRCOphth, FRCSEd, FRCP,\nCertLRS. Sussex Eye Hospital, Eastern Road, Brighton,\nUnited Kingdom BN2 5BF.\nE-mail: cscliu@aol.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>400</fpage><lpage>407</lpage><history><date date-type=\"received\"><day>10</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>25</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Naveed et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><p>The COVID-19 pandemic necessitates implementation of exposure control measures in all facets of the healthcare sector. Healthcare professionals who work in busy ophthalmology clinics and theaters are amidst the highest at-risk of contracting COVID-19. The authors review the up-to-date scientific evidence of SARS-CoV-2 transmission to demystify and explain the exposure control options available for ophthalmic workplace and offer insights from an industrial hygiene standpoint. As the we enter the post-COVID world, these measures will be critical to enhance workplace safety, and thus protect patients and staff alike.</p></abstract><kwd-group><kwd>COVID-19</kwd><kwd> Ophthalmic Workplace</kwd><kwd> Protecting Healthcare Workers</kwd><kwd> SARS-CoV-2</kwd><kwd> Personal Protective Equipment</kwd></kwd-group><counts><table-count count=\"1\"/><ref-count count=\"33\"/><page-count count=\"8\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> INTRODUCTION </title><p>COVID-19, caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), was declared a pandemic by the World Health Organization (WHO) on March 12, 2020.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> It has since displayed its destructive potential by overwhelming the hospital systems of some of the most well-resourced countries and claiming over 360,000 fatalities in its wake.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> Furthermore, the cessation of nonurgent elective work in order to increase capacity to manage the predicted surge of cases, whilst necessary, has had significant impact on the care of patients with other comorbidities by restricting access and delaying treatments.</p><p>Measures including national lockdowns, enforcing social distancing measures, contact tracing, universal masking, shielding of the vulnerable, and quarantining the infected may have slowed the spread of the disease and reduced impact. In fact, most countries have now passed the peak of their COVID-19 cases. However, without a vaccine there is little evidence to suggest that the complete eradication of COVID-19 will be achieved and experts predict recurrent post-pandemic outbreaks. We have to accept that the virus will be a part of our lives for the foreseeable future.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup>\n</p><p>The challenge for healthcare professionals now is to safely resume the necessary healthcare services. The onus is to ensure that this time is used as a valuable opportunity and collective expertise is utilized to innovate genuine improvements for the ultimate benefit of patients.</p></sec><sec sec-type=\"section\"><title> SARS-CoV-2 Description </title><p>SARS-CoV2 is a positive-sense single-stranded RNA virus which infects cells in the lower respiratory tract, gaining entry via the ACE2 receptors, similar to SARS-CoV.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup> Infected individuals can remain asymptomatic or develop symptoms that are predominantly on the respiratory pathological spectrum.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> Most common symptoms experienced are mild in nature and include anosmia, sore throat, cough, fever, and myalgia; however 15% get affected by severe pneumonia leading to acute respiratory distress syndrome requiring intensive care.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>,<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup> The virus has a propensity to affect the elderly, the comorbid, and the immunocompromised more severely; however, this is not exclusive as an increased viral dose and inoculation can also lead to severe disease phenotype.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup>\n</p><p>Early analysis by Liu et al (2020) has shown that viral shedding and viral load found in the nasopharyngeal mucosa are directly proportional to the severity of symptoms experienced.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> It has also been shown that asymptomatic individuals are capable of spreading the virus as well.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>]</sup> SARS-CoV2 is highly efficient in its transmission from person to person for a low infective dose, and primarily spreads via respiratory droplets, aerosols, and contact.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup> The total number of particles and ratio of respiratory aerosols (<inline-formula><mml:math id=\"M1\"><mml:mo><</mml:mo></mml:math></inline-formula> 5µm) to droplets (<inline-formula><mml:math id=\"M2\"><mml:mo>></mml:mo></mml:math></inline-formula> 5µm) is directly proportional to the airway effort and disruption involved in breathing, speaking, coughing, or sneezing.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>,<xref rid=\"B12\" ref-type=\"bibr\">12</xref>,<xref rid=\"B13\" ref-type=\"bibr\">13</xref>]</sup> This is important because larger droplets of respiratory origin do not usually travel more than 2 m, but smaller aerosolized particles can travel further and remain in the air for longer durations.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>,<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup> For example, particles with aerodynamic diameters of 0.5 and 10 µm will settle 5 feet in 41 h and 8.2 min, respectively.<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup> These aforementioned facts make it scientifically plausible that infected individuals constantly spread the virus in their environment, where it lasts in air attached to the aerosols, and also on inanimate surfaces such as plastics for up to 72 h.<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>]</sup>\n</p></sec><sec sec-type=\"section\"><title> Enhancing Safety in Ophthalmology Settings</title><p>Many eye units around the globe had come to a halt during the pandemic. For example, in the UK, the Royal College of Ophthalmologists had recommended the suspension of all non-urgent elective eye operations and postponement of low-risk non-urgent outpatient clinics which lasted approximately 12 weeks.<sup>[<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup> These measures, whilst essential, had a significant impact on the organization and delivery of ophthalmic services. Ophthalmic units now face the challenge of resuming services with a tremendous increase in workload through the added backlog which cannot be ignored.</p><p>Ophthalmology is already one of the busiest outpatient specialties in healthcare. Each patient's journey includes several healthcare personnel interacting to undertake routine objective assessments which is often followed by specialized imaging. The clinical consultation can take an average of 8 min and includes a close proximity slit-lamp examination to systematically inspect the eye and its adnexa. During the Wuhan outbreak of COVID-19, nosocomial transmission was reported to be highest in ENT and Ophthalmology.<sup>[<xref rid=\"B17\" ref-type=\"bibr\">17</xref>]</sup> The standard high-volume practice observed in ophthalmic units is therefore very high-risk and cannot be underestimated in subjecting staff and patients to contracting SARS-CoV-2.</p><p>Thus, the resumption of effective ophthalmic services mandates several key modifications to safely diagnose and treat patients. Exposure mitigation measures in the Ophthalmology need to be specifically tailored including infection control, engineering control, administrative control, and provision of appropriate protective equipment.</p><sec sec-type=\"subsection\"><title>Infection Control </title><p>\n<inline-formula><mml:math id=\"M3\"><mml:mo>•</mml:mo></mml:math></inline-formula>Regular handwashing with soap and running water for 20 sec should be followed by staff. Patients should also gel hands on entry to the unit and before leaving. Upon arrival at home, they should again wash hands</p><p>\n<inline-formula><mml:math id=\"M4\"><mml:mo>•</mml:mo></mml:math></inline-formula>Clinical interactive surfaces should be cleaned with alcohol-based or bleach-based disinfectants. This is also true for slit-lamp and imaging devices.</p><p>\n<inline-formula><mml:math id=\"M5\"><mml:mo>•</mml:mo></mml:math></inline-formula>All personnel in the eye department should be instructed never to touch face. This is to ensure that there is protection of all mucous membranes; eyes, nose, and mouth.</p><p>\n<inline-formula><mml:math id=\"M6\"><mml:mo>•</mml:mo></mml:math></inline-formula>There should be strict non-touch of all surfaces unless unavoidable.</p><p>\n<inline-formula><mml:math id=\"M7\"><mml:mo>•</mml:mo></mml:math></inline-formula>There should be no handshakes between staff and patients.</p></sec><sec sec-type=\"subsection\"><title>Administrative Control</title><sec sec-type=\"subsubsection\"><title>Emergency Clinics</title><p>\n<inline-formula><mml:math id=\"M8\"><mml:mo>•</mml:mo></mml:math></inline-formula>Emergency eye services should continue to run as a priority to provide timely care to those absolute emergencies where there is a risk of loss of sight.</p><p>\n<inline-formula><mml:math id=\"M9\"><mml:mo>•</mml:mo></mml:math></inline-formula>All emergency cases should undergo telephone triage by a senior clinician to ascertain the urgency of review and/or provide reassurance. An effective alternative is to utilize video consultations, such as the NHS's Attend Anywhere, which has reduced ophthalmic A&E attendances by 30%. The success of the video consultations has pushed for its development in other ophthalmic specialties.</p><p>\n<inline-formula><mml:math id=\"M10\"><mml:mo>•</mml:mo></mml:math></inline-formula>It is also recommended that clinicians use a standardized questionnaire during appointment bookings and selection to flag out high-risk patients based on reported symptoms and contact history.</p></sec><sec sec-type=\"subsubsection\"><title>Routine Clinics</title><p>\n<inline-formula><mml:math id=\"M11\"><mml:mo>•</mml:mo></mml:math></inline-formula>Restarting clinical work will require case selection so as to reduce any excessive and avoidable exposure to staff and patients, many of whom are elderly and at an increased risk of becoming infected leading to severe illness with a high mortality rate.</p><p>\n<inline-formula><mml:math id=\"M12\"><mml:mo>•</mml:mo></mml:math></inline-formula>Stratification systems to prioritize patients will need to be developed on the basis of potential harm and urgency due to delay. Patients who have been postponed and now require urgent treatment will need to have virtual reviews with their notes to classify and prioritize their reviews/treatment.</p><p>\n<inline-formula><mml:math id=\"M13\"><mml:mo>•</mml:mo></mml:math></inline-formula>Face-to-face time and slots in clinics still need to be minimized. Virtual clinics and telephone triage should be done in specially designated clinics with the patients' notes available. This can be used to identify patients that necessitate further checks, examination, or procedures in person. Such patients can then be booked into designated face-to-face clinics.</p><p>\n<inline-formula><mml:math id=\"M14\"><mml:mo>•</mml:mo></mml:math></inline-formula>There should be a dedicated patient-accessible eye hospital website detailing information about common ophthalmic conditions and procedures vetted by the ophthalmologists of that institution. There can be supportive video content exploring expectations of different procedures to educate the patient.</p><p>\n<inline-formula><mml:math id=\"M15\"><mml:mo>•</mml:mo></mml:math></inline-formula>Hospitals must invest in IT infrastructure for safe remote prescribing by liaising with local and regional pharmacies. This will aid repeat prescriptions and allow clinicians to review any continuing treatments, such as for glaucoma patients.</p></sec><sec sec-type=\"subsubsection\"><title>Surgical Theatres</title><p>\n<inline-formula><mml:math id=\"M16\"><mml:mo>•</mml:mo></mml:math></inline-formula>General anesthetic in all cases should be considered only when absolutely necessary due to intubation being considered as an aerosol-generating procedure (AGP). Local anesthetic, if possible, should be explained to patients and considered.</p><p>\n<inline-formula><mml:math id=\"M17\"><mml:mo>•</mml:mo></mml:math></inline-formula>Careful consideration needs to be given to cases requiring surgical methods involving exploration of the orbit with high-speed drills should be avoided as it generates aerosols.</p><p>\n<inline-formula><mml:math id=\"M18\"><mml:mo>•</mml:mo></mml:math></inline-formula>Only operating on absolute emergencies where there is a risk of loss of sight during the pandemic and any further waves of infection.</p><p>\n<inline-formula><mml:math id=\"M19\"><mml:mo>•</mml:mo></mml:math></inline-formula>Patients undergoing elective procedures should self-isolate for two weeks before the surgery and undergo COVID testing (Antigen PCR) 48 h before the surgery.</p></sec><sec sec-type=\"subsubsection\"><title>Clinical Pathways</title><p>\n<inline-formula><mml:math id=\"M20\"><mml:mo>•</mml:mo></mml:math></inline-formula>Patient pathways and flow through the department needs to be carefully considered so that least number of stations are visited for the different tests and required examinations. The pathways should also consider being seen by the least number of professionals possible.</p><p>\n<inline-formula><mml:math id=\"M21\"><mml:mo>•</mml:mo></mml:math></inline-formula>Face-to-face clinics should operate on specific time slots with well-staggered time intervals. Patients should come alone, or with one relative/carer.</p><p>\n<inline-formula><mml:math id=\"M22\"><mml:mo>•</mml:mo></mml:math></inline-formula>Patients should be encouraged to arrive at a specific time and wait in car until called in for their appointment. Upon entry, the patients must be screened again for COVID-19 symptoms.</p><p>\n<inline-formula><mml:math id=\"M23\"><mml:mo>•</mml:mo></mml:math></inline-formula>Self-check in computerized stations should be placed at the entrances where patient can register for their appointment and staff can remotely inquire about COVID-19 symptoms. There can also be remote temperature-checking equipment for the patient to self-check for fever. This can also be used to take a history from the patient.</p><p>\n<inline-formula><mml:math id=\"M24\"><mml:mo>•</mml:mo></mml:math></inline-formula>Following the check-in, patient should be allowed to enter the department. All patients should cover their mouth and nose with medical masks, cloth masks, or simply with a piece of cloth (unless contraindicated). Upon entry, they should be directed to the clinic room as quickly as possible without any undue delay and waiting.</p><p>\n<inline-formula><mml:math id=\"M25\"><mml:mo>•</mml:mo></mml:math></inline-formula>Clinic room face-to-face meeting should be primarily examination-based. All examinations must be focused.</p><p>\n<inline-formula><mml:math id=\"M26\"><mml:mo>•</mml:mo></mml:math></inline-formula>All explanation and consultations after examinations should be done remotely to reduce face-to-face time and discussion in clinics.</p><p>\n<inline-formula><mml:math id=\"M27\"><mml:mo>•</mml:mo></mml:math></inline-formula>Conversations should also be kept to a minimum and all patients should be instructed to speak little. There should be no talk during the slit-lamp examination.</p><p>\n<inline-formula><mml:math id=\"M28\"><mml:mo>•</mml:mo></mml:math></inline-formula>Departmental patient numbers should not accrue more than an upper limit, which is defined on the basis of physical space available and ventilation.</p></sec><sec sec-type=\"subsubsection\"><title>Staff</title><p>\n<inline-formula><mml:math id=\"M29\"><mml:mo>•</mml:mo></mml:math></inline-formula>Staff should not accrue in rooms and maintain a distance of at least 1 m at all times.</p><p>\n<inline-formula><mml:math id=\"M30\"><mml:mo>•</mml:mo></mml:math></inline-formula>There should not be any face-to-face meetings, and where possible telephone and e-meetings should be organized instead.</p><p>\n<inline-formula><mml:math id=\"M31\"><mml:mo>•</mml:mo></mml:math></inline-formula>Universal masking throughout the premises should be mandated for all staff (unless contraindicated).</p></sec></sec><sec sec-type=\"subsection\"><title>Environmental Control </title><sec sec-type=\"subsubsection\"><title>Patient separation </title><p>\n<inline-formula><mml:math id=\"M32\"><mml:mo>•</mml:mo></mml:math></inline-formula>There should be a red pathway for COVID positive, symptomatic, and suspect patients who necessitate examination, with a separate entrance and exit and designated room for examination. Red-labelled operating rooms should also be separated.</p><p>\n<inline-formula><mml:math id=\"M33\"><mml:mo>•</mml:mo></mml:math></inline-formula>For all patients who are COVID-negative or asymptomatic, there should be a green stream involving a separate entrance and exit. There should also be designated rooms for examination and operating.</p></sec><sec sec-type=\"subsubsection\"><title>Outpatients </title><p>\n<inline-formula><mml:math id=\"M34\"><mml:mo>•</mml:mo></mml:math></inline-formula>There should be a designated separate entrance and exit for all attending patients.</p><p>\n<inline-formula><mml:math id=\"M35\"><mml:mo>•</mml:mo></mml:math></inline-formula>Two separate doorways should be individually designated and labelled as exit and entrance for clarity of staff and patients alike.</p><p>\n<inline-formula><mml:math id=\"M36\"><mml:mo>•</mml:mo></mml:math></inline-formula>There should be flow markings on the floor, and directions on the walls (similar to supermarkets).</p><p>\n<inline-formula><mml:math id=\"M37\"><mml:mo>•</mml:mo></mml:math></inline-formula>Consider setting up a physical barrier (such as a plexiglass wall) between reception and patient area and adjust ventilation system to have the reception area a positive pressure zone. This way, any direct droplet and airborne particle spread can be blocked from the patient areas.</p><p>\n<inline-formula><mml:math id=\"M38\"><mml:mo>•</mml:mo></mml:math></inline-formula>Waiting room must have a seating arrangement with a gap of at least 1 m between seats.</p></sec><sec sec-type=\"subsubsection\"><title>Clinics and Equipment</title><p>\n<inline-formula><mml:math id=\"M39\"><mml:mo>•</mml:mo></mml:math></inline-formula>Ophthalmic clinics must be conducted in a designated well-ventilated room. Ventilation systems should be fitted with high efficiency particulate air (HEPA) filters to purify air in the clinical areas. If this is not possible, windows can be opened to maintain airflow in spacious rooms.</p><p>\n<inline-formula><mml:math id=\"M40\"><mml:mo>•</mml:mo></mml:math></inline-formula>All slit lamps should be modified to incorporate a clear thick plastic or Perspex shield breath guards with sufficient dimensions to provide cover for the examining ophthalmologist and patient.</p><p>\n<inline-formula><mml:math id=\"M41\"><mml:mo>•</mml:mo></mml:math></inline-formula>In imaging clinics, the machinery can be encompassed by a specialized Perspex box-type enclosure with glove entrances. These enclosures can be linked to the exhaust fan with high efficiency particulate air (HEPA) filter to create negative pressure system for your operation.</p></sec><sec sec-type=\"subsubsection\"><title>Operating Theatres </title><p>\n<inline-formula><mml:math id=\"M42\"><mml:mo>•</mml:mo></mml:math></inline-formula>Operating theatres should be properly ventilated to meet the designed air change requirement or with ambient air recycled with HEPA systems to reduce risk of aerosol spread and deposition in the environment. ASHRAE recommends 20 air changes per hour for operating rooms.<sup>[<xref rid=\"B18\" ref-type=\"bibr\">18</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M43\"><mml:mo>•</mml:mo></mml:math></inline-formula>There should be a designated separate entrance and exit to the theatre.</p></sec></sec><sec sec-type=\"subsection\"><title>Personal Protective Equipment</title><p>Personal protective equipment (PPE) provisions are paramount in reducing transmission of SARS-CoV-2 to the healthcare workers. Staff must also be trained in the proper donning and doffing procedures.</p><sec sec-type=\"subsubsection\"><title>Eye protection </title><p>Eye protection encompasses several different types of safety devices including safety goggles, visors, and face shields. Ideally, eye protection should be offered in the form of visors or goggles that fit snugly by forming a seal around the eyes. There have been case reports suggesting that coronavirus is transmissible through conjunctival tissue as well.<sup>[<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup>\n</p></sec><sec sec-type=\"subsubsection\"><title>Gloves and Gown </title><p>Gloves and gowns are recommended when dealing with high-risk cases, particularly when in an aerosol-generating environment. High-risk areas are cohorted in most hospitals, so the staff should refrain from deploy donning and doffing repeatedly in these areas. PPE donning should only be done in a designated clean area while a separate designated area should be used for PPE removal. This is because doffing can shed virus that may become airborne and contaminate bits of gear, face, or hands. Every surface including used PPE should be considered contaminated. Gloves use should be mandatory to prevent cross-contamination between hand-surface material cross-contamination and when examining ocular and adnexal tissues.</p></sec><sec sec-type=\"subsubsection\"><title>Masks and Respirators</title><p>Formal use of surgical masks has been recommended at least since the early 1900s to avoid droplet contamination during surgical procedures.<sup>[<xref rid=\"B20\" ref-type=\"bibr\">20</xref>]</sup> Respiratory personal protection is paramount as it protects the mucosa of nose and mouth. Nowadays, there are various types of surgical masks and respirators available to the healthcare workforce (Table 1).<sup>[<xref rid=\"B21\" ref-type=\"bibr\">21</xref>,<xref rid=\"B22\" ref-type=\"bibr\">22</xref>]</sup>\n</p><p>Surgical masks comprise of three layers: an inner soft absorbent layer, a middle polypropylene barrier, and an outer hydrophobic fabric which mold to the user's nasal bridge to cover nose, mouth, and chin. They act as a barrier to infectious droplets. They also assist in the maintenance of a sterile field by reducing the spread of droplets from the wearer's nose and mouth.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>,<xref rid=\"B23\" ref-type=\"bibr\">23</xref>,<xref rid=\"B24\" ref-type=\"bibr\">24</xref>]</sup>\n</p><p>Filtering facepiece respirators (FFPs), on the other hand, provide additional benefit to surgical masks by providing an air-tight seal and containing a mechanical filter, which can remove airborne contaminants through interception. Health and Safety Executive and British Safety Industry Federation recommend fit testing to ensure the respirator is suited to the user's facial structure and therefore performs optimally. There are three categories of FFP in Europe: FFP1, FFP2 (equivalent to N95), and FFP3. Class three (FFP3) provides the highest quality of protection and is the only one approved for UK healthcare settings, especially in AGPs, such as intubation and non-invasive ventilation. They must meet industry-standard regulations including strict industry tests with biological aerosols and cannot exceed 2% leakage. FFP3 masks provide 99% efficiency in filtering particles sized above 100 nm, including small airborne droplets.<sup>[<xref rid=\"B22\" ref-type=\"bibr\">22</xref>,<xref rid=\"B24\" ref-type=\"bibr\">24</xref>]</sup>\n</p><p>Based on the scientific rationale provided in this article and established practice in the Far-Eastern countries, masks or facial coverings must be worn by all patients and staff at all times.<sup>[<xref rid=\"B25\" ref-type=\"bibr\">25</xref>]</sup> This reduces the spread of respiratory droplets in the environment and deposition on fomites and therefore protects from inhalation of virus-laden aerosols. Close contact with symptomatic patients who have more respiratory aerosol production necessitates the use of respirators.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Comparison of different commonly used face mask/respirator in the market<inline-formula><mml:math id=\"M44\"><mml:msup><mml:mrow/><mml:mrow><mml:mo>[</mml:mo><mml:mn>21</mml:mn><mml:mo>,</mml:mo><mml:mn>22</mml:mn><mml:mo>]</mml:mo></mml:mrow></mml:msup></mml:math></inline-formula>\n</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Mask Type</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Standards</bold>\n</td><td align=\"center\" colspan=\"3\" rowspan=\"1\"><bold>Filtration Effectiveness</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Surgical Mask</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">China YY 0469</td><td align=\"center\" colspan=\"3\" rowspan=\"1\"><3.0 microns <inline-formula><mml:math id=\"M45\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 95%</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" colspan=\"3\" rowspan=\"1\"><0.1 microns <inline-formula><mml:math id=\"M46\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 30%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">USA ASTM F2100</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Level 1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Level 2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Level 3</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.0 microns <inline-formula><mml:math id=\"M47\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 95%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.0 microns <inline-formula><mml:math id=\"M48\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 98%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.0 microns <inline-formula><mml:math id=\"M49\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 98%</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.1 microns <inline-formula><mml:math id=\"M50\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 95%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.1 microns <inline-formula><mml:math id=\"M51\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 98%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1 microns <inline-formula><mml:math id=\"M52\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 98%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Europe EN 14683</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Type I</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Type II</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Type III</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.0 microns <inline-formula><mml:math id=\"M53\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 95%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.3 microns <inline-formula><mml:math id=\"M54\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 98%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3 microns <inline-formula><mml:math id=\"M55\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 98%</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.1 microns <bold>X</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.1 microns <bold>X</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1 microns <bold>X</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Respirator Mask</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">USA NIOSH (42 CFR 84)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N95/KN95</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">N99/KN99</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">N100/KN100</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">China GB2626</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.3 microns <inline-formula><mml:math id=\"M56\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 95%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3 microns <inline-formula><mml:math id=\"M57\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 99%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3 microns <inline-formula><mml:math id=\"M58\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 99.97%</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Europe EN149 :2001</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">FFP1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">FFP2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">FFP3</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.3 microns <inline-formula><mml:math id=\"M59\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 80%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.3 microns <inline-formula><mml:math id=\"M60\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 94%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.3 microns <inline-formula><mml:math id=\"M61\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 99%</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">3.0 microns: Bacteria Filtration standard (BFE) 0.1 microns: Particle Filtration Efficiency standard (PFE) 0.3 microns: Used to represent the most penetrating particle size <bold>X</bold>: No requirements</td></tr></tbody></table></table-wrap></sec></sec></sec><sec sec-type=\"section\"><title> Conserving and Procuring Personal Protective Equipment</title><p>It is envisioned that by streamlining the service during the COVID-19 pandemic to exclusively treat emergencies and operating on the life-, limb-, or organ-threatening conditions, we would conserve the limited supply of specialized protective equipment necessary to provide safe care. However, if this pandemic lasts for a longer duration, with more and more people being infected, then we may be threatening to fast deplete our supply of resources and PPE kit with the current model of single use or single session use.</p><p>The issue of ensuring a steady supply of PPE equipment will require commissioning production of these items nationally. In the UK, industrial plants that have machinery to produce face shields or masks have already started to offer a helping hand to the government by developing and supplying PPE to hospitals. Similarly, there are also in-house 3D printing technology centers that can be used to make face shields, visors, and goggles. Countries in short supply need to explore the possibility of further importing from China, where they now have spare capacity. As large variation in quality in masks have been reported, PPE procurement should obtain small batch samples to verify PPE effectiveness prior to mass ordering.</p><p>Given the finite resources, it is also paramount that mask reusability and extended wear is explored as a priority. Taiwan was able to successfully limit its public to purchase a maximum of two masks per week, which allowed appropriate distribution to all and enforced reuse.<sup>[<xref rid=\"B26\" ref-type=\"bibr\">26</xref>]</sup> Masks have been shown to be reusable in studies using energetic methods such as germicidal ultraviolet light and microwaved steam.<sup>[<xref rid=\"B27\" ref-type=\"bibr\">27</xref>,<xref rid=\"B28\" ref-type=\"bibr\">28</xref>,<xref rid=\"B29\" ref-type=\"bibr\">29</xref>]</sup> US Food and Drug Administration has given an emergency go-ahead to a novel Battelle Critical Care Decontamination System<inline-formula><mml:math id=\"M62\"><mml:msup><mml:mrow/><mml:mi> TM </mml:mi></mml:msup></mml:math></inline-formula> which uses vaporized hydrogen peroxide to sterilize and does not degrade filter performance of respirators.<sup>[<xref rid=\"B30\" ref-type=\"bibr\">30</xref>]</sup> There are also unlicensed recommendations to utilize autoclaves or ovens to sterilize masks and perhaps that will be effective for a limited number of cycles before the mask efficiency degrades to substandard levels. In these desperate times, we have no choice but to be resourceful and adapt during this pandemic by using the existing scientific knowledge of decontamination with our understanding of SARS-CoV-2 virus.<sup>[<xref rid=\"B29\" ref-type=\"bibr\">29</xref>]</sup>\n</p></sec><sec sec-type=\"section\"><title> Conserving Medical, Nursing, and Allied Healthcare Workforce</title><p>Healthcare workers are at the most risk of repetitive exposure to this pathogen that increases their viral load predisposing them to contracting severe pneumonia and end up hospitalized. There have several fatalities of frontline staff around the world; in Italy, <inline-formula><mml:math id=\"M63\"><mml:mo>∼</mml:mo></mml:math></inline-formula>8% of the total cases affected were healthcare workers.<sup>[<xref rid=\"B31\" ref-type=\"bibr\">31</xref>]</sup> This risk is highest to healthcare workers participating in AGPs and to ENT and Ophthalmology staff who see an increased outpatient load at close quarters.<sup>[<xref rid=\"B17\" ref-type=\"bibr\">17</xref>]</sup>\n</p><p>Risk assessment of BAME, co-morbid, and older members of staff should be prioritized to ensure safe working standards and appropriate job roles are allocated. We also have to ensure that staff are tested regularly to maintain a strong battlefront against this pandemic. It should also be compulsory for staff to self-monitor twice daily temperatures and development of infective symptoms, as was done in National University Hospital, Singapore.<sup>[<xref rid=\"B32\" ref-type=\"bibr\">32</xref>]</sup> Depending on the availability of tests, regular SARS-CoV-2 testing should also be mandated for all working and returning staff to prevent asymptomatic spread in the healthcare workplace.<sup>[<xref rid=\"B33\" ref-type=\"bibr\">33</xref>]</sup>\n</p></sec><sec sec-type=\"section\"><title> Summary</title><p>\n<italic>Veni, vidi, vici</italic>. I came, I saw, I conquered. COVID-19 came and unleashed untold harm to society and will linger on with possibility of further waves unless it self-attenuates, or safe effective vaccines arrive. In the light of current knowledge of COVID-19, we have put together important considerations for protecting patients and staff.</p><p>\n<italic>Primum non nocere</italic> – we need to be certain that we are protecting patients from contracting the coronavirus. At the same time, we also have a duty of care to staff. There is not only a contractual duty but a moral duty to protect medical, nursing, and other healthcare staff. Conserving highly trained healthcare staff is of course a national interest.</p><p>The prevention of spread is done through education, guidelines, rules, and the law. It behooves governments and healthcare regulators to put these in place. In a century's time, we want future ophthalmologists to be able to look back and say their forebears had done well in the year 2020 protecting ophthalmology patients and staff and laid foundations for new pathways and new ways of delivering safe ophthalmic care, thus transforming ophthalmology despite the challenge of high volume and high risk.</p></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>None.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">WHO. WHO announces COVID-19 outbreak a pandemic [Internet]. WHO; 2020. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864060</article-id><article-id pub-id-type=\"pmc\">PMC7431712</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7448</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Association of <italic>TIMP-1</italic> and <italic>COL4A4</italic> Gene Polymorphisms with Keratoconus in an Iranian Population</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Yari</surname><given-names>Davood</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>2</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ehsanbakhsh</surname><given-names>Zohreh</given-names></name><degrees>BS</degrees><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"I4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Validad</surname><given-names>Mohammad-Hosein</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I5\">\n<sup>5</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Langroudi</surname><given-names>Farzaneh Hasanian</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Cellular and Molecular Research Center, Zahedan University of Medical Sciences, Zahedan,\nIran</aff><aff id=\"I2\">\n<sup>2</sup>Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical\nSciences, Zahedan, Iran</aff><aff id=\"I3\">\n<sup>3</sup>Mashhad University of Medical Sciences, Mashhad, Iran</aff><aff id=\"I4\">\n<sup>4</sup>Shariati Hospital, Mashhad University of Medical Sciences, Mashhad, Iran</aff><aff id=\"I5\">\n<sup>5</sup>Department of Ophthalmology, Alzahra Eye Hospital, Zahedan University of Medical Sciences,\nZahedan, Iran</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Davood Yari, PhD. Cellular and Molecular Research\nCenter, Zahedan University of Medical Sciences,\nZahedan 98167, Iran.\nEmail: Davidyari.85@gmail.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>299</fpage><lpage>307</lpage><history><date date-type=\"received\"><day>01</day><month>3</month><year>2019</year></date><date date-type=\"accepted\"><day>14</day><month>3</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Yari et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><sec><title>Purpose</title><p>Keratoconus (KC) is a bilateral and noninflammatory disease, characterized by progressive thinning and anterior protrusion of the cornea and may result in severe visual impairment due to irregular astigmatism. Matrix metalloproteinases (MMP) are the main group of enzymes that degrade extracellular matrix proteins including collagens; Type IV collagen is found in the corneal stroma. MMP enzymatic activity is inhibited by tissue inhibitor of metalloproteinase-1 (TIMP-1). A decrease in TIMP-1 level is associated with the development of KC. In the present study, we investigated the impact of <italic>COL4A4 </italic>rs2228557 C/T and <italic>TIMP-1</italic> rs4898 C/T (X-chromosome) variants on the odds of KC development in a sample of Iranian population.</p></sec><sec><title>Methods</title><p>This case–control study was conducted on 140 patients with KC and 150 healthy control subjects. We used modified methods of Nested-PCR and ARMS-PCR in combination (Nested-ARMS-PCR) and confirmed their validity with RFLP–PCR.</p></sec><sec><title>Results</title><p>Significant differences were noticed between KC patients and healthy individuals regarding the genotype TY or T allele frequencies of rs4898 in the male subjects (OR = 0.43, 95%CI: 0.20–0.92, <italic>P</italic> = 0.03), whereas no significant differences were identified in the female subjects (OR = 1.07, 95%CI: 0.52–2.20, <italic>P</italic> = 0.85). The rs2228557, T allele was associated with KC (OR = 0.69, 95% CI: 0.50–0.97, <italic>P</italic> = 0.035).</p></sec><sec><title>Conclusion</title><p> In the rs2228557 variant, T allele acts as a protective factor from the disease and decreases the risk of KC compared with the C allele. Also, in our investigation about rs4898, we found that TY genotype or T allele decreased the risk of KC compared with the C allele in males and was a protective factor for KC in our population</p></sec></abstract><kwd-group><kwd>Collagen</kwd><kwd> COL4A4</kwd><kwd> Keratoconus</kwd><kwd> Polymorphism</kwd><kwd> TIMP-1</kwd></kwd-group><counts><fig-count count=\"1\"/><table-count count=\"3\"/><ref-count count=\"51\"/><page-count count=\"9\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> INTRODUCTION</title><p>Keratoconus (KC) is defined as a bilateral, non-inflammatory, and progressive disease\ncharacterized by conical protrusion of the cornea. This disease may result in severe visual impairment due to irregular astigmatism and stromal scarring. KC eventually affects both eyes, although the involvement is usually asymmetric. The symptoms of KC-affected patients are different depending on the stage of the disease.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup> Glasses or contact lenses can provide useful vision in the early stage of the disease; nonetheless, corneal transplantation is mandatory for visual rehabilitation in 20% of the patients who are in advanced stage. Corneal thinning is considered as one of the identifying characteristics of KC. Central corneal thickness (CCT) is lower in KC patients by 75 µm as compared to normal controls.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup>\n</p><p>The incidence of KC is approximately 1 per 2,000, and its prevalence is 54.5 per 100,000. This disease occurs in both genders,<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> with different rates among different ethnic groups.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup> KC usually begins in teens, and its progression slows after the age of 30 years.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>KC is a multi-factorial disorder; environmental factors cause KC in genetically susceptible individuals. The environmental factors that may play roles in the pathogenesis of KC include eye rubbing, allergy, connective tissue dysfunction, and contact lens wear. Moreover, subjects with a family history of KC are more susceptible to this disorder.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> Using family-based linkage, several case–control studies have determined various genes that increase the odds of KC development.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> The gene candidates for KC include <italic>LOX</italic>,<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>]</sup>\n<italic>VSX1</italic>,<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup>\n<italic>GPX-1</italic>,<sup>[<xref rid=\"B12\" ref-type=\"bibr\">12</xref>]</sup>\n<italic>TGF-<inline-formula><mml:math id=\"M1\"><mml:mi>β</mml:mi></mml:math></inline-formula>1</italic>,<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>]</sup>\n<italic>COL4A3</italic> and <italic>COL4A4</italic>\n<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup> (polymorphism or mutation), and <italic>TIMP-1</italic>,<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>]</sup>\n<italic>MMP-2</italic>, <italic>MMP-9</italic>\n<sup>[<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup> (gene expression). KC is still an enigmatic disease in many aspects, including inheritance, basic pathophysiology, prevention, associated risk factors, disease development, as well as therapeutic approaches.<sup>[<xref rid=\"B17\" ref-type=\"bibr\">17</xref>]</sup>\n</p><p>Type IV collagen is only present in basement membranes and constitutes their main structural component. Collagen type IV gene, <italic>alpha-4 (COL4A4),</italic> is located in the region 2q35–q37 with a gene span composed of 113 kb and 48 exons.<sup>[<xref rid=\"B18\" ref-type=\"bibr\">18</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup> Type IV collagen is not expressed in cornea in the normal condition and its presence indicates a corneal pathology; therefore, type IV collagen can be a potential candidate in the pathogenesis of KC. In support of this theory, alterations in the expression level of collagen type IV (α-4 chains) were observed in corneas inflicted by KC.<sup>[<xref rid=\"B20\" ref-type=\"bibr\">20</xref>,<xref rid=\"B21\" ref-type=\"bibr\">21</xref>]</sup> Matrix metalloproteinases (MMP) are the major expressed metalloproteases in the cornea. It has been demonstrated that the proteolytic activity of MMP increases in KC. This finding suggests that abnormal MMP activity plays a role in the pathogenesis of KC.<sup>[<xref rid=\"B16\" ref-type=\"bibr\">16</xref>,<xref rid=\"B22\" ref-type=\"bibr\">22</xref>]</sup>\n</p><p>Four types of tissue inhibitor of metalloproteinase (<italic>TIMP1-4</italic>) have been detected; three of which, including <italic>TIMPs-1</italic>, 3, and 4, are nested within an intron in the genes of synapsins. TIMP inhibits collagenases and proteoglycanase called matrix metalloproteinase. All four types of TIMPs have various biological activities apart from their metalloproteinase inhibitory activity. These biological activities include the promotion of cell proliferation, cancer promotion, regulation of angiogenesis, as well as pro- and anti-apoptotic and synaptic flexibility activities, many of which are independent of metalloprotease inhibition. <italic>TIMP-1</italic> is associated with synapsin 1 and its gene is located in X11p11.23–11.4 consisting of six exons. Mature <italic>TIMP-1</italic> is a 28.5 kDa glycoprotein that consists of 184 amino acid residues. The natural precursor contains a signal peptide of 23 residues which are cleaved throughout the protein maturation.<sup>[<xref rid=\"B23\" ref-type=\"bibr\">23</xref>,<xref rid=\"B24\" ref-type=\"bibr\">24</xref>,<xref rid=\"B25\" ref-type=\"bibr\">25</xref>,<xref rid=\"B26\" ref-type=\"bibr\">26</xref>,<xref rid=\"B27\" ref-type=\"bibr\">27</xref>]</sup>\n<italic>TIMP-1</italic> suppresses angiogenesis and controls the balance in the corneal tissue by inhibiting the action of matrix metalloproteinase to protect tissues from permanent damage.<sup>[<xref rid=\"B28\" ref-type=\"bibr\">28</xref>]</sup> Furthermore, it has been demonstrated that increased MMP and decreased TIMP levels are associated with the development of KC.<sup>[<xref rid=\"B29\" ref-type=\"bibr\">29</xref>]</sup>\n</p><p>\n<italic>COL4A4</italic> gene rs2228557 (F1644F) (HGVM1660028) is located in chromosome 2 exon 48, NM_000092.4 region. Several studies examined the association between <italic>COL4A4</italic> and KC and revealed that this gene is associated with KC; however, some other studies failed to find the same relationship in different populations.<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>,<xref rid=\"B22\" ref-type=\"bibr\">22</xref>,<xref rid=\"B30\" ref-type=\"bibr\">30</xref>,<xref rid=\"B31\" ref-type=\"bibr\">31</xref>]</sup>\n</p><p>\n<italic>TIMP-1</italic> gene rs4898 (+372C/T) (HGVM6380940) is located within the intron of the synapsin gene, and single nucleotide polymorphism (SNP) is located in exon 5, the region of NM_006950.3. Some studies investigated the association of rs4898 gene polymorphism with disorders including intracerebral hemorrhage,<sup>[<xref rid=\"B32\" ref-type=\"bibr\">32</xref>]</sup> systemic sclerosis,<sup>[<xref rid=\"B33\" ref-type=\"bibr\">33</xref>]</sup> and severe sepsis.<sup>[<xref rid=\"B34\" ref-type=\"bibr\">34</xref>]</sup>\n</p><p>The present study aimed to evaluate the possible association of <italic>TIMP-1</italic> rs4898 C/T gene polymorphism and <italic>COL4A4</italic> rs2228557 C/T gene polymorphism with the development of KC in a sample of Iranian population.</p></sec><sec sec-type=\"section\"><title> METHODS</title><sec sec-type=\"subsection\"><title>Patients</title><p>The current retrospective case–control study was conducted at the Alzahra Eye Hospital, Zahedan University of Medical Sciences, Zahedan, Iran and recruited 140 unrelated Iranian patients with KC and 150 unrelated healthy controls. The patients were diagnosed with KC after a comprehensive ophthalmic examination using the following criteria: (1) clinical signs of KC (Munson sign, protrusion, Vogts striae, corneal thickness, scarring, Fleischer ring) and abnormal findings in corneal topography (Pentacam AXL, OCULUS INC); (2) the three quantitative videokeratographic indices used for the screening of KC were central corneal power <inline-formula><mml:math id=\"M2\"><mml:mo>></mml:mo></mml:math></inline-formula> 47.2 D, inferior–superior value <inline-formula><mml:math id=\"M3\"><mml:mo>></mml:mo></mml:math></inline-formula> 1.4 D, Sim-K astigmatism <inline-formula><mml:math id=\"M4\"><mml:mo>></mml:mo></mml:math></inline-formula> 1.5 D, and skewed radial axes <inline-formula><mml:math id=\"M5\"><mml:mo>></mml:mo></mml:math></inline-formula> 21°.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>,<xref rid=\"B35\" ref-type=\"bibr\">35</xref>]</sup> Patients with other ocular diseases were excluded from the study. Controls were sex- and age-matched healthy participants who were unrelated to the patients and were selected from a geographic area similar to that of KC subjects.</p><p>The Ethics Committee of Zahedan University of Medical Sciences, Zahedan, Iran approved the study protocol and informed consent was signed by all participants. This study complied with the tenets of the Declaration of Helsinki.</p></sec><sec sec-type=\"subsection\"><title>Analysis of the <italic>TIMP-1</italic> (rs4898), COL4A4 (rs2228557) Polymorphisms</title><p>Blood samples were collected in EDTA-containing tubes and genomic DNA was extracted from the peripheral blood leukocytes using salting out method as previously described.<sup>[<xref rid=\"B36\" ref-type=\"bibr\">36</xref>]</sup> All procedures were performed under a standardized setting to avoid variation in DNA quality. SNP rs2228557 <italic>COL4A4</italic> was evaluated by ARMS–PCR. For the detection of rs4898 <italic>TIMP-1</italic>, we used the combination of Nested-polymerase chain reaction (Nested-PCR)<sup>[<xref rid=\"B37\" ref-type=\"bibr\">37</xref>]</sup> and amplification refractory mutation system-PCR(ARMS-PCR)<sup>[<xref rid=\"B38\" ref-type=\"bibr\">38</xref>]</sup> (Nested-ARMS-PCR). The verification of these methods was accomplished using Restriction Fragment Length Polymorphism (RFLP-PCR).<sup>[<xref rid=\"B39\" ref-type=\"bibr\">39</xref>]</sup>\n</p><p>The rs4898 location was very challengeable for ARMS-PCR; therefore, for the detection of the SNP, we used Nested or hemi-Nested-PCR primers, as mentioned previously.<sup>[<xref rid=\"B40\" ref-type=\"bibr\">40</xref>]</sup> The advantages of this modification include elimination of non-specific products, protection of SNP position for the next steps, low cost, and short duration of the process.</p><p>PCR reactions were performed using PCR master mix (Ampliqon Taq 2x mastermix, Denmark) according to the manufacturer's instructions. For investigation of <italic>COL4A4</italic> (rs2228557), amplification reaction was provided in 20 μL volume including: 1 μL template DNA (<inline-formula><mml:math id=\"M6\"><mml:mo>∼</mml:mo></mml:math></inline-formula>100 ng/μL), 1 μL of each primer (10 pmol/μL), 10 μL mastermix, and 7 μL DNase-free water. The PCR conditions were set as follow: 95°C for 5 min, 30 cycles of 95°C for 30 sec, 55°C for 35 sec, 72°C for 30 sec, and a final extension at 72°C for 5 min. PCR products were detected by electrophoresis on a 2% agarose gel staining by ethidium bromide (Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>A).</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Electrophoresis pattern for the detection of SNPs in <italic>COL4A4</italic> rs2228557 and <italic>TIMP-1</italic> rs4898.\n<bold>(A)</bold> Arms-PCR products of COL4A4 rs2228557, M: DNA marker (100 bp).\n<bold>(B1)</bold>\n<italic>TIMP-1</italic> rs4898, Nested-PCR products, M: DNA marker (100bp), product size (660 and 344bp), respectively. <bold>(B2)</bold> ARMS-PCR products, product size (202bp), (TT, TC, CC) demonstrate genotypes. <bold>(B3)</bold> RFLP-PCR products, M: DNA marker (50bp), product sizes were 344bp for TT, 344bp and 325bp for TC, 325bp for CC in female, 344bp for TY and 325bp for CY in male, (TT, TC, CC) demonstrate female and (TT = TY, CC = CY) demonstrate male genotypes, (Y stands for the Y-chromosome).</p></caption><graphic xlink:href=\"jovr-15-299-g001\"/></fig><p>In the first stage of study for <italic>TIMP-1</italic> rs4898, Nested-PCR reaction was performed in 20 μL volume including: 1 μL template DNA (<inline-formula><mml:math id=\"M7\"><mml:mo>∼</mml:mo></mml:math></inline-formula>100 ng/μL), 1 μL of each primer (10 pmol/μL), 10 μL mastermix, and 7 μL DNase-free water. The PCR conditions were set as follow: 95°C for 5 min, 30 cycles of 95°C for 30 sec, 64°C for 40 sec, 72°C for 30 sec, and a final extension at 72°C for 5 min. In the second phase, the PCR product obtained from the first stage (660 bp) was used as the template and diluted 1:50. Primers for ARMS-PCR were designed to detect the SNP (Table 1). In this step, ARMS-PCR reaction was performed in 20 μL volume including: 1 μL template (1:50 dilution), 1 μL of each primer (10 pmol/μL), 10 μL mastermix, and 7 μL DNase-free water. The PCR conditions were set as follow: 95°C for 5 min, 17 cycles of 95°C for 30 sec, 56°C for 30 sec, 72°C for 30 sec, and a final extension at 72°C for 5 min. PCR products were detected by electrophoresis on a 2% agarose gel staining by ethidium bromide (202bp product). Consequently, SNP (rs4898) <italic>TIMP-1</italic> was successfully detected with the combination of two methods (Nested-PCR and ARMS-PCR) (Figures B1 & B2).</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>The list of primers and methods used for detection of Single Nucleotide Polymorphisms (SNPs) <italic>TIMP-1</italic> rs4898 and <italic>COL4A4</italic> rs2228557</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"4\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\"><bold>TIMP-1(rs4898) T/C</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Primers(5'-3')</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Product size</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"2\" colspan=\"1\">\n<bold>Stage 1Nested-PCR</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">F</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">TGGGGACACCAGAAGTCAAC</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">660 bp</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">R</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">TAAGCTCAGGCTGTTCCAGG</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"3\" colspan=\"1\">\n<bold>Stage 2ARMS-PCR</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">F Common</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">AGGCTCTGATGAGAATGGTCCCA</td><td align=\"left\" rowspan=\"3\" colspan=\"1\">202 bp</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">R (C allele)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CAGATTGTTCCAGGGAGCCAAG</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">R (T allele)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CAGATTGTTCCAGGGAGCCAAA</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"2\" colspan=\"1\">\n<bold>Stage 3RFLP-PCR</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">F</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CCGCCATGGAGAGTGTCTGC</td><td align=\"left\" rowspan=\"2\" colspan=\"1\">344 bp</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">R*</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">AGGCTGTTCCAGGGAGTCGC</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>COL4A4(rs2228557 ) C/T</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"3\" colspan=\"1\">\n<bold>ARMS-PCR</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">F Common</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">TGTCTGAGCCCTAATTCTCT</td><td align=\"left\" rowspan=\"3\" colspan=\"1\">183 bp</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">R (C allele)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">GAGCCAGAAGCTATACTTATTTGAG</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">R (T allele)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">GAGCCAGAAGCTATACTTATTTGAA</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">F, forward; R, reverse; R*, altered reverse</td></tr></tbody></table></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Genotype and allelic frequencies of <italic>COL4A4</italic> rs2228557 and <italic>TIMP-1</italic> rs4898 polymorphisms between keratoconus (KC) patients and healthy controls.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Variants</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>KC Patients n (%)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Controls n (%)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>*OR (95% CI)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold/>\n<italic><bold>P</bold></italic>\n<bold>-value</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>rs2228557,COL4A4</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>CC</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">67 (47.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">61 (40.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ref.</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>CT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">39 (27.9)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37 (24.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.96 (0.54–1.69)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.887</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>TT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">34 (24.3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">52 (34.6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.59 (0.34–1.03)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.067</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Allele</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>C</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">173 (61.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">159 (53)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ref.</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>T</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">107 (38.2)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">141 (47)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.69 (0.50–0.97)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.035</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>rs4898,TIMP-1</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Male</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Male</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>CY</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">46 (75.4)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37 (57)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ref.</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>TY</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15 (24.6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28 (43)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.43 (0.20–0.92)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.038</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold/>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Female</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Female</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>CC</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28 (47.5)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">31 (52.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ref.</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>CT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29 (49.2)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30 (50.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.01 (0.46–2.19)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.97</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>TT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22 (47.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24 (52.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.07 (0.52–2.20)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.854</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Allele</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>C</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">85 (53.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">92 (54.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ref.</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>T</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">73 (46.2)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">78 (45.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.01 (0.66–1.56)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.953</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">Ref., reference; OR, odds ratio; CI, confidence interval; n, number *Adjusted for sex and age. (Y states for the Y-chromosome)</td></tr></tbody></table></table-wrap><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><p>Correlation of clinical and keratometric parameters with COL4A4 (rs2228557) and TIMP-1(rs4898) in keratoconus patients.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"6\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Parameters evaluated</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Patients n (%)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>rs2228557</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold/>\n<italic><bold>P</bold></italic>\n<bold>-value</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>rs4898</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold/>\n<italic><bold>P</bold></italic>\n<bold>-value</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Male</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Female</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>KC ocular</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>OD</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">42 (30.0)</td><td align=\"center\" rowspan=\"3\" colspan=\"1\">0.25</td><td align=\"center\" rowspan=\"3\" colspan=\"1\">0.39</td><td align=\"left\" rowspan=\"3\" colspan=\"1\">0.4</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>OS</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">36 (25.7)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>OU</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">62 (44.3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Level of KC</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>KK 1</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33 (23.6)</td><td align=\"center\" rowspan=\"3\" colspan=\"1\">0.81</td><td align=\"center\" rowspan=\"3\" colspan=\"1\">0.014</td><td align=\"left\" rowspan=\"3\" colspan=\"1\">0.97</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>KK 2</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">45 (32.1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>KK 3</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">62 (44.3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>CXL</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>OD</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">39 (27.9)</td><td align=\"center\" rowspan=\"4\" colspan=\"1\">0.58</td><td align=\"center\" rowspan=\"4\" colspan=\"1\">0.71</td><td align=\"left\" rowspan=\"4\" colspan=\"1\">0.37</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>OS</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">40 (28.6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>OU</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">42 (30.0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Candidate</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19 (13.6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">Correlation of clinical and keratometric parameters with <italic>COL4A4</italic> (rs2228557) and <italic>TIMP-1</italic> (rs4898) in keratoconus patients.\nKC, keratoconus; OD, right eye; OS, left eye; OU, both eyes; CXL, cross-linking surgery\nKK1, 2, 3 are phenotyping classification and show the level progress of keratoconus disease in patients</td></tr></tbody></table></table-wrap><p>RFLP-PCR was applied to validate the method and results of screening of rs4898 polymorphism in <italic>TIMP-1</italic>. As the original sequence of the region surrounding the polymorphism does not make a restriction enzyme site, a site-directed mutagenesis PCR primer (R*) was designed. This primer differs from the referent sequence in two bases and lies close to the polymorphic spot to alter the sequence and provide a restriction enzyme site at product. Thus, the original sequence of the region is TT(C) GTGG, while our PCR product was in fact TT(C) GCGA. The C-variant is a palindrome which forms a site for the Bsp68I (NruI) restriction enzyme (Thermo scientific).<sup>[<xref rid=\"B40\" ref-type=\"bibr\">40</xref>]</sup>\n</p><p>Initially, we amplified PCR product (660bp) using the abovementioned conditions. For alteration in the restriction enzyme site, secondary primers were added to template 1:50 dilution of first stage using the following condition: 1 μL template (1:50 dilution), 1 μL of each primer (10 pmol/μL), 10 μL mastermix, and 7 μL DNase-free water. The PCR conditions were set as follow: 95°C for 5 min, 30 cycles of 95°C for 30 sec, 63°C for 30 sec, 72°C for 30 sec, and a final extension at 72°C for 5 min. PCR products were detected by electrophoresis on a 2% agarose gel with ethidium bromide (344bp product). For optimal results, PCR products were digested at 37°C for 5 h according to the manufacturer's instruction. The restriction of the C-allele PCR product resulted in 325bp and 19bp digest products, whereas the T allele (wild type) products remained unrestricted (Figure B3).</p></sec><sec sec-type=\"subsection\"><title>Statistical analysis</title><p>Statistical analyses were performed using the SPSS software version 19.0 (SPSS Inc., Chicago IL, USA). Frequencies were compared between the study groups using the Chi-square test. Association of gene polymorphisms with KC was investigated and compared between the groups using logistic regression analysis, estimation of odds ratio (OR), and 95% confidence intervals (CI), respectively. A <italic>p</italic>-value <inline-formula><mml:math id=\"M8\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05 was regarded as statistically significant.</p></sec></sec><sec sec-type=\"section\"><title> RESULTS </title><p>A total of 140 patients (61 male and 79 female subjects), aged 28 <inline-formula><mml:math id=\"M9\"><mml:mo>±</mml:mo></mml:math></inline-formula> 12.5 years, included the KC group. The control group consisted of 150 healthy controls (65 male and 85 female subjects), aged 29.8 <inline-formula><mml:math id=\"M10\"><mml:mo>±</mml:mo></mml:math></inline-formula> 15.6 years.</p><p>There was no significant difference between the two groups regarding the age and gender (<italic>P</italic> = 0.20).</p><p>The <italic>Col4A4</italic> rs2228557 C/T variant, T allele was associated with a decrease in the risk of KC development (OR = 0.69, 95% CI: 0.50–0.97, <italic>P</italic> = 0.035), as compared to C allele. Our results indicated that TT was not associated with KC as compared to CC (OR = 0.59, 95% CI = 0.34–1.03, <italic>P</italic> = 0.067) (Table 2).</p><p>Table 2 demonstrates the genotype and allelic frequencies of <italic>TIMP-1 </italic>(rs4898) gene polymorphism in each study group. Since <italic>TIMP-1</italic> is an X-linked gene, the results were compared in male and female subjects separately. This analysis demonstrated that TY genotype or T allele decreased the risk of KC in male subjects as compared to the C allele (OR = 0.43, 95% CI: 0.20–0.92, <italic>P</italic> = 0.03). However, no significant association was found between TT genotype (OR = 1.07, 95% CI: 0.52–2.20, <italic>P</italic> = 0.854) or T allele (OR = 1.01, 95%CI: 0.66–1.56, <italic>P</italic> = 0.95) and KC in female subjects.</p><p>Table 3 illustrates the associations of <italic>COL4A4</italic> (rs2228557) and <italic>TIMP-1</italic> (rs4898) with KC severity. This polymorphism <italic>TIMP1</italic> (rs4898 T/C) located at X chromosome exists in two alleles and their combination results in five possible genotypes. Because men lack a second X-chromosome, the possible genotypes are CY and TY hemizygotes (Y states for the Y-chromosome). As for women, there are CC and TT homozygotes and CT heterozygotes (Table 2). In fact, men just have allele and women have genotype and allele. For this reasons, calculation of <italic>TIMP1</italic> (rs4898) distinctly separated in men and women was done. So, results in men and women can be different.</p></sec><sec sec-type=\"section\"><title> DISCUSSION</title><p>Keratoconus is an eye disorder characterized by bilateral, asymmetrical, noninflammatory, and progressive thinning of the cornea. The shape of cornea progressively alters from the normal round shape to a cone shaped one. Although the etiology of KC is not clear, defective cross-linking between adjacent collagen fibers may play an important role in its pathogenesis.<sup>[<xref rid=\"B41\" ref-type=\"bibr\">41</xref>]</sup> SNPs and gene variants suggest an intricate etiology or the convergence of multiple disease pathways.<sup>[<xref rid=\"B42\" ref-type=\"bibr\">42</xref>]</sup>\n</p><p>Biochemical investigations have suggested that the amount of collagen fibers decrease in KC. Also, the weight of KC corneas was found to be reduced; therefore, it can be assumed that collagenase enzymes might be involved.<sup>[<xref rid=\"B43\" ref-type=\"bibr\">43</xref>]</sup>\n</p><p>In the current study, we investigated the impact of <italic>COL4A4</italic> and <italic>TIMP-1</italic> variants on the risk of KC development in a sample of Iranian population. Our results showed that the T allele reduced the risk of disease development, as compared to the C allele. Results in the distribution of genotypes (CC, CT, TT) in rs2228557 of the <italic>COL4A4</italic> gene between KC patients and controls in the Stabuc-Silih et al's study were different from our results.<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup> Similar to our findings, Kokolakis et al demonstrated that the TT genotype was significantly over-represented in healthy individuals and suggested a protective role for this genotype in the KC development.<sup>[<xref rid=\"B31\" ref-type=\"bibr\">31</xref>]</sup>\n</p><p>The level of <italic>TIMP-1</italic> significantly decreases in KC as compared to normal corneas. Given the fact that KC is not associated with extensive scarring or inflammatory infiltrates, substantial degradation should occur in the extracellular matrix. A decreased level of TIMP-1 increases gelatinase and collagenase activities and apoptosis which are characteristic phenomena in KC. Decreases in <italic>TIMP-1</italic> might play a role in matrix degradation which is a characteristic feature of KC.<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>,<xref rid=\"B44\" ref-type=\"bibr\">44</xref>]</sup> Furthermore, it has been recognized that increased MMP and decreased <italic>TIMP-1</italic> levels are associated with the development of KC.<sup>[<xref rid=\"B29\" ref-type=\"bibr\">29</xref>]</sup>\n</p><p>The <italic>TIMP-1</italic> gene is located in Xp11.3–p11.23 and has three types of polymorphism including <italic>TIMP-1 </italic>(19 C/T) in the 5'-UTR, <italic>TIMP-1</italic> (261 C/T) in exon 4, and <italic>TIMP-1</italic> (372 T/C) (rs4898) in exon 5. The <italic>TIMP-1 </italic>(rs4898) polymorphism is an important site which has been reported in other studies. This variation does not result in changes in the amino acid sequence (F124F). This polymorphism exists in two alleles and their combination results in five possible genotypes. Because male subjects lack a second X-chromosome, the possible genotypes are CY and TY hemizygotes. In female subjects, however, there are CC and TT homozygotes and CT heterozygote genotypes.<sup>[<xref rid=\"B40\" ref-type=\"bibr\">40</xref>]</sup>\n</p><p>Our data suggest that C or T allele is associated with <italic>TIMP-1 </italic>(rs4898) polymorphism in patients or controls. The C allele of the 372T/C polymorphism was more frequently found in female than male controls.<sup>[<xref rid=\"B45\" ref-type=\"bibr\">45</xref>]</sup> However, in other studies, the C allele was detected more frequently in male patients with an abdominal aortic aneurysm.<sup>[<xref rid=\"B46\" ref-type=\"bibr\">46</xref>]</sup> Meijer et al investigated the male subjects with inflammatory bowel disease carrying <italic>TIMP-1</italic> (rs4898) T allele. They reported lower levels of TIMP-1 in surgically resected inflamed tissue, as compared to C allele carriers.<sup>[<xref rid=\"B47\" ref-type=\"bibr\">47</xref>]</sup> In addition, Indelicato et al reported that <italic>TIMP-1</italic> (rs4898) C allele frequency increased in males but not females with systemic sclerosis, as compared to healthy individuals.<sup>[<xref rid=\"B48\" ref-type=\"bibr\">48</xref>]</sup> Along the same lines, Wei et al revealed that C allele carriers of <italic>TIMP-1</italic> (rs4898) run a greater risk of developing ankylosing spondylitis disease.<sup>[<xref rid=\"B49\" ref-type=\"bibr\">49</xref>]</sup> Furthermore, it was found that among cirrhotic patients, males with <italic>TIMP-1</italic> (372C/T) T allele developed cirrhosis at a younger age.<sup>[<xref rid=\"B50\" ref-type=\"bibr\">50</xref>]</sup>\n</p><p>Our findings indicated that <italic>TIMP-1 </italic>(rs4898) was associated with the clinical characteristics of KC only in our male sample population. Nevertheless, the analysis of genotype and allele frequencies revealed no significant differences in female patients as compared to female controls. The T allele decreased the risk of KC, as compared to the C allele in males which can be attributed to the location of <italic>TIMP-1</italic> gene at Xp11.3–p11.23. Males only have one X-chromosome, and the functional difference of genetic polymorphism of <italic>TIMP-1</italic> (rs4898) appears more obvious due to the lack of heterozygotes. We cannot compare our results with the literature because no previous study has evaluated the correlation between the <italic>TIMP-1</italic> variants and the risk of KC development.</p><p>In conclusion, our study showed that in the <italic>COL4A4</italic> rs2228557 C/T variant, the T allele acts as a protective factor against the disease and decreases the risk of KC. In addition, <italic>TIMP-1</italic> rs4898 C/T the TY genotype or T allele in males can decrease the risk of KC in comparison with the C allele. Further studies with a larger sample size and different ethnicities are required to confirm these findings.</p></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>This study was supported by a dissertation grant (M.Sc. thesis of Davood Yari, NO. 6077) from the Deputy for Research, Zahedan University of Medical Sciences.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interests</title><p>There are no conflicts of interest.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Burdon KP, Vincent AL. 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] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864074</article-id><article-id pub-id-type=\"pmc\">PMC7431713</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7462</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Photo Essay</subject></subj-group></article-categories><title-group><article-title>Primary Idiopathic Frosted Branch Angiitis</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Maleki</surname><given-names>Shahin Jahani</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Dourandish</surname><given-names>Maryam</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hosseini</surname><given-names>Seyedeh Maryam</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Eye Research Center, Mashhad University of Medical Sciences, Mashhad, Iran</aff><aff id=\"I2\">\n<sup>2</sup>Retina Research Center, Mashhad University of Medical Sciences, Mashhad, Iran</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Maryam Dourandish, MD. Eye Research Center, Khatam-\nAl-Anbia Eye Hospital, Abutalib Junction, Kolahdouz\nBlvd, Mashhad 919596, Iran.\nEmail: Maryam.dourandeesh.dl@gmail.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>424</fpage><lpage>427</lpage><history><date date-type=\"received\"><day>03</day><month>4</month><year>2018</year></date><date date-type=\"accepted\"><day>05</day><month>12</month><year>2018</year></date></history><permissions><copyright-statement>Copyright © 2020 Maleki et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><counts><fig-count count=\"5\"/><ref-count count=\"4\"/><page-count count=\"4\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> Presentation</title><p>A five-year-old boy presented to the ophthalmology emergency department with a three-day history of sudden onset of severe visual impairment in both eyes. He experienced an upper respiratory tract infection (URTI) two weeks before the presenting complaint. On initial ophthalmic examination, his visual acuity was hand motion with projection OU. He had no relative afferent pupillary defect, and had normal intraocular pressure. The visual reduction was not associated with pain or injection. Slit-lamp examination of both eyes showed mild anterior chamber (AC) reaction (1–2+ cells), moderate bilateral presence of vitreous cells (2–3+ cells), but no keratic precipitates.</p><p>A dilated fundus examination of both eyes revealed mild vitreous haziness and bilateral symmetrical and widespread retinal vasculitis. There was a prominent and florid translucent retinal perivascular infiltration that predominantly affected the venules, starting from the posterior pole and extending up to the periphery. Bilaterally, mild to moderate papillitis and severe macular edema was noted without any obvious retinal hemorrhages (Figure 1).</p><p>Spectral-domain optical coherence tomography (SD-OCT) of both eyes showed high reflectivity in the inner retina suggestive of intracellular edema, and multifocal neurosensory detachments were seen at the posterior pole (Figure 2).</p><p>The patient was admitted for an extensive work-up. Anterior chamber paracentesis was performed to detect the possible presence of viral pathogens by polymerase chain reaction (PCR). Consultations with the pediatrics, rheumatology, infectious diseases, and neurology specialists were requested. Laboratory tests were negative for infectious, rheumatological, and malignant disorders.</p><p>Results of real-time PCR of the aqueous humor sample was negative for cytomegalovirus, herpes simplex virus, varicella zoster, and TB. Brain MRI examination revealed no pathologies such as multiple sclerosis and infiltrative diseases.</p><p>The severe vision-threatening condition was diagnosed to be primary frosted branch angiitis, and empirical treatment with oral prednisolone (1 mg/kg/day) and topical corticosteroids was initiated. On the fourth day of the treatment, a significant therapeutic response was observed. At the end of the second week, vascular sheathing and exudates resolved completely (Figure 3) and visual acuity in both eyes improved to 3/10 (+0.5 LogMAR). SD-OCT image showed complete resolution of the subretinal fluid (Figure 4).</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Bilateral primary idiopathic frosted branch angiitis in a 5-year-old boy. Note the prominent, florid, translucent, retinal perivascular sheathing affecting both the venules and arterioles. The sheathing originates from the posterior pole and extends up to the periphery.</p></caption><graphic xlink:href=\"jovr-15-424-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Spectral domain optical coherence tomography (SD- OCT) of the posterior pole reveals highly reflective retinal layers suggestive of diffuse edema and multifocal exudative retinal detachments.</p></caption><graphic xlink:href=\"jovr-15-424-g002\"/></fig><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>At the end of the 2<inline-formula><mml:math id=\"M1\"><mml:msup><mml:mrow/><mml:mi> nd </mml:mi></mml:msup></mml:math></inline-formula> week of treatment, complete resolution of the vascular sheathing and exudates is observed. Note the pigmentary macular change in both eyes.</p></caption><graphic xlink:href=\"jovr-15-424-g003\"/></fig><fig id=\"F4\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><p>Macular OCT image shows complete resolution of the retinal exudates and subretinal fluids. The central macular thickness of the right and left eyes is 188 and 183 µm, respectively.</p></caption><graphic xlink:href=\"jovr-15-424-g004\"/></fig><fig id=\"F5\" orientation=\"portrait\" position=\"float\"><label>Figure 5</label><caption><p>Fundus photography after 12 months of treatment shows mild granular macular pigmentation.</p></caption><graphic xlink:href=\"jovr-15-424-g005\"/></fig><p>At the three-month follow-up, visual acuity improved to 7/10 (+0.15 LogMAR) in both eyes; no active inflammation or vasculitis was observed. Ophthalmic examinations at the 6 and 12-month follow-ups showed bilateral improvement of vision to 9/10 (+0.04 LogMAR), without any recurrence or significant sequelae. The only abnormal finding was mild pigmentary macular change (Figure 5).</p></sec><sec sec-type=\"section\"><title> Discussion</title><p>Frosted branch angiitis can be idiopathic or secondary to ocular or systemic conditions. Because there are various secondary causes, it is important to perform an extensive work-up to exclude other causes of vasculitis before making a diagnosis of primary idiopathic retinal vasculitis. In the present case, possible underlying conditions such as infectious, rheumatological, or malignant causes were ruled out after extensive evaluation.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup>\n</p><p>This report re-emphasizes the considerable response of this rare disease to corticosteroid therapy without any significant visual consequences.</p></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>Nil</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">He L, Moshfeghi DM, Wong IG. Perivascular exudates in frosted branch angiitis. Ophthal <italic>Surg Laser Imag Retina</italic> 2014;45:443–446.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">Mao F, Wu J, Sun H, You Q, Li D. Frosted branch angiitis in an AIDS patient with cytomegalovirus retinitis. <italic>Int J Infect Dis</italic> 2016;52:9–11.</mixed-citation></ref><ref id=\"B3\"><label>3</label><mixed-citation publication-type=\"other\">Agarwal M, Shrivastav A, Waris A. Tubercular retinal vasculitis mimicking frosted branch angiitis: a case report. <italic>J Ophthal Inflamm Infect</italic> 2018;8:3.</mixed-citation></ref><ref id=\"B4\"><label>4</label><mixed-citation publication-type=\"other\">de Aquino Ferreira BF, Rodriguez EEC, do Prado LL, Gonçalves CR, Hirata CE, Yamamoto JH. Frosted branch angiitis and cerebral venous sinus thrombosis as an initial onset of neuro-Behçet's disease: a case report and review of the literature. <italic>J Med Case Rep</italic> 2017;11:104.</mixed-citation></ref></ref-list></back></article>\n"
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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864063</article-id><article-id pub-id-type=\"pmc\">PMC7431714</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7451</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>The Long-term Visual Outcomes of Primary Congenital Glaucoma</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Esfandiari</surname><given-names>Hamed</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Prager</surname><given-names>Alisa</given-names></name><degrees>MD, MPH</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hassanpour</surname><given-names>Kiana</given-names></name><degrees>MD, MPH</degrees><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kurup</surname><given-names>Sudhi P.</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mets-Halgrimson</surname><given-names>Rebecca</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yoon</surname><given-names>Hawke</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zeid</surname><given-names>Janice Lasky</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mets</surname><given-names>Marilyn B.</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rahmani</surname><given-names>Bahram</given-names></name><degrees>MD, MPH</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Division of Ophthalmology, Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, USA</aff><aff id=\"I2\">\n<sup>2</sup>Department of Ophthalmology, Northwestern University Feinberg School of Medicine, Chicago, USA</aff><aff id=\"I3\">\n<sup>3</sup>Ophthalmic Research Center, Institutue for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>\nAlisa Prager, MD. Department of Ophthalmology,\nNorthwestern University Feinberg School of Medicine,\n645 N Michigan Ave., Chicago, Illinois 60611, USA.\nEmail: alisa.prager@gmail.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>326</fpage><lpage>330</lpage><history><date date-type=\"received\"><day>10</day><month>7</month><year>2019</year></date><date date-type=\"accepted\"><day>02</day><month>1</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Esfandiari et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><sec><title>Purpose</title><p>To evaluate the long-term visual outcomes of ab externo trabeculotomy for primary congenital glaucoma (PCG) at a single pediatric ophthalmology center.</p></sec><sec><title>Methods</title><p> In this retrospective single-center case series, data from 63 eyes of 40 patients who underwent ab externo trabeculotomy between September 2006 and June 2018 were included. The data were analyzed for best corrected visual acuity (BCVA), stereopsis, and surgical success. Kaplan–Meier analysis was performed using the surgical success criteria defined as intraocular pressure (IOP) <inline-formula><mml:math id=\"M1\"><mml:mo>≤</mml:mo></mml:math></inline-formula> 21 mmHg and <inline-formula><mml:math id=\"M2\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 20% below baseline without the need for additional glaucoma surgery.</p></sec><sec><title>Results</title><p>BCVA at the time of diagnosis was 0.37 <inline-formula><mml:math id=\"M3\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.48 logMAR, which changed to 0.51 <inline-formula><mml:math id=\"M4\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.56 logMAR at the final follow-up (<italic>P</italic> = 0.08). Twenty-five percent of patients had BCVA equal to or better than 20/40 at the final visit. The mean refraction at baseline was –4.78 <inline-formula><mml:math id=\"M5\"><mml:mo>±</mml:mo></mml:math></inline-formula> 5.87 diopters, which changed to less myopic refraction of –2.90 <inline-formula><mml:math id=\"M6\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.83 diopters at the final visit. Optical correction was prescribed in 66% of eyes at the final visit. The average final stereopsis was 395.33 sec of arc. The linear regression model showed a significant association between the surgery success rate and final BCVA as well as stereoacuity (<italic>P</italic>-values: 0.04 and 0.03, respectively). Intraocular pressure (IOP) decreased significantly from 29.79 <inline-formula><mml:math id=\"M7\"><mml:mo>±</mml:mo></mml:math></inline-formula> 7.67 mmHg at baseline to 16.13 <inline-formula><mml:math id=\"M8\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.41 mmHg at the final follow-up (<italic>P</italic> = 0.001).</p></sec><sec><title>Conclusion</title><p>Patients with PCG can achieve an acceptable visual acuity and stereoacuity, particularly in cases of timely intervention and close follow-up.</p></sec></abstract><kwd-group><kwd>Ab Externo Trabeculotomy</kwd><kwd> Long-term Outcomes</kwd><kwd> Primary Congenital Glaucoma</kwd><kwd> Stereopsis</kwd><kwd> Visual Function</kwd></kwd-group><counts><table-count count=\"1\"/><ref-count count=\"19\"/><page-count count=\"5\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> INTRODUCTION</title><p>Primary congenital glaucoma (PCG) is the most common type of childhood glaucoma and a major cause of blindness in children.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> PCG is secondary to angle dysgenesis and is primarily a surgical condition.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup> Surgical options include angle surgeries, trabeculectomy, glaucoma drainage devices, and cyclodestructive procedures in advanced cases.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup> Historically, the effect of angle surgeries on the intraocular pressure (IOP) has been considered the main outcome measure in most studies on PCG.</p><p>While surgical outcomes are thoroughly discussed in the literature, not much is known about the visual outcomes of glaucoma surgery in patients with PCG. Studies have shown that unilateral disease, poor vision at the time of diagnosis, multiple surgeries, and ocular comorbidities are associated with poor visual outcomes.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> Even after a timely intervention, these patients need treatment and monitoring for amblyopia, which necessitate frequent visits or examination under anesthesia. The final visual outcome is strongly related to early diagnosis and management of amblyopia. Frequent anesthesia or sedation, complexity of the disease, and the associated visual impairment could have significant impact on children's psychological behavior and make the assessment of visual function more complicated.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> Thus, the ultimate goal of childhood glaucoma management is lifelong control of IOP to maintain visual function.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup>\n</p><p>The purpose of this study was to report the long-term visual outcomes (including Snellen visual acuity and stereopsis) of patients with PCG, treated with ab externo trabeculotomy at a single center.</p></sec><sec sec-type=\"section\"><title> METHODS</title><p>This study was approved by the Institutional Review Board of the Ann & Robert H. Lurie Children's Hospital of Chicago. We followed the tenets of the Declaration of Helsinki and regulations of the Health Insurance Portability and Accountability Act.</p><p>Patients who underwent ab externo trabeculotomy for PCG at the Lurie Children's pediatric ophthalmology center between September 2006 and June 2018 were identified using current procedural terminology (CPT) codes and included in the study. We collected data such as preoperative IOP, baseline ocular biometric characteristics including axial length (AL), central corneal thickness (CCT), corneal diameter, presence of Haab's striae, number of preoperative glaucoma medications, type of surgery, and intra- and postoperative complications. The severity of glaucoma was assessed using three parameters: IOP, corneal diameter, and corneal clarity. Each parameter was given a score of 1–3, and the total score decided the severity of PCG: mild (1–3), moderate (4–6), or severe (7–9) PCG.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> At each postoperative visit, the best corrected visual acuity (BCVA), stereopsis, IOP, glaucoma medications, and complications were noted. Surgical success criteria were defined as IOP <inline-formula><mml:math id=\"M9\"><mml:mo>≤</mml:mo></mml:math></inline-formula> 21 mmHg and <inline-formula><mml:math id=\"M10\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 20% below the baseline without the need for additional glaucoma surgery (except ab externo trabeculotomy). The exclusion criteria were as follows: history of intraocular surgery, combined surgical procedures, any forms of anterior segment dysgenesis, and a follow-up of less than six-months.</p><p>Stereopsis was quantitatively assessed with the Titmus test (Stereo Optical Inc., Chicago, IL) with fly and graded circles. During the examination, children wore their correction under the Polaroid lenses, and the examiner used his index finger to guide fixation. Patients were asked to grab the wings of the fly and point out to the circle that seemed to “jump” out of the book. In the case of a wrong answer, the immediately preceding target was repeated. The last correct target identified was used as the child's stereopsis measurement. Vision was measured with age-appropriate methods and converted to logMAR for statistical analysis. All patients underwent complete ophthalmologic and orthoptic evaluation. Cycloplegic refraction were performed by instilling two drops of 1% cyclopentolate hydrochloride in each eye at 5-min intervals.</p><p>All surgeries were performed under general anesthesia. A fornix-based localized peritomy was created in the temporal or superior quadrant. Triangular or rectangular 3mm limbal base superficial scleral flap was fashioned, followed by a radial incision to expose the Schlemm canal under high magnification. Scleral cut-down was initiated from the blue zone up to the white zone until aqueous was seen oozing out from the cut ends of the canal. The Harms Trabeculotome was then passed into each end of the Schlemm canal to gently cut through the canal in the anterior chamber. The scleral flap and peritomy were sutured with Vicryl sutures.</p><p>All statistical analyses were performed using the SPSS software (SPSS Statistics for Windows, Version 25, IM Corp., Armonk, NY, USA). To compare the change in IOP, we used an interaction analysis within a linear mixed model. To evaluate the baseline differences, we used the <italic>T</italic>-test, Chi-Square, and Fisher's exact tests. Kaplan–Meier survival plots were constructed to assess the long-term survival rates; these were compared using the log-rank test. Linear regression analysis was used to evaluate the factors associated with surgical success.</p></sec><sec sec-type=\"section\"><title> RESULTS</title><p>Sixty-three eyes of 40 patients were included in this study. The mean age at diagnosis was 6.8 <inline-formula><mml:math id=\"M11\"><mml:mo>±</mml:mo></mml:math></inline-formula> 10.6 months and 62.5% of the patients were male. The mean follow-up time was 85.7 <inline-formula><mml:math id=\"M12\"><mml:mo>±</mml:mo></mml:math></inline-formula> 32.9 months (Table 1). The preoperative BCVA was 0.37 <inline-formula><mml:math id=\"M13\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.48, which changed to 0.51 <inline-formula><mml:math id=\"M14\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.56 logMAR at the final follow-up (<italic>P</italic> = 0.08). The baseline visual acuity was measured with fixation and following method in 27 eyes, Teller acuity card in 30, LEA symbols in 4, and HOTV in 2. Twenty-five percent of patients had BCVA <inline-formula><mml:math id=\"M15\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 20/40 at the final visit. The mean refraction of the patients was –4.78 <inline-formula><mml:math id=\"M16\"><mml:mo>±</mml:mo></mml:math></inline-formula> 5.87 diopter at baseline, which changed to less myopic refraction of –2.90 <inline-formula><mml:math id=\"M17\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.83 diopter at the final visit. Sixty-six percent of eyes were prescribed optical correction at the final visit. The average final stereopsis was 395.33 sec of arc (range, 40–800). The linear regression model showed a significant association between surgical success and final BCVA as well as stereoacuity (<italic>P</italic>-values: 0.04 and 0.03, respectively). The final stereoacuity corresponded to the final BCVA of 0.61 logMAR. The average baseline IOP was 29.7 <inline-formula><mml:math id=\"M18\"><mml:mo>±</mml:mo></mml:math></inline-formula> 7.6 mmHg, which decreased to 16.1 <inline-formula><mml:math id=\"M19\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.4 mmHg at the final visit, corresponding to 44% reduction from the baseline. The Kaplan–Meier survival curves indicated a time to failure of 107.7 <inline-formula><mml:math id=\"M20\"><mml:mo>±</mml:mo></mml:math></inline-formula> 9.04 months. Among the baseline parameters, only age at diagnosis of less than three months was associated with a higher failure rate. Of the 63 eyes, 21 met the criteria for mild, 29 for moderate, and 13 for severe PCG. While mild PCGs had significantly better visual acuity and stereopsis at the final follow-up (<italic>P </italic>\n<inline-formula><mml:math id=\"M21\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05), there was no significant difference between the moderate and severe groups. Thirty-five eyes (56%) underwent repeat trabeculotomy to treat a different area of the trabecular meshwork because of inadequately controlled IOP after the first session. Additional glaucoma surgery (glaucoma shunt procedure) was performed in seven patients.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Baseline characteristics of the patients.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Clinical characteristics (Range)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Gender</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25 (62.5%)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Unilateral disease</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Number (%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">17 (42.5%)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Age at the time of diagnosis</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean <inline-formula><mml:math id=\"M22\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD (Months)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.89 <inline-formula><mml:math id=\"M23\"><mml:mo>±</mml:mo></mml:math></inline-formula> 10.68 (0 – 63)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Follow-up</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean <inline-formula><mml:math id=\"M24\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD (Months)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">85.74 <inline-formula><mml:math id=\"M25\"><mml:mo>±</mml:mo></mml:math></inline-formula> 32.95 (3 – 156)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">IOP</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean <inline-formula><mml:math id=\"M26\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD (mmHg)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29.79 <inline-formula><mml:math id=\"M27\"><mml:mo>±</mml:mo></mml:math></inline-formula> 7.67 (11 – 54)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">C/D ratio</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean <inline-formula><mml:math id=\"M28\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.55 <inline-formula><mml:math id=\"M29\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.22 (0.2 – 0.95)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Corneal diameter</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean <inline-formula><mml:math id=\"M30\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD (mm)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.79 <inline-formula><mml:math id=\"M31\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.26 (9.50 – 15.5)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Central corneal thickness</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean <inline-formula><mml:math id=\"M32\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD (Microns)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">585.44 <inline-formula><mml:math id=\"M33\"><mml:mo>±</mml:mo></mml:math></inline-formula> 76.57 (470 – 761)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Axial length</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean <inline-formula><mml:math id=\"M34\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD (mm)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22.37 <inline-formula><mml:math id=\"M35\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.50 (17.50 – 28.50)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Refraction</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mean <inline-formula><mml:math id=\"M36\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD (Diopters)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–4.78 <inline-formula><mml:math id=\"M37\"><mml:mo>±</mml:mo></mml:math></inline-formula> 5.87 (–25.0 – +5.50)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Time of diagnosis by category</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M38\"><mml:mo>≤</mml:mo></mml:math></inline-formula> 3 Months</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18 (31%)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">3 <inline-formula><mml:math id=\"M39\"><mml:mo><</mml:mo></mml:math></inline-formula> Age <inline-formula><mml:math id=\"M40\"><mml:mo>≤</mml:mo></mml:math></inline-formula> 6 months</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">26 (48%)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M41\"><mml:mo>></mml:mo></mml:math></inline-formula> 6 months</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14 (24.1%)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ocular signs</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Presence of Haab striae</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">45 (72.6%)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Buphthalmos</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">30 (83.3%)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ocular symptoms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Tearing</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">30 (93.8%)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Photophobia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">24 (82.8%)</td></tr><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">SD, standard deviation; IOP, intraocular pressure; C/D ratio, cup to disc ratio</td></tr></tbody></table></table-wrap></sec><sec sec-type=\"section\"><title> DISCUSSION</title><p>The main goal of our study was to determine the long-term visual outcome of ab externo trabeculotomy for PCG. In our study, ab externo trabeculotomy resulted in 44% reduction in the IOP with a long-term success rate of 65%. Our success rate is consistent with those in previous reports, which ranged from 45% to 85%.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>,<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> The final visual acuity in our study was 0.51 logMAR and 25% of the patients had corrected vision better than 20/40 at the final visit. The visual outcome in our study is comparable to that of other reports with similar demographic data.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>,<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> However, we found better final visual function than that reported previously in the AlDarrab study in which 55% of the patients had BCVA <inline-formula><mml:math id=\"M42\"><mml:mo><</mml:mo></mml:math></inline-formula> 20/60.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> Poor visual outcomes in their study could be explained by more severe glaucoma population, high prevalence of CYP1B1 mutation, delayed diagnosis, and inadequate follow-up.<sup>[<xref rid=\"B12\" ref-type=\"bibr\">12</xref>]</sup>\n</p><p>Poor vision in children with PCG is multifactorial; high IOP in early life causes structural changes in the eye such as globe enlargement, corneal edema and opacity, tears in the Descemet's membrane (Haab's striae), high refractive error, anisometropia, and glaucomatous optic neuropathy<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>,<xref rid=\"B14\" ref-type=\"bibr\">14</xref>,<xref rid=\"B15\" ref-type=\"bibr\">15</xref>]</sup>. Corneal edema was present in almost 55% of patients at the time of diagnosis, which resolved after surgery in all cases, but accompanying Haab's striae, with or without corneal edema, persisted in two-third of our patients. Myopic refraction was the most frequent refractive error at baseline, which decreased with IOP reduction. The correlation between myopia and glaucoma can be attributed to the increase in the axial diameter of the eye as a consequence of high IOP.<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup> Sixty-six percent of our patients needed optical correction for myopia or myopic astigmatism at the final visit. Astigmatism is usually related to asymmetric expansion of the anterior chamber, corneal scar, and Haab's striae. In most cases, only optical correction was prescribed, but in 7% of the eyes (5 eyes) optical devices were needed. Telescopic systems (angular magnification) were the most common aids used for improving vision. Lower magnifications are beneficial in constricted visual field and low image illumination.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>]</sup> No patient needed optical aid for near vision, probably due to the large range of accommodation in children. Optical devices can improve the visual function and promote daily activities; therefore, they should be employed as early as possible.<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>]</sup>\n</p><p>We did not find any association between the age at diagnosis and final visual acuity. This finding is in contrast to other studies that showed a close relationship between age at PCG diagnosis and visual function.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>]</sup> However, we found a positive association between IOP control and final visual function. It is likely that timely intervention in our cohort and close follow-up reversed the detrimental effect of high IOP on the function and structure of the eye.</p><p>The average final stereopsis in our study was 395.33 sec of arc. The relationship between visual acuity of the amblyopic eye and stereoacuity is complex.<sup>[<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup> In general, worse visual acuity is associated with worse stereoacuity; however, it largely depends on the etiology of amblyopia; strabismic amblyopes have the worst stereoacuity, while anisometropic amblyopes retain some stereopsis.<sup>[<xref rid=\"B17\" ref-type=\"bibr\">17</xref>]</sup> Furthermore, the presence of stereoacuity in patients with PCG can improve the outcomes of amblyopia treatment.<sup>[<xref rid=\"B18\" ref-type=\"bibr\">18</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup> The results of our study show that while stereoacuity is not normal, it is nevertheless functional in many patients.</p><p>Our study was limited by its retrospective nature, variable follow-up duration, and multiple examiners. Additionally, this study was conducted at a single tertiary academic referral center, and the results may not be generalizable to other practice facilities. Baseline visual acuity measurement was not optimal due to age, which made the comparison of pre- and post-surgery measurements less reliable. However, the aim of our study was to present the long-term outcomes of visual function of PCG. Such data for visual acuity are scarce in the literature and there has been no study on stereopsis outcome in PCG.</p><p>In summary, the result of our study showed that patients with PCG can achieve an acceptable visual acuity and stereoacuity with timely intervention and close follow-up. Although our study was not designed to compare the visual function changes after ab externo trabeculotomy, we did not observe any improvement in visual acuity as this parameter could be reliably measured after the procedure. This lack of significant improvement should be discussed preoperatively to gauge the expectations.</p></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>None.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Stamper RL, Lieberman MF, Drake MV, Becker B. Becker-Shaffer’s diagnosis and therapy of the glaucomas. 8th ed. St. Louis: Mosby Elsevier, 2009.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">Esfandiari H, Taranum Basith SS, Kurup SP, Mets-Halgrimson R, Hassanpour K, Yoon H, et al. 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] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864072</article-id><article-id pub-id-type=\"pmc\">PMC7431715</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7460</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Case Report</subject></subj-group></article-categories><title-group><article-title>Orbital Cellulitis Following Uncomplicated Glaucoma Drainage Device Surgery: Case Report and Review of Literature</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Zheng</surname><given-names>Cindy X.</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Uhr</surname><given-names>Joshua H.</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Deaner</surname><given-names>Jordan D.</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Anhalt</surname><given-names>John</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Lin</surname><given-names>Michael M.</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Moster</surname><given-names>Stephen J.</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Razeghinejad</surname><given-names>Reza</given-names></name><degrees>MD</degrees></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Glaucoma Research Center, Wills Eye Hospital, Philadelphia, PA, USA</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Cindy X. Zheng, MD. Wills Eye Hospital Glaucoma\nResearch Center, 840 Walnut St., Suite 1140,\nPhiladelphia, PA 19107, USA.\nE-mail: cindyzheng9@gmail.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>412</fpage><lpage>418</lpage><history><date date-type=\"received\"><day>29</day><month>5</month><year>2019</year></date><date date-type=\"accepted\"><day>31</day><month>8</month><year>2019</year></date></history><permissions><copyright-statement>Copyright © 2020 Zheng et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><sec><title>Purpose</title><p>Orbital cellulitis (OC) is a rare postoperative complication of glaucoma drainage device (GDD) implantation. To date, there have only been 10 reported cases of OC following GDD implantation.</p></sec><sec><title>Case Report</title><p>Here, we report a case of OC in a 57-year-old man who developed pain, proptosis, and limited extraocular motility two days after uneventful Ahmed FP7 implantation in the right eye. Contrast-enhanced computed tomography of the orbits demonstrated fat stranding and a small fluid collection, consistent with OC. He had minimal improvement with intravenous antibiotics and ultimately underwent GDD explantation. A systematic review of the literature showed that the development of OC following GDD implantation can occur in the early or late postoperative period. Immediate hospitalization with intravenous administration of broad-spectrum antibiotics is recommended. Explantation of the infected GDD is often required for source control.</p></sec><sec><title>Conclusion</title><p>OC is a rare postoperative complication of GDD implantation. Prompt evaluation and treatment are required, often combined with GDD explantation.</p></sec></abstract><kwd-group><kwd>Ahmed Tube Shunt</kwd><kwd> Orbital Cellulitis</kwd><kwd> Glaucoma Drainage Device</kwd></kwd-group><counts><fig-count count=\"2\"/><table-count count=\"3\"/><ref-count count=\"14\"/><page-count count=\"7\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> INTRODUCTION</title><p>Glaucoma drainage devices (GDDs) are surgical devices commonly implanted in eyes with refractory glaucoma. The development of orbital cellulitis (OC) following GDD implantation is rare, with only 10 reported cases in the literature.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>,<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B8\" ref-type=\"bibr\">8</xref>,<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> Here, we report a case of OC following the placement of an Ahmed FP7 (New World Medical, Rancho Cucamonga, CA) in a 57-year-old man who showed minimal improvement with intravenous (IV) antibiotics and ultimately underwent GDD explantation.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>External photograph of the affected eye on the day of presentation, demonstrating periorbital edema, erythema, conjunctival injection, and chemosis.</p></caption><graphic xlink:href=\"jovr-15-412-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Contrast-enhanced computed tomography of the orbits showing findings consistent with orbital cellulitis, including proptosis of the right globe, thickening of the sclera and optic nerve insertion, small superior fluid collection, and mild anterior retrobulbar fat stranding (A). An Ahmed tube shunt and Baerveldt tube shunt are visualized in the right and left globes, respectively (B).</p></caption><graphic xlink:href=\"jovr-15-412-g002\"/></fig></sec><sec sec-type=\"section\"><title> CASE REPORT</title><p>A 57-year-old incarcerated man with advanced primary open-angle glaucoma was referred to us because of poorly controlled intraocular pressure (IOP). He had a surgical history of bilateral trabeculectomy and implantation of a Baerveldt tube shunt implanted in the left eye approximately nine years ago. He had a medical history of gastroesophageal reflux disease and was not on any routine medications other than his glaucoma medications. On presentation, his visual acuity (VA) was 20/200 in the right eye and light perception in the left eye. His IOP was 13 mmHg in the right eye and 12 mmHg in the left eye on maximum topical therapy and oral acetazolamide. Due to difficulty tolerating acetazolamide, he agreed to proceed with Ahmed FP7 implantation in the right eye. GDD was implanted uneventfully with Tutoplast processed sclera patch graft (Katena Products Inc., Denville, NJ) in the superonasal quadrant because of conjunctival scarring from prior trabeculectomy. No intraoperative injections or mitomycin C were given. On postoperative day 1, he had a VA of 20/200 and an IOP of 10 mmHg, and the tube shunt was covered and well-positioned.</p><p>The patient presented emergently on postoperative day 4 because of two days of right eye pain, swelling, and blurry vision. He reported that he did not receive his postoperative topical ofloxacin or prednisolone acetate drops from his facility. VA was hand motion and IOP was 20 mmHg. Externally, the right orbit was tense with lid erythema and edema. His right globe was proptotic with limited extraocular motility (Figure 1). There was a small opening in the conjunctiva over the patch graft, located 4 mm posterior to the limbus. A sample of purulent drainage from this opening was swabbed and sent for microbiologic testing. The anterior chamber was deep with rare cells. There was no vitritis. Contrast-enhanced computed tomography (CT) demonstrated soft-tissue thickening, fatty infiltration, and a small fluid collection superiorly (Figure 2).</p><p>He was admitted for administration of IV vancomycin and piperacillin-tazobactam and topical fortified vancomycin and tobramycin. On hospitalization day 2, he received 8 mg of IV dexamethasone. Improvement was minimal with the administration of IV antibiotics for 24 hours; therefore, surgical explantation of the GDD was performed.</p><p>Intraoperatively, there was an area of conjunctival melt over the tube with pockets of purulent material surrounding the valve. To prevent intraocular penetration of the infected material into the anterior chamber, a purse-string suture was passed around the tube entry site in the sclera and was tied-off while a surgical assistant withdrew the tube. The plate and tube were noted to be completely free, presumably because of the surrounding scleritis. The implant was removed, and the area was copiously irrigated with vancomycin and ceftazidime solution. After conjunctival closure with 8-0 Vicryl sutures, subconjunctival injections of vancomycin and ceftazidime were administered. Considering the patient's monocular status with advanced glaucoma in the affected eye and a history of poorly controlled IOP, concomitant MicroPulse transscleral cyclophotocoagulation (Iridex Corp., Mountain View, CA) was performed for 140 sec to the inferior globe at a power of 2000mW and duty cycle of 31.3%.</p><p>Cultures showed light growth of methicillin-susceptible <italic>Staphylococcus aureus</italic> and <italic>Cutibacterium acnes</italic> (formerly <italic>Propionibacterium acnes</italic>). He was discharged two days after the tube shunt explantation with oral moxifloxacin 400 mg and topical fortified vancomycin and gatifloxacin.</p><p>Six months after the surgery, VA was hand motion and IOP was 12 mmHg with three topical glaucoma medications. He had complete resolution of orbital signs.</p></sec><sec sec-type=\"section\"><title> DISCUSSION</title><p>A systematic literature review revealed a total of 11 cases of OC following GDD surgery, including the present case (Table 1). Most patients presented within two days of symptom onset.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>,<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> On presentation, all patients had eyelid erythema and edema, and most patients had chemosis, proptosis or globe displacement, and limited extraocular motility.</p><p>The most common GDD associated with post-implantation OC was the Ahmed valve (n = 7),<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>,<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup> although Molteno,<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> Krupin–Denver,<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> and Baerveldt<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>,<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> implants have also been associated with post-implantation OC. In seven cases, symptoms of OC started in the immediate postoperative period (<inline-formula><mml:math id=\"M1\"><mml:mo>≤</mml:mo></mml:math></inline-formula> 3 months after the surgery).<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>,<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> In the other four cases, OC developed after the postoperative month 3.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> In three of the four cases of delayed-onset OC,<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> the tube was exposed, presumably serving as a conduit for bacteria to travel from the ocular surface into the orbit. In one case of delayed-onset OC, the tube was not specifically exposed; however, the patient had concurrent endophthalmitis.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup> The authors theorized that organisms may have gained entry into the eye from the ocular surface and OC from drainage via the tube.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup>\n</p><p>Table 2 summarizes the management of OC. CT is the imaging modality of choice for OC and was the most common modality used.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>]</sup> All patients were hospitalized and administered IV antibiotics. Although the choice of antibiotic varied, the consensus was to start with broad-spectrum coverage. In the present case, vancomycin was used owing to previous studies showing a high prevalence of methicillin-resistant <italic>Staphylococcus aureus</italic> isolated from ocular infections.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> The antibiotic coverage changed based on infectious disease consultation or culture sensitivities. Topical antibiotics were commonly used.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup>\n</p><p>Although not routinely administered, IV steroids were used in two cases after the administration of IV antibiotics for 24 hours, including our case.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> We administered steroids under the guidance of oculoplastic consultation to reduce orbital inflammation and to improve the ease of access during GDD explantation. Previous studies have shown that steroids can help reduce the cytokine load and improve outcomes in bacterial OC.<sup>[<xref rid=\"B12\" ref-type=\"bibr\">12</xref>,<xref rid=\"B13\" ref-type=\"bibr\">13</xref>]</sup>\n</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Demographics, baseline clinical characteristics, and initial presentation of orbital cellulitis after glaucoma drainage device implantation</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"14\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Case No.</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Author</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Age</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Gender</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Glaucoma diagnosis</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>GDD Type</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Location</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Baseline VA</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Presenting VA</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Interval<inline-formula><mml:math id=\"M2\"><mml:msup><mml:mrow/><mml:mo>*</mml:mo></mml:msup></mml:math></inline-formula></bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Duration<inline-formula><mml:math id=\"M3\"><mml:msup><mml:mrow/><mml:mo>†</mml:mo></mml:msup></mml:math></inline-formula></bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Chemosis</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Proptosis</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>EOM limitation</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Karr<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Congenital</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Molteno</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">ST</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 m</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Yes</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Chaudhry<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">F</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Congenital</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Krupin–Denver</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">ST</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CF</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">LP</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Yes</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Chaudhry</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">F</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Congenital</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ahmed model NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">FF</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8 m</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Yes</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Kassam<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Congenital</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ahmed FP7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8 m</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NR</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Farid<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">F</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Congenital</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ahmed model NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 m</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M4\"><mml:mo><</mml:mo></mml:math></inline-formula> 1 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Yes</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Esporcatte<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Congenital</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ahmed FP7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 m</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NR</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Marcet<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">44</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Uveitic</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ahmed model NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Superior</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CF</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CF</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M5\"><mml:mo><</mml:mo></mml:math></inline-formula> 1d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Yes</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Goldfarb<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">81</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">F</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">POAG</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ahmed model NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">ST</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20/200</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CF</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M6\"><mml:mo><</mml:mo></mml:math></inline-formula> 1d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Yes</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Zheng</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">57</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">POAG</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ahmed FP7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">SN</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20/200</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">HM</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Yes</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Beck<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">53</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">POAG</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Baerveldt 350 mm<inline-formula><mml:math id=\"M7\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20/32</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20/60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 m</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Yes</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Lavina<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">78</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">F</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CACG</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Baerveldt 350 mm<inline-formula><mml:math id=\"M8\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20/400</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NLP</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15 m</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Yes</td></tr><tr><td align=\"left\" colspan=\"13\" rowspan=\"1\">\n<inline-formula><mml:math id=\"M9\"><mml:msup><mml:mrow/><mml:mo>*</mml:mo></mml:msup></mml:math></inline-formula>Interval of time between GDD implantation and presentation of OC\n<inline-formula><mml:math id=\"M10\"><mml:msup><mml:mrow/><mml:mo>†</mml:mo></mml:msup></mml:math></inline-formula>Duration of symptoms prior to presentation\nGDD, glaucoma drainage device; VA, visual acuity; EOM, extraocular movement; M, male; F, female; ST, superotemporal; SN, superonasal; NR, not reported; CF, count fingers; HM, hand motion; LP, light perception; NLP, no light perception; FF, fixes and follows; d, days; m, months</td></tr></tbody></table></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Medical and surgical management of orbital cellulitis after glaucoma drainage device implantation</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"13\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Case No.</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>IV Antibiotics</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Topical antibiotics</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Oral antibiotics, duration</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Tube erosion</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Tube explant</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Time to explant</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Intraoperative antibiotics</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Blood culture</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Endo-phtha-lmitis</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Intravitreal antibiotics</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Culture site</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Culture organism</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Cefuroxime</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Tobramycin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y, NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Irrigation with gentamicin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Neg</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C, D, GDD</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Group A streptococcus, staphylococcus epidermidis</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Gentamicin, cefazolin, switched to flucloxacillin, cefotaxime</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Gentamicin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ceclor for 10 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ceftriaxone, gentamicin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Vancomycin, gentamicin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ceclor for 10 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">IC and SC vancomycin and amikacin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">AC, D</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No growth</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Vancomycin, ceftazidime</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Gatifloxacin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Levofloxacin for 21 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">SC gentamicin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Neg</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Vancomycin, ceftazidime</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C, D, V, S</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Streptococcus pneumoniae</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Vancomycin, ceftazidime, metronidazole</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Moxifloxacin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Irrigation with gentamicin, Povidone; SC vancomycin, ceftazidime</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Neg</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Vancomycin, ceftazidime</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GDD, V</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No growth</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Vancomycin, cefepime</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Gatifloxacin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Amoxicillin- clavulanic acid for 10 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Same day</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Sub-Tenon's vancomycin, ceftazidime</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Neg</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Vancomycin, ceftazidime</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GDD, V</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Staphylococcus epidermidis</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ampicillin-sulbactam</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ciprofloxacin (duration NS)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Vancomycin, ceftriaxone, switched to piperacillin-tazobactam</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Moxifloxacin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ciprofloxacin (duration NS)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">SC vancomycin, ceftazidime</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Neg</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GDD</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Pseudomonas aeruginosa</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Vancomycin, piperacillin-tazobactam</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Vancomycin, tobramycin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Moxifloxacin for 7 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Irrigation with vancomycin, ceftazidime; SC vancomycin, ceftazidime</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C, D</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Staphylococcus aureus, cutibacterium acnes</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Amoxicillin-clavulanic acid</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Levofloxacin</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Flucloxacillin and amoxicillin-clavulanic acid in 10 d</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No growth</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ampicillin-sulbactam</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No growth</td></tr><tr><td align=\"left\" colspan=\"13\" rowspan=\"1\">NS, not specified; NR, not reported; Y, yes; N, no; d, day; Neg, negative; GDD, glaucoma drainage device; IC, intracameral; C, conjunctiva; D, discharge; AC, anterior chamber; V, vitreous; S, sutures; NA not applicable</td></tr></tbody></table></table-wrap><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><p>Outcomes and additional surgical intervention after resolution of orbital cellulitis after glaucoma drainage device implantation</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"6\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Case No.</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Follow-up</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Visual acuity</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Intraocular pressure</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Complication</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Additional surgical intervention</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 year</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Hand motions</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 years</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20/60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 month</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Locate candy bars at 8 inches</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Retinal detachment</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 retina surgeries</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 month</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Fixes and follows</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Elevated intraocular pressure</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cyclophotocoagulation</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Phthisis bulbi</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Phthisis bulbi</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NR</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13 months</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20/200</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Re-exposure of GDD</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Revision of GDD</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 months</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20/100</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6 months</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Hand motions</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cyclophotocoagulation at same time as GDD explantation</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 months</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20/32</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Elevated intraocular pressure</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cyclophotocoagulation</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Count fingers</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">NR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">NA</td></tr><tr><td align=\"left\" colspan=\"6\" rowspan=\"1\">NR, not reported; NA, not applicable; GDD, glaucoma drainage device</td></tr></tbody></table></table-wrap><p>Blood cultures were negative in all tested cases.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup> Presumably, the infection remained localized to the orbit. Since all cases presented within three days of symptom onset, the infection was rapidly managed with antibiotics, thus, bacteremia was less likely to occur.</p><p>The GDD was surgically explanted in 8 of the 11 cases,<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> including all five cases of tube exposure<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>][<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> and all three cases of concurrent endophthalmitis.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> Presumably, erosion allowed bacteria to seed the GDD, and the infection may be difficult to clear without explanting the infected GDD. Explantation was performed in all children aged less than three years.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>][<xref rid=\"B2\" ref-type=\"bibr\">2</xref>][<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> In this age group, it is difficult to perform an examination without the use of general anesthesia; therefore, it may be safer to perform explantation at the initial examination than to subject the patient to repeated episodes of anesthesia. In all cases of explantation, GDDs were most frequently explanted within one to two days of presentation, suggesting that failure to respond to antibiotics can be quickly identified. During explantation, the area surrounding the tube was irrigated with antibiotics,<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup> or subconjunctival or sub-Tenon's injection of antibiotics were performed.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>][<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>][<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup> In three cases, there was a sufficient improvement with IV antibiotics alone; consequently, no surgical interventions were undertaken.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>,<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup>\n</p><p>When IV antibiotics were transitioned to oral antibiotics, fluoroquinolones were most commonly used,<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>][<xref rid=\"B7\" ref-type=\"bibr\">7</xref>][<xref rid=\"B8\" ref-type=\"bibr\">8</xref>,<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> likely owing to their vitreous penetration and relatively broad coverage.<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup> The duration was most commonly 10 days, as seen in four cases.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> The duration of the therapy was likely associated with the severity of presentation and response to therapy. Table 3 details the outcomes and additional procedures that were performed.</p><p>In conclusion, OC is a rare postoperative complication of GDD implantation. Immediate hospitalization with administration of broad-spectrum IV antibiotics is recommended. Explantation of the GDD is often required for source control.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Karr DJ, Weinberger E, Mills RP. An unusual case of cellulitis associated with a Molteno implant in a 1-year-old child. <italic>J Pediatr Ophthalmol Strabismus</italic> 1990;27:107–10.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">Chaudhry IA, Shamsi FA, Morales J. Orbital cellulitis following implantation of aqueous drainage devices. <italic>Eur J Ophthalmol</italic> 2007;17:136–140.</mixed-citation></ref><ref id=\"B3\"><label>3</label><mixed-citation publication-type=\"other\">Kassam F, Lee BE, Damji KF. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864071</article-id><article-id pub-id-type=\"pmc\">PMC7431716</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7459</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Case Report</subject></subj-group></article-categories><title-group><article-title>Uveitis-induced Refractory Ocular Hypotony Managed with High-dose Latanoprost</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Ghassemi</surname><given-names>Fariba</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Niyousha</surname><given-names>Mohammad Reza</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hassanpoor</surname><given-names>Narges</given-names></name><degrees>MD, MPH</degrees><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"I4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Khojasteh</surname><given-names>Hassan</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Retina & Vitreous Service, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran</aff><aff id=\"I2\">\n<sup>2</sup>Retina & Vitreous Service, Nikookari Eye Hospital, Tabriz University of Medical Sciences, Tabriz, Iran</aff><aff id=\"I3\">\n<sup>3</sup>Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran</aff><aff id=\"I4\">\n<sup>4</sup>Department of Ophthalmology, Tabriz University of Medical Sciences, Tabriz, Iran</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Narges Hassanpoor MD, MPH. Ophthalmic Research\nCenter, Shahid Beheshti University of Medical Sciences,\n23 Paidar Fard, Bostan 9, Pasdaran Ave., Tehran, 16666,\nIran. Tel: +98-21-55421006 Fax: +98-21-55416134\nEmail: nargeshassanpoor@gmail.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>408</fpage><lpage>411</lpage><history><date date-type=\"received\"><day>15</day><month>3</month><year>2019</year></date><date date-type=\"accepted\"><day>14</day><month>8</month><year>2019</year></date></history><permissions><copyright-statement>Copyright © 2020 Ghassemi et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><sec><title>Purpose</title><p> To report a case of refractory ocular hypotony due to chronic Behcet's disease with good response to high-dose topical latanoprost.</p></sec><sec><title>Case Report</title><p>We present a 26-year-old man with a known history of Behcet's disease who developed decreasing vision and severe ocular hypotony that was refractory to multiple treatment modalities including subtenon triamcinolone acetonide, ibopamine, pars plana vitrectomy, and silicone oil injection. We decided to try high-dose topical latanoprost for the management of ocular hypotony based on recent reports. After six months, intraocular pressure (IOP) increased by 5 mm Hg, became stable at 7 mm Hg, and remained unchanged at month 24.</p></sec><sec><title>Conclusion</title><p>High-dose topical latanoprost could lead to significant increase in IOP in uveitis-induced refractory ocular hypotony.</p></sec></abstract><kwd-group><kwd>Behcet</kwd><kwd> Hypotony</kwd><kwd> Inflammation</kwd><kwd> Latanoprost</kwd><kwd> Uveitis</kwd></kwd-group><counts><fig-count count=\"1\"/><ref-count count=\"12\"/><page-count count=\"4\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> INTRODUCTION </title><p>Ocular hypotony is one of the complications of uveitis and causes substantial visual loss. It occurs because of hyposecretion of the ciliary body owing to inflammation and increased uveoscleral outflow. In the acute phase of the disease, it is usually reversible; and suppressing the inflammation by corticosteroids or other anti-inflammatory medications may restore the intraocular pressure (IOP). However, in chronic uveitis, long-term inflammation can lead to structural changes such as growth of tractional membranes on the ciliary body and atrophic changes resulting in long-lasting or irreversible hypotony. Chronic ocular hypotony is associated with sight-threatening complications including hypotony maculopathy, choroidal folds, and optic nerve edema.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup> Ocular hypotony is defined as IOP <inline-formula><mml:math id=\"M1\"><mml:mo><</mml:mo></mml:math></inline-formula> 8 mm Hg, and most complications arise when IOP is <inline-formula><mml:math id=\"M2\"><mml:mo><</mml:mo></mml:math></inline-formula> 4 mm Hg.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup>\n</p></sec><sec sec-type=\"section\"><title> CASE REPORT</title><p>In this report, we present a 26-year-old male with a past medical history of Behcet's disease who developed progressive vision loss and severe hypotonia. He had received 15 mg of methotrexate weekly and 7.5 mg of prednisolone daily as well as multiple injections of subtenon triamcinolone acetonide (TA; 40 mg). He had also undergone phacoemulsification and posterior chamber intraocular lens placement for cataract in his both eyes. Pars plana vitrectomy with silicone oil injection was performed in his right eye for hypotony. Visual acuity was 20/400 in his right eye and “hand motion” in his left eye. Ocular hypotony persisted despite all these treatments in the absence of active inflammation. Corneal folds and band keratopathy were noted after few weeks. Fundus was poorly visible but it was remarkable for cystic changes in the macular region. B-scan showed a significant serous choroidal detachment due to severe hypotony in both eyes. To increase the IOP, multiple injections of 40 mg of subtenon and 4 mg of intravitreal TA were administered; however, no improvement was observed in vision, IOP status, and serous choroidal detachment. Visual acuity deteriorated because of persistent hypotony maculopathy. Ibopamine (a dopamine agonist) eye drops were used for three months with an increase in IOP of 2 mm Hg in both the eyes, but no change in vision was detected.</p><p>We discussed the details of our experimental treatment based on published studies with the patient and proceeded with the treatment after obtaining a written consent. Subsequently, high-dose latanoprost eye drops (XALATAN, 0.005%, Pfizer) were administered every 6 hours in both eyes.</p><p>One month later, IOP increased to 4 mm Hg, and at two months, to 7 mm Hg. After two months of latanoprost treatment, we performed a drug rechallenge test by discontinuing latanoprost for four weeks and then resuming the drug to prove its effect on IOP. After 6 months, IOP was stable at 7 mm Hg and remained unchanged even after 24 months. B-scan showed significant improvement in hypotony maculopathy and fluid resolved subretinally (Figures 1B and D). The patient's vision improved to 20/200 in his right eye and “finger counting” at 1.5 m in his left eye.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>(A) Right eye B-scan before latanoprost treatment. (B) Serous choroidal detachment resolution after treatment with high-dose latanoprost in the right eye. (C) Left eye B-scan before latanoprost treatment. (D) Serous choroidal detachment improvement after treatment in the left eye.</p></caption><graphic xlink:href=\"jovr-15-408-g001\"/></fig></sec><sec sec-type=\"section\"><title> DISCUSSION</title><p>Chronic ciliary body inflammation leads to traction on the ciliary body, atrophic changes of this tissue, and subsequent ocular hypotony. Serous choroidal detachment can occur because of chronic hypotony. If ciliary body traction or detachment is visible on ultrasound biomicroscopy, vitrectomy and membranectomy with or without silicone oil injection may be used to improve the IOP. For our patient, although we performed a vitrectomy with silicone oil injection in the right eye, IOP did not change. In such cases, the rise in IOP may be temporary and mandate reinjection of silicone oil in approximately 50% of patients.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup> Ibopamine is a dopamine agonist that can increase aqueous humor secretion and IOP. Administration of ibopamine eye drops increases the IOP by approximately 2 mm Hg.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> In our case, ibopamine improved IOP to a barely measurable 2 mm Hg. Subtenon and intravitreal injections of TA or systemic corticosteroids may increase IOP in chronic hypotony.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>,<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup> However, these treatments did not improve results in our patient.</p><p>Prostaglandins (PGs) play an important role in aqueous humor dynamics. Latanoprost is a selective PGF<inline-formula><mml:math id=\"M3\"><mml:msub><mml:mrow/><mml:mn>2</mml:mn></mml:msub></mml:math></inline-formula> receptor agonist that can reduce IOP by increasing aqueous outflow mostly by increasing the uveoscleral outflow. It is a potent antiglaucoma treatment when applied once daily.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> However, when administered topically at high concentrations or intracamerally, PGs E and F can cause miosis and raise the IOP. At the same time, they increase the protein content and white blood cells in the aqueous humor.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup>\n</p><p>In chronic uveitis, ciliary body atrophy and consequent aqueous hyposecretion may be difficult to overcome; and decreasing aqueous outflow may be a better way to restore the IOP. High-dose latanoprost may cause some grades of inflammation in trabecular meshwork and reduce the aqueous outflow. In cases with no tractional membranes on the ciliary body, it may be more practical to increase the uveoscleral outflow. This could be a mechanism by which high-dose latanoprost can raise the IOP in similar cases as our patient. Another less possible mechanism could be improvement of the serous choroidal detachment due to an increase in uveoscleral outflow.</p><p>In a report of three cases with uveitic glaucoma, latanoprost had a paradoxical impact on IOP.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>]</sup> Latanoprost significantly increased IOP, and a latanoprost rechallenge test—performed by discontinuing and then continuing the drug—proved that latanoprost could increase the IOP. All patients had uveitis; thus, it can be postulated that in the presence of a severely damaged blood-ocular barrier, latanoprost can have a paradoxical impact on IOP. In that report, latanoprost was administered at a therapeutic once daily dose, and it may be assumed that higher doses, as in our case, may have a more prominent impact on IOP. Higher doses of latanoprost may result in more PG release from the impaired blood-ocular barrier and trabecular inflammation, which can overcome the increase in uveoscleral aqueous outflow and result in raised IOP.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>]</sup>\n</p><p>Latanoprost has reportedly been used in uveitic patients without the risk of central macular edema or recurrence of anterior uveitis.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> In patients with anterior and intermediate uveitis, low-dose (once daily) latanoprost did not show higher rates of inflammation recurrence when compared to a fixed combination of dorzolamide and timolol.<sup>[<xref rid=\"B12\" ref-type=\"bibr\">12</xref>]</sup> However, similar findings have not been seen in patients with severe posterior uveitis due to Behcet's disease. Therefore, despite it appearing paradoxical to use latanoprost as an IOP-recovering agent in a uveitic patient, its effect might be dependent on the type, severity, and chronicity of uveitis.</p><p>It can be concluded that, in some cases, high-dose latanoprost can be administered as an adjuvant treatment for refractory hypotony due to chronic inflammation. Prospective clinical trials to further investigate this can be beneficial.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">de Smet MD, Gunning F, Feenstra R. The surgical management of chronic hypotony due to uveitis. <italic>Eye</italic> 2005;19:60–64.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">Tran VT, Mermoud A, Herbort CP. Appraisal and management of ocular hypotony and glaucoma associate with uveitis. <italic>Int Ophthalmol Clin</italic> 2000;40:175–203.</mixed-citation></ref><ref id=\"B3\"><label>3</label><mixed-citation publication-type=\"other\">Kapur R, Birnbaum AD, Goldstein DA, Tessler HH, Shapiro MJ, Ulanski LJ, et al. Treating uveitis-associated hypotony with pars plana vitrectomy and silicone oil injection. <italic>Retina</italic> 2010;30:140–145.</mixed-citation></ref><ref id=\"B4\"><label>4</label><mixed-citation publication-type=\"other\">Pederson JE. Hypotony. In: Tasman W, Jaeger, EA, editors. Duane's clinical ophthalmology. Philadelphia, PA: Lippincott-Raven; 1999; 1–8.</mixed-citation></ref><ref id=\"B5\"><label>5</label><mixed-citation publication-type=\"other\">Ugahary Le, Ganteris E, Veckeneer M, Cohen AC, Jansen J, Mulder PG, et al. Topical ibopamine in the treatment of chronic ocular hypotony attributable to vitreoretinal surgery, uveitis, or penetrating trauma. <italic>Am J Ophthalmol</italic> 2006;141:571–573.</mixed-citation></ref><ref id=\"B6\"><label>6</label><mixed-citation publication-type=\"other\">Sen HN, Drye LT, Goldstein DA, Larson TA, Merrill PT, Pavan PR, et al. Hypotony in patients with uveitis: the Multicenter Uveitis Steroid Treatment (MUST) trial. <italic>Ocul Immunol Inflamm</italic> 2012;20:104–112.</mixed-citation></ref><ref id=\"B7\"><label>7</label><mixed-citation publication-type=\"other\">Pederson JE, Mac Lellan HM. Medical therapy for experimental hypotony. <italic>Arch Ophthalmol</italic> 1982;100:815–817.</mixed-citation></ref><ref id=\"B8\"><label>8</label><mixed-citation publication-type=\"other\">Lim KS, Nau CB, O'Byrne MM, Hodge DO, Toris CB, McLaren JW, et al. Mechanism of action of bimatoprost, latanoprost, and travoprost in healthy subjects. A crossover study. <italic>Ophthalmology</italic> 2008;115:790–795e794.</mixed-citation></ref><ref id=\"B9\"><label>9</label><mixed-citation publication-type=\"other\">Abdel-Latif AA. Release and effects of prostaglandins in ocular tissues. <italic>Prostaglandins Leukot Essent Fatty Acids</italic> 1991;44:71–82.</mixed-citation></ref><ref id=\"B10\"><label>10</label><mixed-citation publication-type=\"other\">Sacca S, Pascotto A, Siniscalchi C. Ocular complications of latanoprost in uveitic glaucoma: three case reports. <italic>J Ocul Pharmacol Ther</italic> 2001;17:107–113.</mixed-citation></ref><ref id=\"B11\"><label>11</label><mixed-citation publication-type=\"other\">Chang JH, McCluskey P, Missotten T, Ferrante P, Jalaludin B, Lightman S. Use of ocular hypotensive prostaglandin analogues in patients with uveitis: does their use increase anterior uveitis and cystoid macular oedema? <italic>Br J Ophthalmol</italic> 2008;92:916–921.</mixed-citation></ref><ref id=\"B12\"><label>12</label><mixed-citation publication-type=\"other\">Markomichelakis NN, Kostakou A, Halkiadakis I, Chalkidou S, Papakonstantinou D, Georgopoulos G. Efficacy and safety of latanoprost in eyes with uveitic glaucoma. <italic>Graefes Arch Clin Exp Ophthalmol</italic> 2009;247:775–780.</mixed-citation></ref></ref-list></back></article>\n"
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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864068</article-id><article-id pub-id-type=\"pmc\">PMC7431717</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7456</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Review Article</subject></subj-group></article-categories><title-group><article-title>Conjunctivitis: A Systematic Review</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Azari</surname><given-names>Amir A.</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Arabi</surname><given-names>Amir</given-names></name><degrees>MD, MPH</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran</aff><aff id=\"I2\">\n<sup>2</sup>Department of Ophthalmology, Torfeh Medical Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Amir A. Azari, MD. Ophthalmic Research Center,\nResearch Institute for Ophthalmology and Vision\nScience, Shahid Beheshti University of Medical\nSciences, No. 23, Paidarfdard St., Boostan 9 St.,\nPasadaran Ave., Tehran 16666, Iran.\nEmail: amirazarimd@gmail.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>372</fpage><lpage>395</lpage><history><date date-type=\"received\"><day>18</day><month>2</month><year>2020</year></date><date date-type=\"accepted\"><day>25</day><month>4</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Azari and Arabi.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><p>Conjunctivitis is a commonly encountered condition in ophthalmology clinics throughout the world. In the management of suspected cases of conjunctivitis, alarming signs for more serious intraocular conditions, such as severe pain, decreased vision, and painful pupillary reaction, must be considered. Additionally, a thorough medical and ophthalmic history should be obtained and a thorough physical examination should be done in patients with atypical findings and chronic course. Concurrent physical exam findings with relevant history may reveal the presence of a systemic condition with involvement of the conjunctiva. Viral conjunctivitis remains to be the most common overall cause of conjunctivitis. Bacterial conjunctivitis is encountered less frequently and it is the second most common cause of infectious conjunctivitis. Allergic conjunctivitis is encountered in nearly half of the population and the findings include itching, mucoid discharge, chemosis, and eyelid edema. Long-term usage of eye drops with preservatives in a patient with conjunctival irritation and discharge points to the toxic conjunctivitis as the underlying etiology. Effective management of conjunctivitis includes timely diagnosis, appropriate differentiation of the various etiologies, and appropriate treatment.</p></abstract><kwd-group><kwd>Allergic</kwd><kwd> Bacterial</kwd><kwd> Conjunctivitis</kwd><kwd> COVID-19</kwd><kwd> Coronavirus</kwd><kwd> Viral</kwd><kwd> Toxic</kwd></kwd-group><counts><fig-count count=\"8\"/><table-count count=\"3\"/><ref-count count=\"170\"/><page-count count=\"24\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> INTRODUCTION</title><p>Conjunctivitis is characterized by inflammation and swelling of the conjunctival tissue, accompanied by engorgement of the blood vessels, ocular discharge, and pain. Many subjects are affected with conjunctivitis worldwide, and it is one of the most frequent reasons for office visits to general medical and ophthalmology clinics. More than 80% of all acute cases of conjunctivitis are reported to be diagnosed by non-ophthalmologists including internists, family medicine physicians, pediatricians, and nurse practitioners.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> This imposes a great economic burden to the healthcare system and occupies a great proportion of the office visits in many medical specialties. It is estimated that the cost of treating bacterial conjunctivitis is $857 million annually in the United States alone.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup>\n</p><p>It has been reported that nearly 60% of all patients with acute conjunctivitis receive antibiotic eye drops; and the vast majority receive their prescription from a non-ophthalmologist physician. For example, 68% of patients who visited a physician at an emergency room received antibiotic eye drops while this figure dropped to 36% for those who saw an ophthalmologist.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> Interestingly, patients from a higher socioeconomic status were more likely to receive and fill a prescription for their conjunctivitis.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup>\n</p><p>There are several ways to categorize conjunctivitis; it may be classified based on etiology, chronicity, severity, and extend of involvement of the surrounding tissue. The etiology of conjunctivitis may be infectious or non-infectious. Viral conjunctivitis followed by bacterial conjunctivitis is the most common cause of infectious conjunctivitis, while allergic and toxin-induced conjunctivitis are among the most common non-infectious etiologies. In terms of chronicity, conjunctivitis may be divided into acute with rapid onset and duration of four weeks or less, subacute, and chronic with duration longer than four weeks.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup> Furthermore, conjunctivitis may be labeled as severe when the affected individuals are extremely symptomatic and there is an abundance of mucopurulent discharge. Conjunctivitis may be associated with the involvement of the surrounding tissue such as the eyelid margins and cornea in blepharoconjunctivitis and viral keratoconjunctivitis, respectively.</p><p>Additionally, conjunctivitis may be associated with systemic conditions, including immune-related diseases [e.g., Reiter's, Stevens-Johnson syndrome (SJS), and keratoconjunctivitis sicca in rheumatoid arthritis], nutritional deprivation (vitamin A deficiency), and congenital metabolic syndromes (Richner-Hanhart syndrome and porphyria)<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> (Table 1).</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Guideline to help differentiate the major etiologies in conjunctivitis</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"2\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Clinical history and exam findings</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Most probable etiologies</bold>\n</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Alarming signs and symptoms</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decreased vision, severe pain, painful pupillary reaction, anisocoria, orbital signs</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Uveitis, scleritis, keratitis, glaucoma, orbital, or parasellar pathology</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Chronicity</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Sudden onset, lasting less than four weeks</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Infectious conjunctivitis, allergic conjunctivitis, acute systemic reactions (SJS/TEN)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Insidious onset, chronic course</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Conjunctivitis associated with systemic diseases, toxic conjunctivitis, allergic conjunctivitis</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Recurrent course</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Allergic conjunctivitis, conjunctivitis associated with systemic diseases</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Associated symptoms</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Skin lesions, arthropathy, genito-perineal involvement, oropharyngeal lesions</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Conjunctivitis associated with systemic diseases, infectious diseases</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Drug history</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Long-term eye drop usage</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Toxic conjunctivitis, allergic conjunctivitis</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Recent initiation of a systemic medication</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Acute systemic reactions (SJS/TEN)</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">SJS, Stevens-Johnson syndrome; TEN, toxic epidermal necrolysis</td></tr></tbody></table></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Selected non-conjunctivitis etiologies of red eye</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Differential diagnosis</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Symptoms</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Exam findings</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Dry eyes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Burning and FB sensation. Symptoms are usually transient, worse with reading or watching TV due to decreased blinking. Symptoms are worse in dry, cold, and windy environments due to increased evaporation</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Bilateral redness, superficial punctate keratopathy, meibomian glands dysfunction, decreased tear break-up time, small tear meniscus</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Blepharitis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Similar to dry eyes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Redness greater at the margins of eyelids, inflammation, telangiectasia, and crust around eyelashes</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Pterygium</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Recurrent ocular redness</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Visible conjunctival extension over the cornea</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Hordeolum, chalazion</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Eyelid pain and swelling</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Palpable eyelid mass, may be tender or not</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Anterior segment tumors</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Variable</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Variable</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Corneal abrasion, keratitis, corneal foreign body</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">FB sensation, relevant history including contact lens usage and occupational exposure</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Corneal epithelial defects, corneal infiltration, corneal FB</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Contact lens overwear</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Relevant history</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Corneal epithelial defect</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Subconjunctival hemorrhage</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ocular redness</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Blood under conjunctiva</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Scleritis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decreased vision, moderate to severe pain</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Redness, bluish scleral hue</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Iritis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Photophobia, pain, blurred vision. Symptoms are usually bilateral</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Decreased vision, poorly reacting pupils, constant eye pain radiating to temple and brow. Redness, severe photophobia, presence of inflammatory cells in the anterior chamber</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Angle closure glaucoma</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Headaches, nausea, vomiting, ocular pain, decreased vision, light sensitivity, and seeing haloes around lights. Symptoms are usually unilateral.</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Firm eye upon palpation, ocular redness with limbal injection. Appearance of a hazy/steamy cornea, moderately dilated pupils that are unreactive to light.</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Carotid cavernous fistula</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Chronic red eye, may have a history of head trauma</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Dilated tortuous vessels (corkscrew vessels), bruits upon auscultation with a stethoscope</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Endophthalmitis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Severe pain, photophobia, may have a history of eye surgery or ocular trauma</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Redness, puss in the anterior chamber and photophobia</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Cellulitis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Pain, double vision, and fullness</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Redness and swelling of lids, may have restriction of the eye movements, may have a history of preceding sinusitis (usually ethmoiditis)</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">FB, foreign body; TV, television</td></tr></tbody></table></table-wrap><p>It is extremely important to differentiate conjunctivitis from other causes of “red eye” associated with severe sight- or life-threatening consequences such as acute angle closure glaucoma, uveitis, endophthalmitis, carotid-cavernous fistula, cellulitis, and anterior segment tumors.</p></sec><sec sec-type=\"section\"><title> METHODS</title><p>The scientific literature published as of February 2020 was thoroughly reviewed by searching PubMed, the ISI web of knowledge database, and the Cochrane library using relevant keywords. The following keywords were used: \"bacterial conjunctivitis\", \"viral conjunctivitis,\" \"allergic conjunctivitis\", \"treatment of bacterial conjunctivitis\", and \"treatment of viral conjunctivitis\". No language restriction was applied.</p><p>Articles published between March 2013 and February 2020 were screened and those that provided the best evidence-based information were included in this review. A total of 167 articles were finally included. The first study was published in 1964 and the last study was published in 2020.</p></sec><sec sec-type=\"section\"><title> History and clinical examination</title><sec sec-type=\"subsection\"><title>How to diagnose conjunctivitis</title><p>Conjunctival injection or “red eye” is a shared presentation for many ophthalmic diseases, and it accounts for up to 1% of all primary care office visits.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> The clinicians, whether ophthalmologist or not, must be aware that “red eye” may be the presenting sign for serious eye conditions such as uveitis, keratitis, or scleritis, or it may be secondary to more benign conditions that are limited just to the conjunctival tissue (e.g., conjunctivitis or subconjunctival hemorrhage). Traditionally, it was believed that more harmful ophthalmic disorders are associated with disturbances in vision, disabling pain, and photophobia.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> However, in a recent large meta-analysis,<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> anisocoria and mild photophobia were significantly associated with “serious eye conditions”; the presence of these two signs could discover 59% of cases of “serious eye conditions”, including anterior uveitis and keratitis. Table 2 provides a summary of the main etiologies of “red eye” and their clinical characteristics.</p></sec><sec sec-type=\"subsection\"><title>How to distinguish infectious conjunctivitis from non-infectious conjunctivitis </title><p>Obtaining history from patients who present with conjunctivitis is crucial in order to arrive at the correct diagnosis. A focused ocular history should include the following: onset and duration of symptoms; laterality; impairment of vision; presence of itching; contact lens wear history; presence of fellow travelers such as recent upper respiratory infection, sinusitis, and lymphadenopathy; previous episodes of conjunctivitis; systemic allergies and medication; and history of exposure to chemical agents.</p><p>The presence of constitutional signs such as fever, malaise, fatigue, and contact with individuals with conjunctivitis helps to further narrow down the differential diagnosis. Physical examination, including checking for palpable lymph nodes, especially in the periauricular and submandibular areas, is of great importance. Ophthalmic examination should be performed to determine the type of discharge. Closer examination using a slit-lamp biomicroscope to evaluate the ocular surface structures including the palpebral conjunctiva for the presence of pseudomembranes, symblepharon, papilla or follicles, and the corneal tissue for the presence of opacities and infiltrates is absolutely essential.</p><p>Some of the clinical signs and symptoms that are used to help diagnose infectious conjunctivitis include the following: eye discharge, conjunctival injection, presence of red eye(s), eyelashes being stuck together in the morning, grittiness of the eye(s), eyelid or conjunctival edema, and history of contact with individuals with conjunctivitis.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup>\n</p><p>Allergic conjunctivitis may be underdiagnosed and undertreated.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> It is presented with itching, chemosis, and redness in the absence of any significant corneal involvement.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> The degree of conjunctival swelling is often out of proportion to conjunctival hyperemia. The main findings in vernal keratoconjunctivitis (VKC) are the presence of giant papillae in the superior tarsal conjunctiva accompanied by severe itching,<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>]</sup> while the presence of conjunctival scar and anterior subcapsular cataract supports the diagnosis of atopic keratoconjunctivitis (AKC).<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup>\n</p><p>Another similar condition, chronic toxic conjunctivitis, may present with watery discharge, an initial papillary conjunctival reaction followed by a follicular reaction, punctate epithelial erosion of the cornea, and eyelid dermatitis.<sup>[<xref rid=\"B12\" ref-type=\"bibr\">12</xref>,<xref rid=\"B13\" ref-type=\"bibr\">13</xref>,<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup>\n</p></sec><sec sec-type=\"subsection\"><title>How to distinguish bacterial conjunctivitis from viral conjunctivitis </title><p>Predicting the underlying etiology of conjunctivitis based on the presenting signs and symptoms may often result in an inaccurate diagnosis. In one study, centers with expertise in ocular surface disease had an accuracy rate of only 48% in making the correct diagnosis of adenoviral conjunctivitis.<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>]</sup> Several other studies demonstrated that bacterial pathogens are only isolated in 50% of cases of suspected bacterial conjunctivitis.<sup>[<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup> In addition, one study reported that up to 52% of presumed cases of viral conjunctivitis were culture-positive for bacteria.<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>]</sup>\n</p><p>Traditionally, the following associations between the clinical history and the etiology of conjunctivitis were believed to be true; these principles were presented in many textbooks and were used to select patients in many clinical trials.<sup>[<xref rid=\"B17\" ref-type=\"bibr\">17</xref>]</sup> For example, according to the major text books in ophthalmology (e.g., Krachmer, Duane, and Kanski), involvement of one eye followed by the involvement of the second eye within 24–48 hours is indicative of bacterial infection, while if the second eye becomes infected after 48 hours with an accompanying enlarged periauricular lymph node, a viral etiology should be considered. According to the same textbooks, a papillary conjunctival reaction or pseudomembranous conjunctivitis strongly suggests a bacterial origin for conjunctivitis while follicular conjunctival reaction is more likely to indicate a viral etiology.</p><p>There are many other associations between the etiology of conjunctivitis and symptoms that are thought to be true, but lack strong clinical evidence. For example, association between lack of itching and bacterial conjunctivitis have come under scrutiny in the recent years. Other associations that once thought to be true but lack evidence include: recent upper respiratory tract infection and lymphadenopathy in favor of viral conjunctivitis; sinusitis, fever, malaise, and fatigue in association with bacterial conjunctivitis; and previous history of conjunctivitis with bilateral involvement of the eyes in favor of viral and allergic but not bacterial conjunctivitis.</p><p>A meta-analysis in 2003 failed to find any clinical studies correlating the signs and symptoms of conjunctivitis with its underlying etiology.<sup>[<xref rid=\"B17\" ref-type=\"bibr\">17</xref>]</sup> Following the above meta-analysis, a prospective study was conducted and found that combination of three signs, bilateral mattering of the eyelids, lack of itching, and no previous history of conjunctivitis were strong predictors of bacterial conjunctivitis.<sup>[<xref rid=\"B18\" ref-type=\"bibr\">18</xref>]</sup> Having both eyes matter and their eyelashes adhere together in the morning was a stronger predictor for positive bacterial culture, and either itching or a previous episode of conjunctivitis made a positive bacterial culture less likely. In addition, types of the discharge (purulent, mucus, or watery) or other symptoms were not specific to any particular class of conjunctivitis.</p><p>A more recent meta-analysis, which analyzed the clinical data of 622 patients from three clinical trials,<sup>[<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup> found that patients with purulent discharge or mild to moderate red eye were less likely to benefit from topical antibiotics; this finding reiterates lack of meaningful correlation between signs and symptoms and the underlying etiology in most cases of conjunctivitis. Another recent study in 2013 found a strong likelihood of positive bacterial culture results in patients with the “gluing of the eyelids” upon waking up in the morning, and the age above 50 at presentation.<sup>[<xref rid=\"B20\" ref-type=\"bibr\">20</xref>]</sup>\n</p></sec><sec sec-type=\"subsection\"><title> How do laboratory findings help us?</title><p>Clinicians may collect discharge samples from eyes with conjunctivitis and send them for microbiological evaluation. Conjunctival cultures are generally reserved for cases of suspected infectious neonatal conjunctivitis, recurrent conjunctivitis, conjunctivitis recalcitrant to therapy, conjunctivitis presenting with severe purulent discharge, and cases suspicious for gonococcal or chlamydial infection.<sup>[<xref rid=\"B21\" ref-type=\"bibr\">21</xref>]</sup> Swabs from the discharge are better to be taken before the initiation of antimicrobial therapy. The swabs are then plated in various growth mediums in the laboratory for obtaining cultures. Sabouraud agar plates are used to identify fungus, and it should be utilized in patients with chronic blepharitis and those who are immunocompromised. Anaerobic culture plates may also be helpful, especially in patients with a history of previous surgery or trauma.<sup>[<xref rid=\"B22\" ref-type=\"bibr\">22</xref>]</sup> If antimicrobial therapy has already been started, they should be stopped 48 hours prior to obtaining cultures. In a five-year review of 138 pediatric ocular surface infections, the most common organisms were coagulase-negative <italic>staphylococci</italic>, followed by <italic>Pseudomonas aeruginosa</italic> and <italic>Staphylococcus aureus</italic>.<sup>[<xref rid=\"B23\" ref-type=\"bibr\">23</xref>]</sup>\n</p><p>Nucleic acid amplification techniques, requiring special swabs, may be used in diagnosing viral infections, where a multitude of polymerase chain reaction (PCR) tests for detection of viruses are available.</p><p>Although primary studies from in-office rapid antigen testing for adenoviruses report 89% sensitivity and up to 94% specificity,<sup>[<xref rid=\"B21\" ref-type=\"bibr\">21</xref>]</sup> the results of more recent studies point toward a high specificity but only moderate sensitivity ranging from 39.5% to 50%.<sup>[<xref rid=\"B24\" ref-type=\"bibr\">24</xref>]</sup> Accordingly, it may be suggested that negative Adeno-Plus test results should be confirmed by real-time PCR owing to its suboptimal sensitivity.</p><p>For those suspected of having allergic conjunctivitis, skin scratch test or intradermal injection of common allergens, and assays for detecting elevated <italic>in vitro</italic> levels of specific serum IgE may be used; however, the diagnosis of allergic conjunctivitis remains a clinical one.</p></sec></sec><sec sec-type=\"section\"><title> Viral conjunctivitis</title><p>Viral conjunctivitis is the most common overall cause of infectious conjunctivitis, and it is usually secondary to inoculation of the ocular surface with the adenoviruses.<sup>[<xref rid=\"B25\" ref-type=\"bibr\">25</xref>,<xref rid=\"B26\" ref-type=\"bibr\">26</xref>]</sup> Less frequently, other viruses may be the underlying etiology in viral conjunctivitis; amongst them, herpes simplex virus (HSV), varicella zoster virus (VZV), and enterovirus have been the subject of investigation.<sup>[<xref rid=\"B27\" ref-type=\"bibr\">27</xref>]</sup>\n</p><sec sec-type=\"subsection\"><title>Adenoviral conjunctivitis</title><p>As the leading cause of infectious conjunctivitis worldwide, up to 90% of viral conjunctivitis cases are caused by adenoviruses.<sup>[<xref rid=\"B28\" ref-type=\"bibr\">28</xref>]</sup> Recent advances in genome sequencing of human adenoviruses (HAdV) have identified over 72 unique HAdV genotypes classified into seven different species (HAdV-A through HAdV-G), with HAdV-D species having the most members and the strongest association with viral conjunctivitis.<sup>[<xref rid=\"B29\" ref-type=\"bibr\">29</xref>,<xref rid=\"B30\" ref-type=\"bibr\">30</xref>]</sup>\n</p><p>Perhaps the most common form of infection by the adenoviruses in children is pharyngoconjunctival fever (PCF) caused by HAdV types 3, 4, and 7.<sup>[<xref rid=\"B31\" ref-type=\"bibr\">31</xref>,<xref rid=\"B32\" ref-type=\"bibr\">32</xref>,<xref rid=\"B33\" ref-type=\"bibr\">33</xref>]</sup> This condition is usually characterized by the presence of fever, pharyngitis, periauricular lymphadenopathy, and acute follicular conjunctivitis. Additional ocular surface findings include edema, hyperemia, and petechial hemorrhages of the conjunctiva as a result of interaction between pro-inflammatory cytokines and conjunctival vasculature.<sup>[<xref rid=\"B32\" ref-type=\"bibr\">32</xref>]</sup> This condition is self-limited, often resolving spontaneously in two–three weeks without any treatment.</p><p>The most severe ocular manifestation of adenoviral infection is the epidemic keratoconjunctivitis (EKC); this condition affects both the conjunctiva and cornea, leaving behind long-lasting and permanent ocular surface changes and visual disturbances. Ocular manifestations of EKC include conjunctival discharge, follicular conjunctivitis, corneal subepithelial infiltrates (SEI), corneal scarring, development of conjunctival membranes and pseudomembranes, and symblepharon formation (Figures 1 and 2).</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Adenoviral conjunctivitis presenting as bilateral watery eyes.</p></caption><graphic xlink:href=\"jovr-15-372-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Pseudomembrane formation in a patient with adenoviral conjunctivitis.</p></caption><graphic xlink:href=\"jovr-15-372-g002\"/></fig><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>Subepithelial infiltrations in a patient with adenoviral conjunctivitis.</p></caption><graphic xlink:href=\"jovr-15-372-g003\"/></fig><p>Classically, serotypes 8, 19, 37, and less frequently serotype 4 were believed to be associated with EKC, but more recently, HAdV-D53 and HAdV-D54 have been identified in several outbreaks and are thought to be responsible for the majority of EKC cases.<sup>[<xref rid=\"B30\" ref-type=\"bibr\">30</xref>]</sup>\n</p><p>Pseudomembranes, which are sheets of fibrin-rich exudates without blood or lymphatic vessels, may be encountered in the tarsal conjunctiva of the EKC patients.<sup>[<xref rid=\"B35\" ref-type=\"bibr\">35</xref>]</sup> Depending on the intensity of inflammation, true conjunctival membranes may also form in EKC. True membranes, once form, can lead to the development of subepithelial fibrosis and symblepharon; additionally, they tend to bleed severely upon removal.<sup>[<xref rid=\"B36\" ref-type=\"bibr\">36</xref>]</sup>\n</p><p>Cornea is another tissue that may become adversely affected in EKC. Replication of the virus in the corneal epithelium may cause superficial punctate keratopathy, followed by focal areas of epithelial opacities.<sup>[<xref rid=\"B37\" ref-type=\"bibr\">37</xref>]</sup> Focal SEI in the anterior stroma of the cornea appears approximately 7–10 days following the initial involvement of the eyes with EKC<sup>[<xref rid=\"B38\" ref-type=\"bibr\">38</xref>]</sup>\n(Figure 3). These opacities may persist for years, and they may be associated with visual disturbance, photophobia, and astigmatism. The incidence of SEI formation in EKC has been reported to vary from 49.1 to 80%.<sup>[<xref rid=\"B39\" ref-type=\"bibr\">39</xref>]</sup> An immunologic reaction to the replicating adenoviruses in anterior stromal keratocytes is hypothesized to be the underlying mechanism for the formation of SEIs. The observation that these opacities recur following discontinuation of steroids supports the hypothesis.<sup>[<xref rid=\"B40\" ref-type=\"bibr\">40</xref>]</sup>\n</p><p>Adenovirus conjunctivitis is very contagious and it may be transmitted up to 50% of the time according to some reports.<sup>[<xref rid=\"B41\" ref-type=\"bibr\">41</xref>,<xref rid=\"B42\" ref-type=\"bibr\">42</xref>]</sup> The virus may spread through contaminated fingers, medical devices, contaminated water at the swimming pools, or by sharing of personal items; as many as 46% of individuals with viral conjunctivitis had positive viral culture grown from their hands according to one study.<sup>[<xref rid=\"B43\" ref-type=\"bibr\">43</xref>]</sup> The adenovirus is a very hardy organism, and it is reported to be resistant to 70% isopropyl alcohol and 3% hydrogen peroxide.<sup>[<xref rid=\"B44\" ref-type=\"bibr\">44</xref>]</sup> The American Academy of Ophthalmology recommends using a 1:10 dilute bleach solution (sodium hypochlorite) to disinfect the office equipment and instruments against common infectious agents encountered in eye care clinics including the adenoviruses.<sup>[<xref rid=\"B45\" ref-type=\"bibr\">45</xref>]</sup>\n</p><p>Due to the highly contagious nature of viral conjunctivitis, frequent hand washing, meticulous disinfection of medical instruments, and isolation of conjunctivitis patients from the rest in the healthcare provider's office has been recommended.<sup>[<xref rid=\"B46\" ref-type=\"bibr\">46</xref>]</sup> The incubation period for the adenovirus is approximately 5–12 days, while the infected individuals can transmit the disease for up to 14 days from the time they are infected.<sup>[<xref rid=\"B41\" ref-type=\"bibr\">41</xref>]</sup>\n</p><p>There is no single effective treatment modality for viral conjunctivitis; however, use of frequent artificial tears, antihistamines containing eye drops, or cold-compresses seem to alleviate many of the clinical symptoms that are associated with this condition.<sup>[<xref rid=\"B47\" ref-type=\"bibr\">47</xref>,<xref rid=\"B48\" ref-type=\"bibr\">48</xref>]</sup> Topical and oral antiviral medications do not appear to be useful.<sup>[<xref rid=\"B47\" ref-type=\"bibr\">47</xref>,<xref rid=\"B48\" ref-type=\"bibr\">48</xref>]</sup> In addition, antibiotic eye drops do not play a role in treating viral conjunctivitis and may even obscure the clinical picture by inducing ocular surface toxicity.<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>,<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup> Other concerns with using antibiotic drops include increased bacterial resistance and the possibility of spreading the disease to the contralateral eye by cross-contamination through the infected bottles.<sup>[<xref rid=\"B42\" ref-type=\"bibr\">42</xref>]</sup>\n</p><p>Membranes or pseudomembranes may be peeled at the slit-lamp by using a pair of jeweler forceps or cotton swab after anesthetizing the ocular surface. This is done to alleviate patient discomfort and prevent future scar formation.</p><p>Monotherapy against viral conjunctivitis with Povidone-iodine 2% have been investigated in a pilot study. The authors discovered that topical administration of Povidone-iodine 2% four times a day for one week led to complete resolution of the disease in three-quarters of the eyes.<sup>[<xref rid=\"B49\" ref-type=\"bibr\">49</xref>]</sup>\n</p><p>The American Academy of Ophthalmology suggests that topical corticosteroids play an important role in the treatment of conjunctivitis, but they should be used judiciously and with caution in selected cases.<sup>[<xref rid=\"B47\" ref-type=\"bibr\">47</xref>]</sup> Indications for steroid usage in viral conjunctivitis are membrane formation and sub-epithelial infiltration associated with severe photophobia and decreased vision. Prolonging the duration of adenoviral conjunctivitis, exacerbation of HSV keratitis, and an increase in intraocular pressure are the main adverse effects of indiscriminate use of topical corticosteroids.</p><p>Prolongation of viral shedding following monotherapy with corticosteroids has been reported;<sup>[<xref rid=\"B50\" ref-type=\"bibr\">50</xref>]</sup> however, combination therapies with corticosteroids and anti-infective agents (i.e., antibiotics) have proven to be effective in treating viral and bacterial conjunctivitis.<sup>[<xref rid=\"B51\" ref-type=\"bibr\">51</xref>,<xref rid=\"B52\" ref-type=\"bibr\">52</xref>]</sup>\n</p><p>Ophthalmic formulations of PVP-I/dexamethasone are widely investigated. PVP-I 0.4%/dexamethasone 0.1% suspension, PVP-I 1.0%/dexamethasone 0.1%, and PVP-I 0.6%/dexamethasone 0.1% have been used, and the results suggest that the combination therapies reduce patient symptoms and eradicate the virus effectively.<sup>[<xref rid=\"B50\" ref-type=\"bibr\">50</xref>,<xref rid=\"B53\" ref-type=\"bibr\">53</xref>,<xref rid=\"B54\" ref-type=\"bibr\">54</xref>,<xref rid=\"B55\" ref-type=\"bibr\">55</xref>]</sup>\n</p><p>Ongoing phase 3, randomized, double-masked, controlled studies will further clarify the efficacy and safety of combined PVP-I/dexamethasone in adenoviral conjunctivitis (ClinicalTrials.gov identifiers: NCT0299855441 and NCT0299854142) and bacterial conjunctivitis (ClinicalTrials.gov identifiers: NCT03004924).</p><p>Use of 1 and 2% cyclosporine-A (CsA) eye drops have been advocated for the treatment of SEIs, and it has been demonstrated to be effective in improving patient symptoms and reducing the amounts of infiltrates.<sup>[<xref rid=\"B30\" ref-type=\"bibr\">30</xref>,<xref rid=\"B56\" ref-type=\"bibr\">56</xref>]</sup> However, Jeng et al suggested that it might be difficult to wean patients completely off CsA once they have started it; in their study, when CsA was stopped, SEIs returned, necessitating reinstitution of the CsA eye drops.<sup>[<xref rid=\"B57\" ref-type=\"bibr\">57</xref>]</sup> This finding is in contrast with the Reinhard's pilot study, where no recurrence was observed after discontinuation of the CsA drops.<sup>[<xref rid=\"B58\" ref-type=\"bibr\">58</xref>]</sup> In a small study consisting of 39 patients, administration of 1% cyclosporine-A (four times a day) during the acute phase of viral conjunctivitis and continuing it thereafter for 21 days lowered the incidence of corneal opacities significantly.<sup>[<xref rid=\"B59\" ref-type=\"bibr\">59</xref>]</sup> A case-controlled double-blinded randomized clinical trial is needed to investigate the effectiveness of cyclosporine-A and to formulate an ideal tapering regiment for this medication.</p><p>The use of topical tacrolimus eye drops has also been investigated for the treatment of SEIs secondary to adenoviral keratoconjunctivitis. When tacrolimus eye drops or ointments were used for an average of six months, a significant reduction in the size and numbers of SEIs was observed in 60% of the cases, while in 31.76% of the eyes, SEIs were eliminated after one year.<sup>[<xref rid=\"B60\" ref-type=\"bibr\">60</xref>]</sup> There was also a statistically significant improvement in the visual acuity of the patients with the use of topical tacrolimus.</p></sec><sec sec-type=\"subsection\"><title>Herpetic conjunctivitis</title><p>It is estimated that 1.3–4.8% of all cases of acute conjunctivitis are caused by HSV infection.<sup>[<xref rid=\"B61\" ref-type=\"bibr\">61</xref>,<xref rid=\"B62\" ref-type=\"bibr\">62</xref>,<xref rid=\"B63\" ref-type=\"bibr\">63</xref>]</sup> HSV often causes a unilateral follicular conjunctivitis, which may be accompanied by a thin watery discharge and associated vesicular lesions on the skin of the eyelids. Treatment consists of topical antiviral agents, including ganciclovir, idoxuridine, vidarabine, and trifluridine. The purpose of the treatment is to reduce virus shedding and the chance of the development of keratitis.</p><p>Ocular involvement with herpes zoster virus, especially when the first and second branches of the trigeminal nerve are involved, can lead to conjunctivitis in 41.1% of cases, eyelid lesions in 45.8%, uveitis in 38.2%, and corneal lesions such as SEIs, pseudodendrites, and nummular keratitis in another 19.1%.<sup>[<xref rid=\"B64\" ref-type=\"bibr\">64</xref>,<xref rid=\"B65\" ref-type=\"bibr\">65</xref>]</sup>\n</p></sec><sec sec-type=\"subsection\"><title>Acute hemorrhagic conjunctivitis</title><p>Acute hemorrhagic conjunctivitis (AHC) is an extremely contagious form of viral conjunctivitis. It manifests by foreign body sensation, profuse tearing, eyelid edema, dilatation of conjunctival vessels, chemosis, and subconjunctival hemorrhage. In a small proportion of patients, fever, fatigue, and leg pain may ensue. Two picornaviruses, namely enterovirus 70 (EV70) and coxsackievirus A24 variant (CA24v), as well as certain subtypes of adenoviruses are believed to be the responsible pathogens.<sup>[<xref rid=\"B66\" ref-type=\"bibr\">66</xref>,<xref rid=\"B67\" ref-type=\"bibr\">67</xref>,<xref rid=\"B68\" ref-type=\"bibr\">68</xref>]</sup> Like the other forms of conjunctivitis, AHC is also believed to be transmitted primarily by hand-to-eye-to-hand contact and infected fomites.<sup>[<xref rid=\"B69\" ref-type=\"bibr\">69</xref>]</sup> The condition is self-limited and the symptoms diminish gradually during the first week of infection and completely resolves after 10–14 days.<sup>[<xref rid=\"B69\" ref-type=\"bibr\">69</xref>]</sup> Medical intervention aims primarily at controlling the large outbreaks as well as instituting preventative measures to protect the vulnerable groups, such as children, elderly, pregnant women, and immunocompromised individuals, by encouraging frequent handwashing and reducing contact with the affected individuals.<sup>[<xref rid=\"B68\" ref-type=\"bibr\">68</xref>]</sup>\n</p></sec><sec sec-type=\"subsection\"><title>Miscellaneous viral conjunctivitis</title><p>Infection with Molluscum contagiosum (MC) is characterized by multiple umblicated and papular skin lesions caused by Pox-2 virus. Skin-to-skin contact and sexual intercourse are the main routes of transmission. Shedding of the viral proteins from the eyelid lesions into the tear film leads to chronic follicular conjunctival reaction, punctate keratopathy, and subepithelial pannus. Rarely, primary MC lesions are found in the conjunctiva.<sup>[<xref rid=\"B70\" ref-type=\"bibr\">70</xref>]</sup>\n</p><p>Ebola hemorrhagic fever is a fatal disease caused by the species of ebolavirus. Conjunctival injection, subconjunctival hemorrhage, and tearing have been reported in the affected individuals.<sup>[<xref rid=\"B71\" ref-type=\"bibr\">71</xref>]</sup> Conjunctival injection, which is often bilateral and present in up to 58% of cases, has been identified in both the acute and late stages of this disease and may play an important role in the early diagnosis of this potentially deadly condition.<sup>[<xref rid=\"B72\" ref-type=\"bibr\">72</xref>]</sup> While human-to-human transmission through bodily fluids can spread the infection, the natural reservoir is thought to be the fruit bat.<sup>[<xref rid=\"B73\" ref-type=\"bibr\">73</xref>]</sup>\n</p><p>Coronaviruses include a broad family of viruses that normally affect animals, although some strains can spread from animals to humans.<sup>[<xref rid=\"B74\" ref-type=\"bibr\">74</xref>]</sup> The most recently isolated strain of coronavirus, “2019-nCoV”, has made the headlines since it was first recognized in December 2019 in China. COVID-19 has been reported to cause fever, cough, shortness of breath, and even death.<sup>[<xref rid=\"B75\" ref-type=\"bibr\">75</xref>,<xref rid=\"B76\" ref-type=\"bibr\">76</xref>]</sup> Some reports have suggested that this virus can cause conjunctivitis and be transmitted via the conjunctival secretions of the infected individuals.<sup>[<xref rid=\"B76\" ref-type=\"bibr\">76</xref>]</sup> All healthcare professionals including the ophthalmologists should be vigilant in approaching patients with conjunctivitis and respiratory symptoms, especially if they report a recent history of travel to high risk regions.<sup>[<xref rid=\"B76\" ref-type=\"bibr\">76</xref>]</sup>\n</p></sec></sec><sec sec-type=\"section\"><title> Bacterial conjunctivitis</title><p>While in adults, bacterial conjunctivitis is less common than viral conjunctivitis, in children, it is encountered more frequently.<sup>[<xref rid=\"B77\" ref-type=\"bibr\">77</xref>]</sup> Bacterial conjunctivitis can result from either a direct contact with infected individuals or from abnormal proliferation of the native conjunctival flora.<sup>[<xref rid=\"B78\" ref-type=\"bibr\">78</xref>]</sup> Contaminated fingers,<sup>[<xref rid=\"B41\" ref-type=\"bibr\">41</xref>]</sup> oculogenital spread,<sup>[<xref rid=\"B47\" ref-type=\"bibr\">47</xref>]</sup> and contaminated fomites<sup>[<xref rid=\"B79\" ref-type=\"bibr\">79</xref>]</sup> are common routes of transmission. In addition, certain conditions such as compromised tear production, disruption of the natural epithelial barrier, abnormality of adnexal structures, trauma, and immunosuppressed status increase the likelihood of contracting bacterial conjunctivitis.<sup>[<xref rid=\"B47\" ref-type=\"bibr\">47</xref>]</sup>\n</p><p>Acute bacterial conjunctivitis is most often caused by <italic>Staphylococcus</italic> species, <italic>Haemophilus influenza</italic>, <italic>Streptococcus</italic> species, <italic>Moraxella catarrhalis</italic>, and gram-negative intestinal bacteria.<sup>[<xref rid=\"B80\" ref-type=\"bibr\">80</xref>]</sup> In younger children, minor epidemics may occur secondary to <italic>H. influenza</italic> or <italic>S. pneumonia</italic>. Acute bacterial conjunctivitis manifests by foreign body sensation and increased ocular secretion in addition to moderate conjunctival hyperemia (Figure 4).</p><p>Several studies on bacterial conjunctivitis<sup>[<xref rid=\"B81\" ref-type=\"bibr\">81</xref>,<xref rid=\"B82\" ref-type=\"bibr\">82</xref>]</sup> demonstrate that sticky eyelids and itching may be present in approximately 90% of the affected individuals; these findings are followed by the less frequently encountered signs and symptoms such as purulent secretion and ocular burning. <italic>H. influenza</italic> conjunctivitis may be associated with acute otitis media and upper respiratory tract infection.<sup>[<xref rid=\"B80\" ref-type=\"bibr\">80</xref>]</sup>\n</p><p>In more than 60% of cases, spontaneous cure occurs within one–two weeks,<sup>[<xref rid=\"B83\" ref-type=\"bibr\">83</xref>]</sup> and serious complications are extremely rare.<sup>[<xref rid=\"B84\" ref-type=\"bibr\">84</xref>]</sup> However, presence of a large population of bacteria on the conjunctiva exposes the patient to a higher risk of keratitis, particularly in conditions associated with corneal epithelial defects, such as dry eye.<sup>[<xref rid=\"B80\" ref-type=\"bibr\">80</xref>]</sup>\n</p><p>Although topical antibiotics reduce the duration of the disease, no difference in the outcome is seen between the treatment and placebo groups. In a meta-analysis,<sup>[<xref rid=\"B81\" ref-type=\"bibr\">81</xref>]</sup>, consisting of 3,673 patients from 11 randomized clinical trials, antibiotic treatment increased the rate of clinical improvement by 10% compared to placebo. Both “2 to 5” and “6 to 10” day regiments were included in this analysis. Although, highly virulent bacteria can potentially inflict serious damage to the ocular surface and the eye,<sup>[<xref rid=\"B78\" ref-type=\"bibr\">78</xref>]</sup>, no sight-threatening complications were reported in any of the placebo groups in the aforementioned meta-analysis.<sup>[<xref rid=\"B85\" ref-type=\"bibr\">85</xref>]</sup>\n</p><p>All broad-spectrum antibiotic eye drops seem to be effective in treating bacterial conjunctivitis and it is unlikely that there is a significant difference among various antibiotics in achieving clinical cure. Factors that influence antibiotic choice are local availability, patient allergies, resistance patterns, and cost.</p><p>From a large systematic review, it was concluded that topical antibiotics were more effective in achieving clinical and microbial cure when patients had positive bacterial cultures.<sup>[<xref rid=\"B21\" ref-type=\"bibr\">21</xref>]</sup> However, no significant difference has been reported in clinical cure rate when different frequencies of the antibiotics were administered.<sup>[<xref rid=\"B86\" ref-type=\"bibr\">86</xref>,<xref rid=\"B87\" ref-type=\"bibr\">87</xref>]</sup> Due to lengthening the course of the illness and potentiating the infection, topical steroids should be avoided<sup>[<xref rid=\"B47\" ref-type=\"bibr\">47</xref>]</sup> (Table 3).</p><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><p>Ophthalmic drug therapies for acute bacterial conjunctivitis.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"2\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Antibiotic agents</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Treatment</bold>\n</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Aminoglycosides</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Gentamicin</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ointment: 4 <inline-formula><mml:math id=\"M1\"><mml:mo>×</mml:mo></mml:math></inline-formula>/d for 1 wk Solution: 1-2 drops 4 <inline-formula><mml:math id=\"M2\"><mml:mo>×</mml:mo></mml:math></inline-formula>/d for 1 wk</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Tobramycin</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ointment: 3 <inline-formula><mml:math id=\"M3\"><mml:mo>×</mml:mo></mml:math></inline-formula>/d for 1 wk</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Fluoroquinolones</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Besifloxacin</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1 drop 3 <inline-formula><mml:math id=\"M4\"><mml:mo>×</mml:mo></mml:math></inline-formula>/d for 1 wk</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ciprofloxacin</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ointment: 3 <inline-formula><mml:math id=\"M5\"><mml:mo>×</mml:mo></mml:math></inline-formula>/d for 1 wk Solution: 1-2 drops 4 <inline-formula><mml:math id=\"M6\"><mml:mo>×</mml:mo></mml:math></inline-formula>/d for 1 wk</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Gatifloxacin</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3 <inline-formula><mml:math id=\"M7\"><mml:mo>×</mml:mo></mml:math></inline-formula>/d for 1 week</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Levofloxacin</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1-2 drops 4 <inline-formula><mml:math id=\"M8\"><mml:mo>×</mml:mo></mml:math></inline-formula>/d for 1 wk</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Moxifloxacin</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3 <inline-formula><mml:math id=\"M9\"><mml:mo>×</mml:mo></mml:math></inline-formula>/d for 1 wk</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ofloxacin</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1-2 drops 4 <inline-formula><mml:math id=\"M10\"><mml:mo>×</mml:mo></mml:math></inline-formula>/d for 1 wk</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Macrolides</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Azithromycin</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 <inline-formula><mml:math id=\"M11\"><mml:mo>×</mml:mo></mml:math></inline-formula>/d for 2 d; then 1 drop daily for 5 d</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Erythromycin</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 <inline-formula><mml:math id=\"M12\"><mml:mo>×</mml:mo></mml:math></inline-formula>/d for 1 wk</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Sulfonamides</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Sulfacetamide</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ointment: 4 <inline-formula><mml:math id=\"M13\"><mml:mo>×</mml:mo></mml:math></inline-formula>/d and at bedtime for 1 wk Solution: 1-2 drops every 2-3 h for 1 wk</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Combination</bold>\n<bold>drops</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Trimethoprim/polymyxin B</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1 or 2 drops 4 <inline-formula><mml:math id=\"M14\"><mml:mo>×</mml:mo></mml:math></inline-formula>/d for 1 wk</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></table-wrap><fig id=\"F4\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><p>Thick purulent discharge in a patient with acute bacterial conjunctivitis.</p></caption><graphic xlink:href=\"jovr-15-372-g004\"/></fig><fig id=\"F5\" orientation=\"portrait\" position=\"float\"><label>Figure 5</label><caption><p>Spectrum of allergic conjunctivitis. CS, corticosteroid</p></caption><graphic xlink:href=\"jovr-15-372-g005\"/></fig><fig id=\"F6\" orientation=\"portrait\" position=\"float\"><label>Figure 6</label><caption><p>Cobblestone appearance of large conjunctival papillae in a patient with VKC (left). Limbal VKC with Horner-Trantas dots in another patient (right).</p></caption><graphic xlink:href=\"jovr-15-372-g006\"/></fig><fig id=\"F7\" orientation=\"portrait\" position=\"float\"><label>Figure 7</label><caption><p>Some systemic and dermatological conditions associated with conjunctivitis.</p></caption><graphic xlink:href=\"jovr-15-372-g007\"/></fig><fig id=\"F8\" orientation=\"portrait\" position=\"float\"><label>Figure 8</label><caption><p>Symblepharon formation in a patient with ocular cicatricial pemphigoid.</p></caption><graphic xlink:href=\"jovr-15-372-g008\"/></fig><sec sec-type=\"subsection\"><title>Methicillin-resistant S. aureus conjunctivitis</title><p>The term methicillin-resistant <italic>S. aureus</italic> (MRSA) refers to <italic>Staphylococcus aureus</italic> species that are resistant to methicillin antibiotic; however, nowadays the term is used to describe resistance to all β-lactam antimicrobials.<sup>[<xref rid=\"B88\" ref-type=\"bibr\">88</xref>]</sup> Growing in prevalence, 3–64% of all ocular <italic>Staphylococcus</italic> conjunctival infections are MRSA conjunctivitis.<sup>[<xref rid=\"B89\" ref-type=\"bibr\">89</xref>]</sup> Suspected cases need to be treated with fortified vancomycin eye drops or ointments.<sup>[<xref rid=\"B90\" ref-type=\"bibr\">90</xref>]</sup> Culture-directed administration of antimicrobials, effective dosing, considering the local resistance patterns, and appropriate antiseptic strategies should be applied to restrict the spread of MRSA conjunctivitis.<sup>[<xref rid=\"B91\" ref-type=\"bibr\">91</xref>]</sup>\n</p></sec><sec sec-type=\"subsection\"><title>Chlamydial conjunctivitis </title><p>\n<italic>Chlamydia trachomatis</italic> may cause a variety of ocular surface infections including trachoma, neonatal conjunctivitis, and inclusion conjunctivitis. Serotype D-K are causative agents for neonatal conjunctivitis and adult inclusion conjunctivitis, while trachoma is caused by serotypes A, B, Ba, and C.<sup>[<xref rid=\"B92\" ref-type=\"bibr\">92</xref>]</sup>\n</p><p>Inclusion conjunctivitis is reported to cause 1.8–5.6% of all cases of acute conjunctivitis,<sup>[<xref rid=\"B61\" ref-type=\"bibr\">61</xref>,<xref rid=\"B62\" ref-type=\"bibr\">62</xref>,<xref rid=\"B93\" ref-type=\"bibr\">93</xref>]</sup> where the majority of cases are unilateral and have concurrent genital infection.<sup>[<xref rid=\"B94\" ref-type=\"bibr\">94</xref>]</sup> Patients often present with mild mucopurulent discharge and follicular conjunctivitis persisting for weeks to months.<sup>[<xref rid=\"B77\" ref-type=\"bibr\">77</xref>]</sup> Up to 54% of men and 74% of women are reported to have simultaneous genital infection.<sup>[<xref rid=\"B95\" ref-type=\"bibr\">95</xref>]</sup> The disease is frequently acquired via oculogenital spread.<sup>[<xref rid=\"B47\" ref-type=\"bibr\">47</xref>]</sup> Treatment with systemic antibiotics such as oral azithromycin and doxycycline is efficacious, while addition of topical antibiotics is not beneficial. Treatment of sexual partners and looking for the evidence of coinfection with gonorrhea must be instituted.</p><p>As the leading cause of infectious blindness in the world, trachoma affects 40 million individuals worldwide; this infection is prevalent in areas with poor hygiene. Although mucopurulent discharge is the initial presenting sign, in the later stages, scarring of the eyelids, conjunctiva, and cornea may lead to loss of vision. A single dose of oral azithromycin (20 mg/kg) in addition to oral tetracycline or erythromycin for three weeks is very effective. Patients may also be treated with topical antibiotic ointments, such as tetracycline and erythromycin, for six weeks.<sup>[<xref rid=\"B96\" ref-type=\"bibr\">96</xref>,<xref rid=\"B97\" ref-type=\"bibr\">97</xref>]</sup>\n</p><p>In newborns, chlamydia can cause conjunctivitis following passage through an infected birth canal. The acute phase, which typically begins between days 5 and 14 following vaginal delivery, is characterized by purulent discharge, erythema and edema of the eyelids and conjunctiva.<sup>[<xref rid=\"B98\" ref-type=\"bibr\">98</xref>]</sup> More prevalent than gonococcal conjunctivitis (GC), neonatal conjunctivitis secondary to <italic>C. trachomatis</italic> is considered the most frequent infectious cause of neonatal conjunctivitis worldwide.<sup>[<xref rid=\"B98\" ref-type=\"bibr\">98</xref>,<xref rid=\"B99\" ref-type=\"bibr\">99</xref>,<xref rid=\"B100\" ref-type=\"bibr\">100</xref>]</sup>\n</p><p>Although the chlamydial conjunctivitis has a mild course, scarring of the cornea and/or conjunctiva have been reported in untreated cases.<sup>[<xref rid=\"B101\" ref-type=\"bibr\">101</xref>]</sup> It is important to note that up to 20% of the neonates who are exposed to chlamydia may develop pneumonia; in these, 50% demonstrate a previous history of conjunctivitis.<sup>[<xref rid=\"B102\" ref-type=\"bibr\">102</xref>]</sup>\n</p><p>A recent meta-analysis supports the superiority of traditional treatment with systemic erythromycin at 50 mg/kg per day (given in four divided doses for two weeks), in comparison to topical antibiotic therapy alone.<sup>[<xref rid=\"B103\" ref-type=\"bibr\">103</xref>]</sup> A recent study evaluating the efficacy of azithromycin in neonatal chlamydial conjunctivitis<sup>[<xref rid=\"B104\" ref-type=\"bibr\">104</xref>]</sup> demonstrated superiority of erythromycin over azithromycin; however, risk of pyloric stenosis related to the use of erythromycin may reduce its clinical use in neonates in the future.<sup>[<xref rid=\"B103\" ref-type=\"bibr\">103</xref>]</sup> Additionally, less-frequent dose of azithromycin may improve compliance.<sup>[<xref rid=\"B105\" ref-type=\"bibr\">105</xref>]</sup>\n</p></sec><sec sec-type=\"subsection\"><title>Gonococcal conjunctivitis (GC)</title><p>Typically viewed as a condition affecting the neonates, GC, however, affects other age groups as well.<sup>[<xref rid=\"B106\" ref-type=\"bibr\">106</xref>]</sup>\n<italic>Neisseria gonorrhoeae</italic> is a common cause of hyperacute conjunctivitis in neonates and sexually active adults.<sup>[<xref rid=\"B78\" ref-type=\"bibr\">78</xref>]</sup> Ocular infection with <italic>N. gonorrhea</italic> is associated with a high prevalence of corneal perforation.<sup>[<xref rid=\"B80\" ref-type=\"bibr\">80</xref>]</sup> GC should be considered as the causative agent in neonates who present with conjunctivitis in days 2 to 5 after delivery.<sup>[<xref rid=\"B106\" ref-type=\"bibr\">106</xref>]</sup> In both neonatal and non-neonatal populations, eye exam may reveal conjunctival injection and chemosis along with copious mucopurulent discharge; a tender globe with periauricular lymphadenopathy may also be associated with this type of conjunctivitis.<sup>[<xref rid=\"B106\" ref-type=\"bibr\">106</xref>]</sup>\n</p><p>The suggested treatment for neonates include single dose of ceftriaxone (25 to 50 mg/kg), or cefotaxime (100 mg/kg IV or IM), in addition to hourly saline irrigation of the ocular surface.<sup>[<xref rid=\"B106\" ref-type=\"bibr\">106</xref>,<xref rid=\"B107\" ref-type=\"bibr\">107</xref>,<xref rid=\"B108\" ref-type=\"bibr\">108</xref>]</sup> Non-neonates can be treated with combination of 1 gm of IM ceftriaxone given in a single dose and 1 gm of oral azithromycin (which is used to treat the frequently encountered chlamydial coinfection). Irrigation of the ocular surface with saline solution is not necessary in adults.<sup>[<xref rid=\"B106\" ref-type=\"bibr\">106</xref>]</sup>\n</p></sec></sec><sec sec-type=\"section\"><title> Allergic conjunctivitis</title><p>Ocular allergy can affect the entire ocular surface including conjunctiva, eyelids, and cornea. According to the immunological mechanism responsible for the final clinical picture, Leonardi et al have classified ocular allergic conditions into three main categories:<sup>[<xref rid=\"B109\" ref-type=\"bibr\">109</xref>]</sup> IgE-mediated reactions, including seasonal allergic conjunctivitis (SAC) and perennial allergic conjunctivitis (PAC); combined IgE and non-IgE-mediated reactions, including VKC and AKC; and non-IgE-mediated reactions, including giant papillary conjunctivitis (GPC) and contact dermatoconjunctivitis (CDC) (Figure 5).</p><sec sec-type=\"subsection\"><title>Seasonal allergic conjunctivitis (SAC) and perennial allergic conjunctivitis (PAC)</title><p>SAC and PAC are considered as the most prevalent allergic ocular conditions, affecting 15–20% of the population.<sup>[<xref rid=\"B110\" ref-type=\"bibr\">110</xref>]</sup> The pathogenesis is predominantly an IgE-mediated hypersensitivity reaction, and allergen-specific IgE antibodies are found in almost all cases of SAC and PAC.<sup>[<xref rid=\"B111\" ref-type=\"bibr\">111</xref>]</sup> Activation of mast cells contributes to increased levels of histamine, prostaglandins, and leukotrienes in the tear film. This phase, which is known as the early response phase, clinically lasts 20–30 min.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup>\n</p><p>SAC, also known as hay fever conjunctivitis, is seen in all age groups. The ocular manifestations occur predominantly during the spring and summer months when pollens from the trees and plants are released into the air. PAC on the other hand can occur throughout the year with exposure to more common allergens such as animal hair, mites, and feathers.<sup>[<xref rid=\"B112\" ref-type=\"bibr\">112</xref>]</sup> Clinical signs and symptoms are similar in SAC and PAC, and include itching and burning of the eyes, tearing, and rhinorrhea. Corneal involvement is rarely seen.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup>\n</p></sec><sec sec-type=\"subsection\"><title>Vernal keratoconjunctivitis (VKC)</title><p>VKC is known as the disease of young males who live in warmer climates.<sup>[<xref rid=\"B113\" ref-type=\"bibr\">113</xref>,<xref rid=\"B114\" ref-type=\"bibr\">114</xref>]</sup> Although VKC is frequently diagnosed in children, adults can also be affected with this condition.<sup>[<xref rid=\"B115\" ref-type=\"bibr\">115</xref>]</sup> A mixture of IgE and non-IgE reaction in response to nonspecific stimuli, such as wind, dust, and sunlight is often elucidated in this condition. Accordingly, skin tests and serum IgE antibody tests to well-known allergens are generally negative.<sup>[<xref rid=\"B116\" ref-type=\"bibr\">116</xref>]</sup> Both clinical and histological findings support the concomitant role of T-helper 2 and IgE in the pathogenesis of VKC.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>,<xref rid=\"B117\" ref-type=\"bibr\">117</xref>]</sup> Recently, IL-17 has been reported to be linked to VKC, where its serum levels can serve as a marker for the severity of the disease.<sup>[<xref rid=\"B118\" ref-type=\"bibr\">118</xref>,<xref rid=\"B119\" ref-type=\"bibr\">119</xref>]</sup> High percentage of antinuclear antibodies (ANA) positivity and family history of autoimmune disorders in patients with VKC suggests a strong link between this condition and other autoimmune disorders including atopy.<sup>[<xref rid=\"B120\" ref-type=\"bibr\">120</xref>,<xref rid=\"B121\" ref-type=\"bibr\">121</xref>]</sup>\n</p><p>Typical seasonal patterns as well as perennial forms have been reported in patients affected with VKC.<sup>[<xref rid=\"B122\" ref-type=\"bibr\">122</xref>]</sup> Presence of papillary hyperplasia is essential for the diagnosis of VKC, and its presence allows for the differentiation of VKC from other related entities such as SAC and PAC.<sup>[<xref rid=\"B123\" ref-type=\"bibr\">123</xref>]</sup>\n</p><p>Conjunctival injection, profuse tearing, severe itching, and photophobia are the main clinical signs and symptoms that are associated with VKC. There are three clinical forms of VKC that include limbal, palpebral, and mixed type.<sup>[<xref rid=\"B112\" ref-type=\"bibr\">112</xref>]</sup> Limbal type is characterized by limbal papillary reaction and gelatinous thickening of the limbus; when the disease is active, Horner-Trantas dots are usually present at the superior limbal margins.<sup>[<xref rid=\"B112\" ref-type=\"bibr\">112</xref>]</sup> The hallmark of the palpebral VKC is the presence of giant papillae, with consequent cobblestone appearance. The mixed type has the features of palpebral and limbal VKC simultaneously (Figure 6).</p><p>The corneal pathology that is seen in VKC is partly caused by the mechanical trauma from the tarsal conjunctival papillae and the inflammatory responses secondary to the release of cytokines. The inflammatory mediators are believed to be released by the eosinophils and mast cells that are infiltrated into the conjunctival tissue.<sup>[<xref rid=\"B124\" ref-type=\"bibr\">124</xref>,<xref rid=\"B125\" ref-type=\"bibr\">125</xref>]</sup> In up to 6% of patients, corneal ulcers (i.e., shields ulcer) and plaques may develop, leading to the exacerbation of the clinical symptoms and worsening of the vision.<sup>[<xref rid=\"B126\" ref-type=\"bibr\">126</xref>,<xref rid=\"B127\" ref-type=\"bibr\">127</xref>]</sup> These ulcers are usually found as oval lesions with elevated margins surrounding a chronic epithelial defect covered by eosinophilic and epithelial debris in the upper parts of the cornea.<sup>[<xref rid=\"B128\" ref-type=\"bibr\">128</xref>]</sup> Keratoconus is another entity that is highly associated with VKC affecting nearly 15% of the patients with this condition.<sup>[<xref rid=\"B129\" ref-type=\"bibr\">129</xref>]</sup>\n</p></sec><sec sec-type=\"subsection\"><title>Atopic keratoconjunctivitis (AKC)</title><p>AKC is characterized by chronic allergic disease of the eyelid, cornea, and conjunctiva. It is considered the ocular component of atopic dermatitis (AD), and roughly 95% of the patients with AKC have concomitant AD;<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>,<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> however, less than half of patients with AD have involvement of their ocular tissue.<sup>[<xref rid=\"B130\" ref-type=\"bibr\">130</xref>]</sup> Many cytokines are released from the epithelial cells of the conjunctiva as well as the inflammatory cells that have infiltrated the conjunctival tissues in AKC. This causes constant remodeling of the ocular surface connective tissue leading to mucus metaplasia, scar formation, and corneal neovascularization.<sup>[<xref rid=\"B131\" ref-type=\"bibr\">131</xref>]</sup>\n</p><p>AKC is typically diagnosed in the second and third decades of life, although scattered cases are seen in the early childhood as well as in the fifth decade of life.<sup>[<xref rid=\"B132\" ref-type=\"bibr\">132</xref>]</sup> Age of the onset, duration of the disease, and clinical presentations may help clinicians to distinguish this condition from VKC.<sup>[<xref rid=\"B132\" ref-type=\"bibr\">132</xref>]</sup>\n</p><p>Clinical manifestation of AKC includes epiphora, itching, redness, and decreased vision. Presentation is often bilateral; however, unilateral disease has been reported.<sup>[<xref rid=\"B133\" ref-type=\"bibr\">133</xref>]</sup> The eyelid skin may be edematous with a sandpaper-like texture. Conjunctival injection and chemosis range from mild to severe, and conjunctival scarring is common.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> Trantas dots and giant papillae may or may not be present. In contrast to VKC, AKC is associated with conjunctival fibrosis and corneal vascularization and opacities. An early cataract surgery is not uncommon in AKC patients, as this condition is associated with formation of “atopic cataracts” at a relatively young age. Shield-like cataracts, as well as nuclear, cortical and even posterior subcapsular cataracts may also occur. Nearly 50% of AKC patients test negative for common allergens.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup>\n</p></sec><sec sec-type=\"subsection\"><title>Giant papillary conjunctivitis (GPC)</title><p>Similar to vernal conjunctivitis, GPC is characterized by papillary hypertrophy of the superior tarsal conjunctiva.<sup>[<xref rid=\"B134\" ref-type=\"bibr\">134</xref>]</sup> Although GPC is primarily considered as a complication of contact lens usage, this condition has also been reported in association with corneal foreign bodies, filtering blebs, ocular prostheses, exposed sutures, limbal dermoids, and tissue adhesives.<sup>[<xref rid=\"B135\" ref-type=\"bibr\">135</xref>,<xref rid=\"B136\" ref-type=\"bibr\">136</xref>,<xref rid=\"B137\" ref-type=\"bibr\">137</xref>]</sup> The classic signs of GPC consist of excessive mucous secretion associated with decreased contact lens tolerance.<sup>[<xref rid=\"B137\" ref-type=\"bibr\">137</xref>]</sup> Mast cells and eosinophils may be found in the conjunctiva; however, there are no increases in the levels of IgE or histamines in the tears of patients with GPC.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup>\n</p><p>GPC can occur with both hydrogel and rigid contact lenses, and it has been reported with either hydroxyethyl methacrylate (HEMA), silicone polymers, or the new gas permeable polymers.<sup>[<xref rid=\"B134\" ref-type=\"bibr\">134</xref>]</sup> However, it is less frequent with rigid contact lenses. Mechanical injuries due to contact lens wear and inflammatory reactions secondary to surface proteins of the lens can contribute to the chronic inflammatory damage of the ocular surface<sup>[<xref rid=\"B110\" ref-type=\"bibr\">110</xref>,<xref rid=\"B138\" ref-type=\"bibr\">138</xref>]</sup> seen in this condition.</p></sec><sec sec-type=\"subsection\"><title>Contact allergy</title><p>CDC is a classic example of type-IV delayed hypersensitivity reaction that occurs through interaction of antigens with T cells followed by release of cytokines.<sup>[<xref rid=\"B139\" ref-type=\"bibr\">139</xref>]</sup> Low molecular weight allergens combine with host proteins to form the final allergens capable of exerting immune response. Some of the known allergens for CDC include poison ivy, poison oak, neomycin, nickel, latex, atropine and its derivatives.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> Primary sensitization phase describes the process through which memory T cells derive from resident T cells of the ocular tissue, while the following elicitation phase includes the interaction between these memory cells and allergens.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> IL-17-producing Th cells and regulatory T cells also play a role in the pathogenesis of CDC.<sup>[<xref rid=\"B140\" ref-type=\"bibr\">140</xref>]</sup>\n</p><p>Similar to AKC, contact allergy involves the conjunctiva, cornea, and eyelids. The condition may be associated with itching, lid swelling, follicular reaction, and even cicatrization in later stages of the disease. The corneal involvement may be in the form of punctate keratitis, pseudodendritic keratitis, and grayish stromal infiltrates.<sup>[<xref rid=\"B112\" ref-type=\"bibr\">112</xref>,<xref rid=\"B141\" ref-type=\"bibr\">141</xref>]</sup>\n</p></sec><sec sec-type=\"subsection\"><title>Treatment</title><p>Avoidance of the allergens is the main stay of treatment for many forms of allergies including allergic conjunctivitis. Artificial tears provide a barrier function, dilute various allergens, and flush the ocular surface clean from many inflammatory mediators.</p><p>The treatment options for allergic conjunctivitis include lubricating eye drops, anti-histamines, and mast cell stabilizers.<sup>[<xref rid=\"B142\" ref-type=\"bibr\">142</xref>,<xref rid=\"B143\" ref-type=\"bibr\">143</xref>]</sup> Many studies have demonstrated the superiority of topical antihistamines and mast cell stabilizers compared to placebo in alleviating the symptoms of allergic conjunctivitis; in addition, it has been demonstrated that antihistamines are more beneficial than mast cell stabilizers for providing short-term relief.<sup>[<xref rid=\"B144\" ref-type=\"bibr\">144</xref>]</sup> Several eye drop preparations with dual action (antihistamine and mast cell-stabilizing effects) including olopatadine, ketotifen, azelastine, and epinastine have been introduced to market in the recent years. These agents can provide simultaneous histamine receptor antagonist effects, stabilize mast-cell membranes, and modify the action of eosinophils.<sup>[<xref rid=\"B145\" ref-type=\"bibr\">145</xref>]</sup> Mast cell stabilizers require a loading period of several weeks, and therefore, they are better to be administered before the antigen exposure.</p><p>Oral antihistamines are commonly used for alleviating the ocular symptoms in patients with allergic conjunctivitis. Second generation antihistamines are preferred due to their fewer adverse systemic side effects.<sup>[<xref rid=\"B146\" ref-type=\"bibr\">146</xref>]</sup> Unfortunately, oral antihistamines induce ocular drying, which can significantly worsen the symptoms of allergic conjunctivitis.<sup>[<xref rid=\"B147\" ref-type=\"bibr\">147</xref>]</sup>\n</p><p>Steroids should be used judiciously and only in selected cases. Topical and oral administration, in addition to supratarsal injections are often required if the condition is severe; unfortunately, any route of corticosteroid administration is associated with formation of cataracts and elevated intraocular pressure.<sup>[<xref rid=\"B112\" ref-type=\"bibr\">112</xref>]</sup> Non-steroidal anti-inflammatory drugs such as ketorolac and diclofenac can also be added to the treatment regimen to provide additional benefits. Moreover, other steroid-sparing agents such as cyclosporine-A and tacrolimus are effective in treating severe and chronic forms of ocular allergies.</p><p>Allergen-specific immunotherapy, which has gained popularity in the recent years, works by inducing clinical tolerance to a specific allergen. This appears to be an effective treatment options for those with allergic rhinoconjunctivitis who demonstrate specific IgE antibodies.<sup>[<xref rid=\"B148\" ref-type=\"bibr\">148</xref>]</sup> Traditionally, immunotherapy is performed via subcutaneous injections; however, sublingual immunotherapy (SLIT) has drawn the attention among allergists as an alternative. SLIT has been shown to effectively reduce the ocular and nasal signs and symptoms of allergic conjunctivitis, with a greater benefit toward improving the nasal symptoms.<sup>[<xref rid=\"B112\" ref-type=\"bibr\">112</xref>]</sup>\n</p></sec></sec><sec sec-type=\"section\"><title> Conjunctivitis associated with systemic diseases</title><p>Conjunctivitis may be the initial presentation for many systemic diseases; therefore, a thorough history and systemic evaluation in selected cases may help in early diagnosis of many potentially disabling and even life-threatening conditions. A summary of systemic diseases associated with conjunctivitis is provided in Figure 7.</p><sec sec-type=\"subsection\"><title>Reactive arthritis</title><p>Conjunctivitis is one of the most common ocular manifestations of reactive arthritis; other associated ocular entities include uveitis, episcleritis, scleritis, and keratitis.<sup>[<xref rid=\"B149\" ref-type=\"bibr\">149</xref>]</sup> Conjunctivitis in reactive arthritis entities manifests itself as conjunctival hyperemia with purulent discharge. Occurring in nearly one third of the patients, conjunctivitis is an essential component of the “Reiter's triad”.<sup>[<xref rid=\"B150\" ref-type=\"bibr\">150</xref>]</sup> Conjunctivitis usually happens early in the course of reactive arthritis and it may even precede it in some instances; given its mild initial clinical presentation, it is often missed. The signs and symptoms usually abate within one to four weeks; however, in some cases, progression to more severe ocular surface problems may ensue.<sup>[<xref rid=\"B151\" ref-type=\"bibr\">151</xref>]</sup>\n</p></sec><sec sec-type=\"subsection\"><title>Rosacea</title><p>Ocular surface may also be involved in the inflammatory course of ocular rosacea. The clinical findings include a follicular and papillary conjunctival reaction in association with interpalpebral conjunctival hyperemia. In addition, cicatrization of the conjunctival tissue, mimicking trachoma, may be seen in these patients. Conjunctival scarring secondary to entropion and trichiasis has been reported to occur in approximately 10% of the cases. Conjunctival granuloma, pinguecula, phlyctenule, and peripheral corneal infiltration and phlyctenule are amongst some of the other findings associated with ocular rosacea.<sup>[<xref rid=\"B152\" ref-type=\"bibr\">152</xref>]</sup>\n</p></sec><sec sec-type=\"subsection\"><title>Graft-versus-host disease</title><p>Conjunctival involvement is rarely seen in acute graft-versus-host disease (GVHD); however, its presence indicates more severe systemic involvement and a poor prognosis. Conjunctival involvement in GVHD ranges from mild conjunctival injection to pseudomembranous and cicatrizing conjunctivitis.<sup>[<xref rid=\"B153\" ref-type=\"bibr\">153</xref>,<xref rid=\"B154\" ref-type=\"bibr\">154</xref>]</sup> In acute GVHD, conjunctivitis is often ulcerative and manifests itself with numerous alternating episodes of conjunctival hemorrhage and exudative discharge. Sterile purulent discharge, pseudomembrane formation, and scarring are amongst the other findings in this condition.<sup>[<xref rid=\"B153\" ref-type=\"bibr\">153</xref>]</sup> In the chronic form of GVHD, one-fourth to three-fourth of the patients suffer from dry eyes, where its severity correlates with the severity of GVHD.<sup>[<xref rid=\"B155\" ref-type=\"bibr\">155</xref>]</sup> Frequently, keratoconjunctivitis sicca persists after remission of GVHD.<sup>[<xref rid=\"B156\" ref-type=\"bibr\">156</xref>]</sup>\n</p><p>Four stages of conjunctival GVHD have been described in the literature. Stage 1 is marked by simple conjunctival injection. Stage 2 is characterized by an exudative response, which may lead to conjunctival chemosis. Stage 3 is characterized by pseudomembrane formation; majority of the patients are diagnosed at this stage of the diseases. Stage 4 is manifested by scarring and cicatrization of the conjunctival tissue.<sup>[<xref rid=\"B153\" ref-type=\"bibr\">153</xref>,<xref rid=\"B156\" ref-type=\"bibr\">156</xref>]</sup>\n</p></sec><sec sec-type=\"subsection\"><title>Ocular cicatricial pemphigoid</title><p>Ocular cicatricial pemphigoid is a rare condition. Patients are often in their fifth and sixth decades of life at presentation, and females are up to three times more frequently affected than males.<sup>[<xref rid=\"B157\" ref-type=\"bibr\">157</xref>]</sup> Chronic inflammation, loss of conjunctival goblet cells along with an abnormal mucosal epithelial turn-over leads to desiccation of the ocular surface in this condition<sup>[<xref rid=\"B158\" ref-type=\"bibr\">158</xref>]</sup> (Figure 8). Disruption of conjunctival immune network increases the risk of ocular surface infection.<sup>[<xref rid=\"B158\" ref-type=\"bibr\">158</xref>]</sup> Recurrent infectious conjunctivitis and trichiasis may lead to keratinization of the surface epithelium.<sup>[<xref rid=\"B158\" ref-type=\"bibr\">158</xref>]</sup> Definitive diagnosis requires direct immunofluorescence, where deposits of immunoglobulins and/or complements produce areas of linear hyperfluorescence at the epithelial basement membrane. Systemic immunosuppression along with frequent lubrication is often needed to adequately control this condition.</p></sec><sec sec-type=\"subsection\"><title>Stevens-Johnson syndrome and toxic epidermal necrolysis</title><p>Ophthalmic manifestations of the acute stages of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) range from conjunctival hyperemia to near-complete sloughing of palpebral conjunctiva and lid margins.<sup>[<xref rid=\"B159\" ref-type=\"bibr\">159</xref>]</sup> Acute ocular involvement is reported to occur in up to 88% of the cases.<sup>[<xref rid=\"B159\" ref-type=\"bibr\">159</xref>]</sup> It remains unclear whether the severity of ocular involvement is any different between SJS and TEN.<sup>[<xref rid=\"B160\" ref-type=\"bibr\">160</xref>]</sup> Long-term adverse consequences following the acute stage of ocular surface disease include severe dry eyes, symblepharon formation, corneal limbal stem cell deficiency, and corneal scarring.<sup>[<xref rid=\"B160\" ref-type=\"bibr\">160</xref>]</sup>\n</p></sec></sec><sec sec-type=\"section\"><title> Toxic conjunctivitis</title><p>It has been recently realized that long-term use of topical eye medications may induce ocular surface changes including dry eyes, conjunctival inflammation, ocular surface fibrosis, and scarring.<sup>[<xref rid=\"B161\" ref-type=\"bibr\">161</xref>,<xref rid=\"B162\" ref-type=\"bibr\">162</xref>]</sup> Another area where the side effects of topical eye drops cause significant ocular morbidity is their use in glaucoma and in patients who have undergone glaucoma surgery. Subclinical infiltration of the conjunctival epithelium and substantia propria by inflammatory cells has also been reported.<sup>[<xref rid=\"B163\" ref-type=\"bibr\">163</xref>,<xref rid=\"B164\" ref-type=\"bibr\">164</xref>]</sup> The published literature during the past decade has pointed to the deleterious effects of benzalkonium chloride (BAK), which is used as a preservative in eye drops, on the ocular surface.<sup>[<xref rid=\"B165\" ref-type=\"bibr\">165</xref>]</sup>\n</p><p>Allergic reactions are the most clinically noticeable side effect of the eye drops; however, they are far less frequent and harmful than their adverse toxic side effects.<sup>[<xref rid=\"B166\" ref-type=\"bibr\">166</xref>]</sup> The allergic reaction to eye drops includes simple conjunctival congestion, papillary conjunctivitis, and GPC.<sup>[<xref rid=\"B165\" ref-type=\"bibr\">165</xref>]</sup> The signs and symptoms usually manifest a few days after starting the offending eye drop and tend to resolve quickly when the medication is stopped.<sup>[<xref rid=\"B166\" ref-type=\"bibr\">166</xref>]</sup>\n</p><p>Observational studies have confirmed the high prevalence of dry eyes in glaucoma patients related to the number of eye drops being used. This ranges from 11% in those who use only one eye drop to 43% in those who use two or three different eye drops.<sup>[<xref rid=\"B167\" ref-type=\"bibr\">167</xref>]</sup> Similarly, a cross-sectional study evaluating the ocular surface in 101 patients being treated for glaucoma reported that approximately 60% of them were symptomatic in at least one eye.<sup>[<xref rid=\"B168\" ref-type=\"bibr\">168</xref>]</sup> In a survey performed on 300 patients in the US between 2001 and 2004, adverse side effects were reported to be the second most common reason for switching eye drops.<sup>[<xref rid=\"B169\" ref-type=\"bibr\">169</xref>]</sup>\n</p><p>Increase in fibroblast density in the conjunctiva, and development of subconjunctival fibrosis has been reported in patients who use antiglaucoma drops chronically.<sup>[<xref rid=\"B165\" ref-type=\"bibr\">165</xref>]</sup> In a series of 145 patients, Thorne et al reported that exposure to antiglaucoma eye drops was the primary reason for development of pseudopemphigoid.<sup>[<xref rid=\"B170\" ref-type=\"bibr\">170</xref>]</sup>\n</p><p>Despite the indisputable data and the findings from multiple observational studies on the harmful side effects of BAK, it is still used as the main preservative ingredient in most eye drop preparations due to lack of a better alternative.<sup>[<xref rid=\"B165\" ref-type=\"bibr\">165</xref>]</sup> Limiting the exposure to preservatives may diminish the toxic side effects of eye drops; this will likely lead to higher patient compliance and result in a more favorable clinical outcome, especially in those who need to be on antiglaucoma medications.</p></sec><sec sec-type=\"section\"><title> Summary</title><p>Approximately 1% of all patient visits to their primary care physician is conjunctivitis related, and the estimated cost of infectious conjunctivitis to the healthcare is more than $800 million annually in the US alone.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> The first step in approaching a patient with presumed conjunctivitis is to rule out serious ocular conditions that present with “red eye”, mimicking conjunctivitis. This must be done with obtaining a thorough history and performing a detailed ophthalmologic and physical examination. Ancillary laboratory testing and imaging are also important components of evaluating these patients. Various studies have demonstrated that obtaining a thorough history is essential to narrow down the differential diagnosis and discover the underlying etiology for the conjunctivitis, while relying solely on the presenting signs and symptoms can be misleading and often leads to an inaccurate diagnosis. Viral conjunctivitis followed by bacterial conjunctivitis are the most common causes of infectious conjunctivitis.<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>,<xref rid=\"B25\" ref-type=\"bibr\">25</xref>,<xref rid=\"B81\" ref-type=\"bibr\">81</xref>]</sup> The majority of viral conjunctivitis cases are due to adenoviruses,<sup>[<xref rid=\"B28\" ref-type=\"bibr\">28</xref>]</sup> and the use of rapid antigen test to diagnose adenoviral conjunctivitis may present an appropriate strategy to avoid overuse of antibiotics. Bacterial pathogens are isolated in half of the cases of conjunctivitis,<sup>[<xref rid=\"B61\" ref-type=\"bibr\">61</xref>]</sup> and approximately 60% of culture-positive cases are known to be self-limited.<sup>[<xref rid=\"B80\" ref-type=\"bibr\">80</xref>]</sup> Cultures should be obtained from the conjunctival swabs of patients that do not respond to therapy, and those suspected to have chlamydial infection and hyperacute conjunctivitis.<sup>[<xref rid=\"B47\" ref-type=\"bibr\">47</xref>]</sup> Treatment with topical antibiotics is usually recommended for suspected cases of chlamydial and gonococcal conjunctivitis and contact lens wearers.<sup>[<xref rid=\"B61\" ref-type=\"bibr\">61</xref>,<xref rid=\"B80\" ref-type=\"bibr\">80</xref>]</sup> The majority of cases of allergic conjunctivitis are due to seasonal allergies. Antihistamines and mast cell stabilizers are widely used for treating allergic conjunctivitis. Steroids must be used judiciously and only when indicated. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"editorial\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864077</article-id><article-id pub-id-type=\"pmc\">PMC7431718</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7465</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Letter to Editor</subject></subj-group></article-categories><title-group><article-title>How Much of Hazardous Blue Light is Transmitted By Spectacle Lenses?</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Rahmani</surname><given-names>Saeed</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Nazari</surname><given-names>Mohammadreza</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Baghban</surname><given-names>Alireza Akbarzadeh</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ghassemi-Broumand</surname><given-names>Mohammad</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Department of Optometry, School of Rehabilitation, Shahid Beheshti University of Medical Sciences, Tehran, Iran</aff><aff id=\"I2\">\n<sup>2</sup>Proteomics Research Center, Department of Biostatistics, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Saeed Rahmani, PhD. Department of Optometry, School\nof Rehabilitation Sciences, Shahid Beheshti University\nof Medical Sciences, Opposite to Bou-Ali Hospital,\nDamavand Ave., Tehran, Iran.\nE-mail: medicalopto@yahoo.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>435</fpage><lpage>437</lpage><permissions><copyright-statement>Copyright © 2020 Rahmani et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><counts><fig-count count=\"2\"/><ref-count count=\"5\"/><page-count count=\"3\"/></counts></article-meta></front><body><p>Dear Editor,</p><p>The first region of the visible light spectrum is called blue light. Blue light is beneficial to humans in color vision, night vision, and circadian rhythms.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> However, this type of light raises concerns as it carries high energy and can cause ocular damages, such as photic retinopathy. In addition to the sun, there are several artificial sources of blue light emission, such as light-emitting diodes (LEDs), light bulbs, and fluorescent light tubes. With the increasing use of digital blue-rich LED-backlight displays, such as in mobile devices and tablets, users' eyes are more exposed to blue light.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup> Blue light can also induce eyestrain, however, the blue light-blocking lenses may reduce eye fatigue.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup>\n</p><p>Currently, some lens manufactures claim that their products can alleviate eyestrain and ocular discomfort associated with the use of digital devices.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> This raises important questions about the efficacy of blue light-control lenses. Therefore, eight blank-white spectacle lenses (four with and four without blue light-blocking property) were collected from different optical companies. A spectrophotometer (Cecil Instrument, UK) was used to measure the blue light transmission. Three ranges of blue light were evaluated: 400–450 nm, 455–500 nm, and 400–500 nm. For the statistical analysis, non-parametric Mann–Whitney test was employed. A <italic>P-value</italic>\n<inline-formula><mml:math id=\"M1\"><mml:mo>≤</mml:mo></mml:math></inline-formula> 0.05 was considered statistically significant.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Blue light transmission of lenses with and without blue light-blocking property in different wavelengths.</p></caption><graphic xlink:href=\"jovr-15-435-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Comparison between spectral transmittance of lenses with and without blue light-blocking property.</p></caption><graphic xlink:href=\"jovr-15-435-g002\"/></fig><p>The mean transmission of blue light through lenses with and without blue light-blocking coating in the range of 400–455 nm were 58.76 <inline-formula><mml:math id=\"M2\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.01% and 83.10 <inline-formula><mml:math id=\"M3\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.71%, respectively. The differences were statistically significant (<italic>P =</italic> 0.02). The harmful portion of blue light is accumulated in this range as previous studies have shown that using filters capable of 50% reduction in 430 nm blue light transmission can prevent approximately 80% of photochemical damage to the retina. Notably, there is currently no strict guideline for blue light-blocking coatings.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup>\n</p><p>The mean transmission of blue light through lenses with and without blue light-blocking coating in the range of 455–500 nm were 95.58 <inline-formula><mml:math id=\"M4\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.46% and 96.00 <inline-formula><mml:math id=\"M5\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.57%, respectively. The differences were not statistically significant (<italic>P</italic> = 0.39). Higher wavelengths, that is, 455–500 nm, are considered useful light for color vision and circadian rhythm.</p><p>The mean transmission of blue light through lenses with and without blue light-blocking coating in the range of 400–500 nm were 75.33 <inline-formula><mml:math id=\"M6\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.51% and 88.40 <inline-formula><mml:math id=\"M7\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.63%, respectively. The differences were statistically significant (<italic>P</italic> = 0.02). The lenses with blue light-blocking property could reduce the blue light transmission by approximately 25% in the wavelength range of 400–500 nm. Thus, the filtering value was twice the amount in the lenses without blue light-blocking property (Figures 1 and 2).</p><p>Finally, the spectacle lenses with blue light-blocking property could effectively attenuate hazardous lights. It is recommended to use the spectacle lenses that are equipped with blue light-blocking coating to reduce the risk of ocular diseases attributed to hazardous blue light.</p><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ack><title> Acknowledgments</title><p>The authors thank the Research Affairs of the Shahid Beheshti University of Medical Sciences for their support.</p></ack><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Giannos SA, Kraft ER, Lyons LJ, Gupta PK. Spectral evaluation of eyeglass blocking efficiency of ultraviolet/high-energy visible blue light for ocular protection. <italic>Optom Vis Sci </italic>2019;96:513–522.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">Leung TW, Li RW, Kee CS. Blue-light filtering spectacle lenses: optical and clinical performances. <italic>PLOS ONE </italic>2017;12:e0169114.</mixed-citation></ref><ref id=\"B3\"><label>3</label><mixed-citation publication-type=\"other\">Smith BT, Belani S, Ho AC. Ultraviolet and near-blue light effects on the eye. <italic>Int Ophthalmol Clin </italic>2005;45:107–115.</mixed-citation></ref><ref id=\"B4\"><label>4</label><mixed-citation publication-type=\"other\">Ide T, Toda I, Miki E, Tsubota K. Effect of blue light-reducing eye glasses on critical flicker frequency. <italic>Asia Pac J Ophthalmol (Phila) </italic>2015;4:80–85.</mixed-citation></ref><ref id=\"B5\"><label>5</label><mixed-citation publication-type=\"other\">Downie LE. Blue-light filtering ophthalmic lenses: to prescribe, or not to prescribe? <italic>Ophthalmic Physiol Opt </italic>2017;37:640–643.</mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864075</article-id><article-id pub-id-type=\"pmc\">PMC7431719</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7463</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Photo Essay</subject></subj-group></article-categories><title-group><article-title>Cytomegalovirus Retinitis in a Patient on Long-term Mycophenolate Mofetil Treatment for Myasthenia Gravis</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Patel</surname><given-names>Shyam</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Robin</surname><given-names>Alexander</given-names></name><degrees>BS</degrees></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Department of Ophthalmology, Cook County Hospital, Chicago, IL, USA</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Shyam Patel, MD. Department of Ophthalmology, Cook\nCounty Hospital, 1900 West Polk St., Ste. 617, Chicago,\n60612, IL, USA.\nEmail: spatel0687@gmail.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>428</fpage><lpage>431</lpage><history><date date-type=\"received\"><day>01</day><month>4</month><year>2018</year></date><date date-type=\"accepted\"><day>19</day><month>6</month><year>2019</year></date></history><permissions><copyright-statement>Copyright © 2020 Patel et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><counts><fig-count count=\"3\"/><ref-count count=\"5\"/><page-count count=\"4\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> PRESENTATION</title><p>A 64-year-old man with myasthenia gravis (MG) presented with blurry vision in his left eye (OS). His visual acuity was 20/20 in the right eye and 20/50 OS. His intraocular pressures, pupils, and anterior segment were normal. He had 1+ vitritis and vaso-occlusive appearance of the retina with sclerotic vessels and hemorrhages in the inferonasal quadrant OS (Figure 1), consistent with features of cytomegalovirus (CMV) retinitis. He was immunocompromised secondary to mycophenolate mofetil (MMF) administered for MG, with 0.6% lymphocytes (normal: 26.0–46.0%), a lymphocyte count of 0.1<inline-formula><mml:math id=\"M1\"><mml:mo>×</mml:mo></mml:math></inline-formula>10<inline-formula><mml:math id=\"M2\"><mml:msup><mml:mrow/><mml:mn>3</mml:mn></mml:msup></mml:math></inline-formula> cells/μL, and a leukocyte count of 11.3<inline-formula><mml:math id=\"M3\"><mml:mo>×</mml:mo></mml:math></inline-formula>10<inline-formula><mml:math id=\"M4\"><mml:msup><mml:mrow/><mml:mn>3</mml:mn></mml:msup></mml:math></inline-formula> cells/μL. He was receiving 1000 mg of MMF BID. The human immunodeficiency virus (HIV) test result was negative, and no further workup for immunosuppression, including cancer, was conducted.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>(A) A funduscopic photo of the left eye shows fluffy white lesions with intraretinal hemorrhage predominantly in the inferonasal quadrant. (B) A funduscopic photo of the left inferonasal quadrant five weeks after treatment initiation.</p></caption><graphic xlink:href=\"jovr-15-428-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>(A) OCT of the macula of the left eye before treatment initiation showing vitreomacular adhesion and few vitreous cells. (B) OCT of the macula of the left eye after 10 weeks from treatment initiation showing a fine epiretinal membrane.</p></caption><graphic xlink:href=\"jovr-15-428-g002\"/></fig><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>The inferonasal fundus photo of the left eye at 10 weeks showing preretinal fibrosis with a corresponding OCT (over the white lesion, see Arrow) showing an atrophic retina with resolved vitritis. A corresponding infrared image showing the line of scan of the OCT.</p></caption><graphic xlink:href=\"jovr-15-428-g003\"/></fig><p>The patient was administered with 0.05 mL ganciclovir (4 mg/0.1 mL) and 0.10 mL foscarnet (2.4 mg/0.1 mL) intravitreal injections on diagnosis. Subsequently, his symptoms improved, and a 10-week course of oral valganciclovir (900 mg BID for 21 days followed by 900 mg QD for seven weeks) was administered. There was also a decrease in the dosage and eventual cessation of MMF with initiation of intravenous immunoglobulins. His lymphocytes improved to 9.8% (lymphocyte count, 0.7<inline-formula><mml:math id=\"M5\"><mml:mo>×</mml:mo></mml:math></inline-formula>10<inline-formula><mml:math id=\"M6\"><mml:msup><mml:mrow/><mml:mn>3</mml:mn></mml:msup></mml:math></inline-formula> cells/μL; leukocyte count, 6.9<inline-formula><mml:math id=\"M7\"><mml:mo>×</mml:mo></mml:math></inline-formula>10<inline-formula><mml:math id=\"M8\"><mml:msup><mml:mrow/><mml:mn>3</mml:mn></mml:msup></mml:math></inline-formula> cells/μL).</p><p>On valganciclovir discontinuation in week 10, the patient had a visual acuity of 20/25 OS with no inflammation and improvements in retinal hemorrhages and lesions. An epiretinal membrane was observed on macular optical coherence tomography (Figure 2). The inferonasal retina showed inactive whitish atrophy (Figure 3).</p></sec><sec sec-type=\"section\"><title> DISCUSSION</title><p>CMV retinitis is the most common ocular opportunistic infection associated with acquired immune deficiency syndrome.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> The prevalence of CMV retinitis in HIV patients has decreased since the advent of highly active antiretroviral therapy (HAART).<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> However, the rate of CMV retinitis in non-HIV patients is increasing, likely due to the use of aggressive immunosuppressive agents.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> CMV is an infectious complication frequently associated with MMF.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup>\n</p><p>Visual prognosis of CMV infection in non-HIV patients is similar to that in HIV patients with poor visual outcomes associated with retinal detachments and macular involvement.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> CMV retinitis in patients with concomitant HIV infection lacks vitreous involvement.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> In contrast, vitritis is more commonly associated with non-HIV-related CMV retinitis infections.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> This is consistent with our patient's presentation.</p><p>CMV retinitis treatment in HIV patients involves HAART and antiviral therapy.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> In non-HIV patients, different etiologies of an immunocompromised state must be considered. Commonly used treatment strategies include systemic ganciclovir, foscarnet, valganciclovir, and intravitreal ganciclovir. Our patient received one initial intravitreal injection each of ganciclovir and foscarnet, as well as oral valganciclovir. Intravitreal injections are important for the treatment of vision-threatening CMV infections and were used in our case.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup> Nevertheless, the mainstay treatment of CMV retinitis remains systemic antivirals, and it is not always necessary to start with intravitreal injections. The combination of intravitreal ganciclovir and foscarnet is effective in treating CMV retinitis.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup>\n</p><p>In summary, we presented a patient with MG who developed CMV retinitis due to immunosuppression as a result of MMF treatment. He was treated successfully with intravitreal and systemic antivirals.</p></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>Nil.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Kim D, Jo J, Joe S, Kim J, Yoon Y, Lee J. Comparison of visual prognosis and clinical features of cytomegalovirus retinitis in HIV and non-HIV patients. <italic>Retina</italic> 2017;37:376–381.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">Iu L, Fan M, Lau J, Chan T, Kwong Y, Wong I. Long-term follow-up of Cytomegalovirus Retinitis in non-HIV immunocompromised patients: clinical features and visual prognosis. <italic>Am J Ophthalmol </italic>2016;165:145–153.</mixed-citation></ref><ref id=\"B3\"><label>3</label><mixed-citation publication-type=\"other\">Meier-Kriesche HU, Friedman G, Jacobs M, Mulgaonkar S, Vaghela M, Kaplan B. Infectious complications in geriatric renal transplant patients: comparison of two immunosuppressive protocols.<italic> Transplantation</italic> 1999;68:1496–1502.</mixed-citation></ref><ref id=\"B4\"><label>4</label><mixed-citation publication-type=\"other\">Pearce W, Yeh S, Fine H. Management of Cytomegalovirus Retinitis in HIV and non-HIV patients. <italic>Ophthalmic Surg Lasers Imaging Retina</italic> 2016;47:103–107.</mixed-citation></ref><ref id=\"B5\"><label>5</label><mixed-citation publication-type=\"other\">Velez G, Roy CE, Whitcup SM, Robinson MR. High-dose intravitreal ganciclovir and foscarnet for cytomegalovirus retinitis. <italic>Am J Ophthalmol </italic>2001;131:396–397.</mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"editorial\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864056</article-id><article-id pub-id-type=\"pmc\">PMC7431720</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7444</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Editorial</subject></subj-group></article-categories><title-group><article-title>The Development Pathway for Biosimilar Biotherapeutics</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Zarbin</surname><given-names>Marco</given-names></name><degrees>MD, PhD</degrees></contrib></contrib-group><aff id=\"I1\">Institute of Ophthalmology and Visual Science, New Jersey Medical School, Rutgers University, Newark, USA</aff><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>273</fpage><lpage>274</lpage><permissions><copyright-statement>Copyright © 2020 Zarbin.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><counts><ref-count count=\"8\"/><page-count count=\"2\"/></counts></article-meta></front><body><p>Many physicians have experience using generic drugs in their practice. Generics tend to be small molecules with a relatively simple structure and are identical to licensed reference products. Generic compounds typically are synthesized using organic medicinal chemistry. Variations in the manufacturing process are unlikely to have a major impact on the final product, an outcome that is verified through analytical characterization of the generic.</p><p>A biosimilar biotherapeutic is a protein. Biosimilar products are quite different from generic drugs. A biosimilar has similar quality, safety, and efficacy to a licensed reference product, but it is not necessarily identical to the reference product with regard to these properties.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> In contrast to generic drugs, biosimilars tend to have complex structures and may differ from the reference product in their primary amino acid sequence and other features such as glycosylation and PEGylation that alter their tertiary structure (i.e., protein folding) as well as their immunogenicity.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> These differences arise from the manufacturing process, which tends to be much more complex than that of generic drugs. Typically, a biosimilar protein is synthesized by transfecting a target cell with a DNA sequence that encodes the desired product. Often, transfected mammalian cells are required to produce complex proteins, but these cells typically have lower yields than bacterial hosts. The initial product must be purified to remove undesired proteins. As one might expect, the use of different expression systems can be associated with different post-translational protein modifications.</p><p>Changes in the manufacturing process are thus critical (and essential to avoid patent infringement on proprietary biomanufacturing processes), as they may alter protein structure and function. Analytical characterization of these compounds is not straightforward and, in any case, is not expected to reveal structure and properties identical to the reference product if different vectors, expression systems, purification steps, and excipients are used in the manufacturing process. Analysis of the immunogenicity of a biosimilar, for example, is a critical aspect of evaluating the therapeutic modality, whereas a generic drug is expected to be identical to the reference product in this regard. The development process and quality control for biosimilars are challenging,<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup> which helps to explain why the manufacturing costs for biosimilars and generic drugs are quite different, averaging $100–200 million/molecule for the former and $3–5 million/molecule for the latter. Accordingly, the price reduction for biosimilars versus generic drugs is less and might be on the order of 20–30%.</p><p>Clinical trials of biosimilars must demonstrate safety and efficacy comparable to the reference product regarding pharmacokinetic, pharmacodynamic, and immunogenic properties. If phase 3 studies are successful and a biosimilar is approved for one indication, it is approved for all other indications for which the reference product is approved, provided there is adequate scientific justification.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> In general, there is an expectation that a patient can switch from a biosimilar to the reference product and vice versa with no lapse in therapeutic efficacy or increased risk. Switching studies demonstrating interchangeability of biosimilars and reference products have not been required for marketing approval by the European Medicine Agency,<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> whereas they are required by the US Food and Drug Administration (US FDA) (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.fda.gov/media/124907/download\">https://www.fda.gov/media/124907/download</ext-link>). In order to assist physicians in identifying biosimilars versus the reference product and avoid inadvertent product substitution, the US FDA determined that each biosimilar's name should comprise a core name hyphenated with a four-letter suffix representing the developer.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup>\n</p><p>Studies such as the one reported by Lashay and coworkers in this issue of the <italic>Journal of Ophthalmic and Vision Research</italic> constitute an essential step toward adopting use of Stivant, a biosimilar to bevacizumab, for non-approved ophthalmic indications.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> This work has been executed expertly and provides reassurance that Stivant may well be an appropriate substitute for intravitreal bevacizumab, which is more expensive. The authors qualify their results with great care, but it may be worth emphasizing a few points. First, the rabbit retina is merangiotic and has no fovea. Apart from the inability to identify drug effects on foveal function, these and other features of the rabbit eye may lead to differences in the intraocular and systemic pharmacokinetic profile of the drug associated with intravitreal injection in human patients. Also, although the dose administered was much higher than that anticipated for human subjects, a dose response curve was not undertaken. While we may conclude that a dose of 1.25 mg in a normal size human eye is likely to be safe, we do not know the upper bound of a safe dose based on the data provided. Naturally, these animals had healthy eyes. We do not know whether the safety profile observed in this work will be the same in eyes with damaged retina, retinal pigment epithelium, and/or choroid, as will be encountered in patients with diabetic retinopathy, retinal vein occlusion, and age-related macular degeneration. Of course, these same limitations apply to studies using bevacizumab in rabbits, but biosimilars can differ in subtle ways from the licensed product they mimic. Nonetheless, the data provided by Lashay and coworkers are positive and justify additional studies that will enable Stivant to be deployed for clinical use in patients with retinal vascular diseases. Ultimately, efforts such as these will enable us to provide sight-saving therapy to many more patients through the cost savings realized by the use of biosimilars. I commend the authors for this excellent work and look forward to additional progress in this area. We and our patients will benefit enormously from their efforts.</p><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>Nil.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Harvey RD. Science of Biosimilars. <italic>J Oncol Pract</italic> 2017;13:17s–23s.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">Tsuruta LR, Lopes dos Santos M, Moro AM. Biosimilars advancements: Moving on to the future<italic>.</italic>\n<italic>Biotechnol Prog</italic> 2015;31:1139–1149.</mixed-citation></ref><ref id=\"B3\"><label>3</label><mixed-citation publication-type=\"other\">Sharma A, Kumar N, Kuppermann BD, Bandello F, Loewenstein A. Biotherapeutics and immunogenicity: ophthalmic perspective<italic>.</italic>\n<italic>Eye</italic> 2019;33:1359–1361.</mixed-citation></ref><ref id=\"B4\"><label>4</label><mixed-citation publication-type=\"other\">Sharma A, Kumar N, Kuppermann BD, Francesco B, Lowenstein A. Ophthalmic biosimilars: Lessons from India. <italic>Indian J Ophthalmol</italic> 2019;67;1384–1385.</mixed-citation></ref><ref id=\"B5\"><label>5</label><mixed-citation publication-type=\"other\">Macdonald JC, Hartman H, Jacobs IA. Regulatory considerations in oncologic biosimilar drug development. <italic>MAbs</italic> 2015;7:653–661.</mixed-citation></ref><ref id=\"B6\"><label>6</label><mixed-citation publication-type=\"other\">Scavone C, Rafaniello C, Berrino L, Rossi F, Capuano A. Strengths, weaknesses and future challenges of biosimilars' development. An opinion on how to improve the knowledge and use of biosimilars in clinical practice. <italic>Pharmacol Res</italic> 2017;126:138–142.</mixed-citation></ref><ref id=\"B7\"><label>7</label><mixed-citation publication-type=\"other\">CBER/CDER. Nonproprietary naming of biological products: guidance for industry<italic>.</italic> Rockville, MD: US Food and Drug Administration; 2017.</mixed-citation></ref><ref id=\"B8\"><label>8</label><mixed-citation publication-type=\"other\">Lashay A, Faghihi H, Mirshahi A, Khojasteh H, Khodabande A, Riazi-Esfahani H, et al. Safety of intravitreal injection of Stivant, a biosimilar to bevacizumab, in rabbit eyes. <italic>J Ophthalmic Vis Res</italic> 2020;15:341–350.</mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864058</article-id><article-id pub-id-type=\"pmc\">PMC7431721</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7446</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>In Vivo Corneal Microstructural Changes in Herpetic Stromal Keratitis: A Spectral Domain Optical Coherence Tomography Analysis</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Rodriguez-Garcia</surname><given-names>Alejandro</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Alfaro-Rangel</surname><given-names>Raul</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Bustamante-Arias</surname><given-names>Andres</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Hernandez-Camarena</surname><given-names>Julio C.</given-names></name><degrees>MD, PhD</degrees></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Tecnologico de Monterrey, School of Medicine and Health Sciences, Institute of Ophthalmology and Visual Sciences, Cornea and External Diseases Service,\nMonterrey, Mexico</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Alejandro Rodriguez-Garcia, MD. Centro Medico\nZambrano Hellion (Piso 1, Ote.) Av., Batallon de San\nPatricio No. 112. Col. Real de San Agustin. San Pedro\nGarza Garcia, N.L. México. CP. 66278.\nE-mail: immuneye@gmail.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>279</fpage><lpage>288</lpage><history><date date-type=\"received\"><day>14</day><month>6</month><year>2019</year></date><date date-type=\"accepted\"><day>05</day><month>2</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Rodriguez-Garcia et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><sec><title>Purpose</title><p>To describe and analyze the microstructural changes in herpetic stromal keratitis (HSK) observed <italic>in vivo</italic> by spectral-domain ocular coherence tomography (SD-OCT) at different stages of the disease.</p></sec><sec><title>Methods</title><p>A prospective, cross-sectional, observational, and comparative SD-OCT analysis of corneas with active and inactive keratitis was performed, and the pathologic differences between the necrotizing and non-necrotizing forms of the disease were analyzed.</p></sec><sec><title>Results</title><p>Fifty-three corneas belonging to 43 (81.1%) women and 10 (18.8%) men with a mean age of 41.0 years were included for analysis. Twenty-four (45.3%) eyes had active keratitis, and 29 (54.7%) had inactive keratitis; the majority (83.0%) had the non-necrotizing form. Most corneas (79.1%) with active keratitis showed stromal edema and inflammatory infiltrates. Almost half of the active lesions affected the visual axis, were found at mid-stromal depth, and had a medium density. By contrast, corneas with inactive keratitis were characterized by stromal scarring (89.6%), epithelial remodeling (72.4%), and stromal thinning (68.9%). In contrast to non-necrotizing corneas, those with necrotizing HSK showed severe stromal scarring, inflammatory infiltration, and thinning. Additionally, most necrotizing lesions (77.7%) affected the visual axis and had a higher density (<italic>P</italic> = 0.01).</p></sec><sec><title>Conclusion</title><p>Active HSK is characterized by significant epithelial and stromal thickening and the inactive disease manifests epithelial remodeling at sites of stromal thinning due to scarring. Necrotizing keratitis is characterized by distorted corneal architecture, substantial stromal inflammatory infiltration, and thinning. <italic>In vivo</italic> SD-OCT analysis permitted a better understanding of the inflammatory and repair mechanisms occurring in this blinding corneal disease.</p></sec></abstract><kwd-group><kwd>Corneal Infection</kwd><kwd> Herpetic Keratitis</kwd><kwd> HSV-1</kwd><kwd> SD-OCT</kwd><kwd> Stromal Edema</kwd></kwd-group><counts><fig-count count=\"3\"/><table-count count=\"3\"/><ref-count count=\"32\"/><page-count count=\"10\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> INTRODUCTION</title><p>Herpetic keratitis is the major cause of corneal blindness in many developed countries, with the stromal form representing almost one-third (29.5%) of all cases.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> Herpetic stromal keratitis (HSK) accounts for up to 44% of recurrences, and the risk for recurrent infection increases after multiple attacks of keratitis.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup> Therefore, HSK represents a significant burden of ocular disease, being the most feared presentation of herpetic corneal infection due to its severe damage to the cornea.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> Most cases of HSK present as non-necrotizing.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> Non-necrotizing HSK or immune stromal keratitis is characterized by stromal inflammation leading to scarring, thinning, and vascularization of the cornea.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> The direct viral antigen stimulation of HSV-1-specific CD4<inline-formula><mml:math id=\"M1\"><mml:mrow><mml:mi>α</mml:mi><mml:mi>β</mml:mi></mml:mrow></mml:math></inline-formula> T-lymphocytes probably drives stromal inflammation. Other likely pathogenic mechanisms are autoantigens unmasked and mimicked by HSV-1 corneal infection, bystander cytokine activation of CD4<inline-formula><mml:math id=\"M2\"><mml:mrow><mml:mi>α</mml:mi><mml:mi>β</mml:mi></mml:mrow></mml:math></inline-formula> T-cells, or a combination of all these mechanisms.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>,<xref rid=\"B10\" ref-type=\"bibr\">10</xref>,<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> On the other hand, the necrotizing form is characterized by ulcerations, dense leukocytic stromal infiltration, and necrosis that may rapidly progress to corneal perforation.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>,<xref rid=\"B10\" ref-type=\"bibr\">10</xref>]</sup> Both viral antigens and replicating virions have been implicated in the pathogenesis of this form of keratitis.<sup>[<xref rid=\"B12\" ref-type=\"bibr\">12</xref>,<xref rid=\"B13\" ref-type=\"bibr\">13</xref>]</sup>\n</p><p>Most of our knowledge of the pathologic alterations seen in HSK is based on direct slit-lamp observations and histopathologic findings from fixed tissues.<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>,<xref rid=\"B15\" ref-type=\"bibr\">15</xref>]</sup> Recently, <italic>in vivo</italic> confocal microscopy analysis of herpetic keratitis has shown that a significant and gradual decrease in superficial epithelial cell density is correlated with decreased corneal innervation.<sup>[<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup> The same imaging technique has also been used to monitor the inflammatory process of HSK.<sup>[<xref rid=\"B17\" ref-type=\"bibr\">17</xref>]</sup> Spectral-domain optical coherence tomography (SD-OCT) provides noncontact <italic>in vivo</italic> corneal cross-sectional, high-resolution images that allow a detailed delineation of the cornea.<sup>[<xref rid=\"B18\" ref-type=\"bibr\">18</xref>]</sup> The high speed of the technique permits the acquisition of high-definition frames with few motion artifacts.<sup>[<xref rid=\"B19\" ref-type=\"bibr\">19</xref>,<xref rid=\"B20\" ref-type=\"bibr\">20</xref>]</sup> Enlarged sections of averaged images permit the discrimination of all corneal layers.<sup>[<xref rid=\"B21\" ref-type=\"bibr\">21</xref>]</sup> SD-OCT also enables more accurate measurements of the corneal thickness at a particular point, or in a 6 mm diameter pachymetry map even in the presence of corneal irregularity or opacity.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>,<xref rid=\"B22\" ref-type=\"bibr\">22</xref>]</sup>\n</p><p>The primary purpose of the present study was to describe and analyze the microstructural corneal alterations observed by SD-OCT during the active and inactive stages of HSK and to compare those changes between non-necrotizing and necrotizing keratitis. This analysis adds to our understanding of the inflammatory and repair mechanisms of the cornea in this sight-threatening disease.</p></sec><sec sec-type=\"section\"><title> METHODS</title><p>This is a prospective, cross-sectional, descriptive, comparative, and observational study of patients diagnosed with HSK based on clinical findings. The inclusion criteria for the disease diagnosis were based on previous medical history, clinical manifestations, and therapeutic response to herpes-specific antiviral therapy. Past medical histories of mucocutaneous herpetic vesicular eruption, oral herpetic stomatitis, and herpetic blepharoconjunctivitis were all considered for the diagnosis. Previous or current clinical manifestations consisting of herpetic epithelial and/or stromal keratitis, characterized by dendritic or geographic ulceration, inflammatory stromal infiltration, stromal edema, and hypoesthesia were required for inclusion in the study. Corneal examination parameters performed on all patients included a refractive power analysis (OPD-Scan III, Nidek Co., LTD. Japan), a detailed comparative correlative slit lamp exam, including colored photographs and schematic drawings of the corneal lesions, as well as qualitative esthesiometry and fluorescein and lissamine green staining. Therapeutic response to conventional antiviral therapy was also considered necessary for the diagnosis.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B8\" ref-type=\"bibr\">8</xref>][<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup>\n</p><p>The exclusion criteria consisted of an unwillingness to participate in the study, any previous history of corneal pathology such as trauma, chemical burns, bacterial, fungal, adenoviral, or protozoan infection, and other forms of inflammatory or infectious external disease. Corneas with disciform keratitis, endotheliitis, anterior uveitis, or neurotrophic keratitis were excluded from the analysis. Additionally, cases with a doubtful diagnosis or with atypical HSK defined as corneas where dendritic or geographical ulcers, epithelial infiltration, and hypoesthesia were not evident were excluded from the analysis.</p><p>Before inclusion in the study, all patients read and signed an informed consent form previously approved by the Research and Ethics Committees of our institution according to the tenets of the Declaration of Helsinki.</p><p>Corneas included for analysis were divided according to the pathophysiologic classification into immunologic, or non-necrotizing, and infectious, or necrotizing, HSK.</p><p>Corneas were also categorized as inactive when corneal opacity and scarring due to herpetic keratitis was evident, and no signs or symptoms of inflammation or infection were present. Active disease was considered when patients experienced related symptoms, including red eye, blurred vision, foreign body sensations, photophobia, tearing, and pain. Additionally, signs of active inflammation, such as ciliary injection, corneal ulceration, active vascularization, inflammatory infiltration, stromal edema or melting, and keratic precipitates were observed under slit-lamp examination.</p><p>SD-OCT (RTVue-100Ⓡ, Optovue, Inc., Fremont, CA, USA) analysis was performed by the same technician (SIS) to each eye using a corneal module adaptor (CAM) L-lens (15 µm) and S-lens (10 µm), depending on whether the full extent or the details of corneal lesions, respectively, were analyzed as requested in a drawing scheme. While the wide-angle CAM L-lens provides a scan width of up to 6 mm and a transverse resolution of 15 μm, the high magnification CAM S-lens provides a scan width of up to 4 mm and a transverse resolution of 10 μm. Additionally, the corneal adaptor module software (version 5.5) automatically processes five consecutive sets of eight high-definition meridional scans, of which the three most consistent sets are used to provide a 6 mm scan diameter pachymetry map and the minimum corneal thickness point.<sup>[<xref rid=\"B20\" ref-type=\"bibr\">20</xref>]</sup> Specific measurements of corneal epithelial and stromal thicknesses were also performed manually at the site of the lesions. Cursors were always placed perpendicular to the anterior corneal surface at the point of measurement. Four thickness measurements were considered for analysis: the central corneal thickness (CCT), minimal corneal thickness (MCT), corneal thickness at the site of the lesion (CTL), and corneal epithelial thickness over the stromal lesion (CETL).</p><p>Multiple high-resolution meridional scans were performed for each corneal lesion related to HSK according to a drawing scheme prepared from direct observations under the slit lamp. Representative scan images were selected for morphologic analysis by one of us (ARG) based on representative slit-lamp observations and corneal color photographs registered just before sending the patients for SD-OCT analysis. Corneal lesions consisting of inflammatory infiltrates and stromal edema from eyes with active keratitis and scars or leukomas from eyes with inactive disease were studied.</p><p>Stromal edema assessed by SD-OCT was defined as a hypodense area (grade I, <inline-formula><mml:math id=\"M3\"><mml:mo><</mml:mo></mml:math></inline-formula> 33%) within the involved corneal stroma, accompanied by a <inline-formula><mml:math id=\"M4\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 20% increase in thickness compared to the adjacent unaffected stroma. Stromal thinning was defined as a <inline-formula><mml:math id=\"M5\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 15% thickness reduction compared to an immediate adjacent healthy area. Inflammatory infiltrate was described as a hyperdense lesion (grades II, 34–66%, and III, <inline-formula><mml:math id=\"M6\"><mml:mo>></mml:mo></mml:math></inline-formula> 67%) within the involved corneal stroma, accompanied by a <inline-formula><mml:math id=\"M7\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 10% increase in the thickness of an adjacent unaffected area.</p><p>The following measurement parameters were analyzed: localization (central = 5 mm zone, or paracentral/peripheral = 5–12 mm zone); percentage of surface extension (small <inline-formula><mml:math id=\"M8\"><mml:mo>≤</mml:mo></mml:math></inline-formula> 25%, medium = 25–45%, and large <inline-formula><mml:math id=\"M9\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 45%); depth (superficial <inline-formula><mml:math id=\"M10\"><mml:mo>≤</mml:mo></mml:math></inline-formula> 150 µm, medial = 150–350 µm, and deep <inline-formula><mml:math id=\"M11\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 350 µm); and density, which was analyzed using the color scale provided by the SD-OCT device. For this purpose, a classification system was created to quantify the density of each lesion by examining the warm tones (white, red, orange) that predominated in the image, which corresponded to the percentage of backscattering and reflective properties of the tissue analyzed (grade I, <inline-formula><mml:math id=\"M12\"><mml:mo><</mml:mo></mml:math></inline-formula> 33%; grade II, 34–66%; and grade III, <inline-formula><mml:math id=\"M13\"><mml:mo>></mml:mo></mml:math></inline-formula> 67%).</p><p>Statistical analysis was performed using the SPSS software version 23.0 (SPSS Inc. Chicago, IL. USA). A Shapiro-Wilk test was performed to explore the normality of variable distribution; observing that the pachymetric measurements, density, and depth values of the corneal lesions were not distributed normally, nonparametric tests (Mann–Whitney U-test) were used to compare the difference in medians. A <italic>P</italic>-value <inline-formula><mml:math id=\"M14\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05 was considered as statistically significant.</p><sec sec-type=\"subsection\"><title>Ethical Consideration</title><p>Ethics Committee Approval No. CONBIOETICA19CEI00820130520)</p><p>Research Committee Approval No. 13C119039138</p><p>COFEPRIS Authorization No. 13CEI19039139</p></sec></sec><sec sec-type=\"section\"><title> RESULTS</title><p>SD-OCT scanning was performed on 53 corneas of patients with unilateral HSK. These corneas belonged to 43 (81.1%) women and 10 (18.9%) men. The mean age at presentation was 41.0 years (range, 7 to 77 years).</p><p>Most eyes (83.1%) had immune or non-necrotizing HSK, and only nine (16.9%) eyes had the infectious or necrotizing form. Regarding the inflammatory status at the time of analysis, 24 (45.3%) eyes had active keratitis and 29 (54.7%) had inactive keratitis.</p><p>In the group of patients with active keratitis, 17 (70.8%) corneas belonged to women and 7 (29.1%) to men with a mean age of 42.2 years (range, 9–77 years). The most frequent symptoms found in these patients were red eye, tearing, and photophobia, and the most frequent signs were red eye, stromal edema, and hypoesthesia.</p><p>Meanwhile, in the inactive keratitis group, 25 (86.2%) corneas belonged to women and only 4 (13.7%) to men with a younger mean age of 39.9 years (range, 7–70 years). Apart from nonspecific symptoms of dry eye and discomfort, most of these patients were asymptomatic at the time of the analysis, and only those with corneal scarring along the visual axis or irregular astigmatism complained of blurred vision.</p><sec sec-type=\"subsection\"><title>SD-OCT Microstructural Analysis for Active Keratitis</title><p>Table 1 shows the frequency of the SD-OCT morphologic alterations from corneas with active keratitis. The most common findings were stromal edema and inflammatory infiltrates seen in 79.1% of eyes each, followed by epithelial thickening in 66.6% and stromal scarring in 62.5% of eyes. Of the seven corneas with active necrotizing HSK, five (71.4%) showed epithelial ulceration over the stromal infiltration. Half of the HSK lesions affected the visual axis, and the other 50% were located in the paracentral and peripheral cornea. Additionally, nearly half of the active corneal lesions were found at mid-stromal depth, had a medium density, and covered an area between 25 and 45% of the corneas (Table 2).</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Frequency of SD-OCT morphologic changes of herpetic stromal keratitis according to inflammatory status and type of keratitis.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"7\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Corneal structure change (cross-sectional analysis)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Active HSK No. Eyes (%) (<italic><bold>n</bold></italic><bold> = 24)</bold></bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Inactive HSK No. Eyes (%) (<italic><bold>n</bold></italic><bold> = 29)</bold></bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold/>\n<italic><bold>P</bold></italic>\n<bold>-value</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Non-necrotizing keratitis No. Eyes (%) (<italic><bold>n</bold></italic><bold> = 44)</bold></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Necrotizing keratitis No. Eyes (%) (<italic><bold>n</bold></italic><bold> = 9)</bold></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold/>\n<italic><bold>P</bold></italic>\n<bold>-value</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Epithelial remodeling</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (4.1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21 (72.4)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M15\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19 (43.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3 (33.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.02*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Epithelial thickening</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16 (66.6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (3.4)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M16\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13 (29.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (44.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.387</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Epithelial damage (ulceration)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5 (20.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M17\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5 (55.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M18\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Stromal thinning (<inline-formula><mml:math id=\"M19\"><mml:mo><</mml:mo></mml:math></inline-formula> 450 µm)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6 (25.0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20 (68.9)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.004*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20 (45.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7 (77.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.080</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Stromal edema (<inline-formula><mml:math id=\"M20\"><mml:mo>></mml:mo></mml:math></inline-formula> 570 µm)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19 (79.1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (3.4)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M21\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15 (34.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (44.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.652</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Stromal inflammatory infiltration</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19 (79.1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (6.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M22\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (31.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7 (77.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.011*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Stromal scarring (<inline-formula><mml:math id=\"M23\"><mml:mo>></mml:mo></mml:math></inline-formula> 35% surface)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15 (62.5)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26 (89.6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.042*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">34 (77.2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9 (100.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.095</td></tr><tr><td align=\"left\" colspan=\"7\" rowspan=\"1\">SD-OCT, spectral-domain optical coherence tomography; HSK, herpetic stromal keratitis\n<inline-formula><mml:math id=\"M24\"><mml:msup><mml:mrow/><mml:mo>*</mml:mo></mml:msup></mml:math></inline-formula>\n<italic>P</italic>-value <inline-formula><mml:math id=\"M25\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05 was considered as statistically significant (Mann–Whitney U-test for two independent samples).</td></tr></tbody></table></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>SD-OCT cross-sectional analysis of most representative corneal lesions (scar, leukoma, or infiltrate) seen in eyes with herpetic stromal keratitis.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"7\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Lesion(s) parameter measured</bold>\n</td><td align=\"center\" colspan=\"3\" rowspan=\"1\"><bold>Disease Activity</bold>\n</td><td align=\"center\" colspan=\"3\" rowspan=\"1\"><bold>Type of Keratitis</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Active HSK No. Eyes (%) (<italic><bold>n</bold></italic><bold> = 24)</bold></bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Inactive HSK No. Eyes (%) (<italic><bold>n</bold></italic><bold> = 29)</bold></bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold/>\n<italic><bold>P-</bold></italic>\n<bold>value</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Non necrotizing keratitis No. Eyes (%) (<italic><bold>n</bold></italic><bold> = 44 )</bold></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Necrotizing keratitis No. Eyes (%) (<italic><bold>n</bold></italic><bold> = 9 )</bold></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold/>\n<italic><bold>P-</bold></italic>\n<bold>value</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Localization:</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">- Central (visual axis)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12 (50.0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15 (51.7)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.901</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20 (45.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7 (77.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.08</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">- Paracentral/peripheral</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12 (50.0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (48.3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.901</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24 (54.6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (22.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.08</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Density:</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">- Low (grade-I, < 33%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (29.1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 (34.4)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.464</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17 (36.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.040*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">- Medium (grade-II, 34–66%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11 (45.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13 (44.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.942</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21 (45.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3 (33.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.956</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">- High (grade-III, > 67%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6 (25.0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6 (20.6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.777</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8 (17.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6 (66.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.010*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Depth:</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">- Superficial (< 150 µm)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (29.2)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 (34.5)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.683</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17 (38.6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.025*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">- Medial (150–350 µm)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11 (45.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13 (44.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.942</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21 (47.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3 (33.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.434</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">- Deep (> 350 µm)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6 (25.0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6 (20.7)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.712</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6 (13.6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6 (66.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.001*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Extension:</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">- Small (< 25% surface)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (16.6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6 (20.6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.712</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 (22.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.116</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">- Medium (25–45% surface)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13 (54.1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21 (72.4)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.172</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30 (68.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 (44.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.18</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">- Large (> 45% surface)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (29.1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (6.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.033*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (9.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5 (55.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.001*</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">SD-OCT, spectral-domain optical coherence tomography; HSK, herpetic stromal keratitis\n<inline-formula><mml:math id=\"M26\"><mml:msup><mml:mrow/><mml:mo>*</mml:mo></mml:msup></mml:math></inline-formula>\n<italic>P</italic>-value <inline-formula><mml:math id=\"M27\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05 was considered as statistically significant (Mann–Whitney U test for two independent samples).</td></tr></tbody></table></table-wrap><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><p>Mean pachymetry measurements by SD-OCT of corneas with herpetic stromal keratitis according to inflammatory status and type of keratitis.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"7\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" colspan=\"3\" rowspan=\"1\"><bold>Disease Activity</bold>\n</td><td align=\"center\" colspan=\"3\" rowspan=\"1\"><bold>Type of Keratitis</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Measurement Parameter (µm)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Eyes with active keratitis (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 24)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Eyes with Inactive keratitis (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 29)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold/>\n<italic><bold>P-</bold></italic>\n<bold>value</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Non- necrotizing keratitis (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 44)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Necrotizing keratitis (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 9)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold/>\n<italic><bold>P-</bold></italic>\n<bold>value</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>CCT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">561.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">482.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M28\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">519.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">492.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">O.405</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>MCT</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">483.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">436.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.007*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">468.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">396.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.002*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>CTL</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">644.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">439.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M29\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">528.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">473.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.711</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>CETL</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">68.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">78.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.048*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">72.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">47.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.043*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Stromal thinning (irregular astigmastism</bold>\n<inline-formula><mml:math id=\"M30\"><mml:msup><mml:mrow/><mml:mo>†</mml:mo></mml:msup></mml:math></inline-formula>\n<bold>) No eyes (%)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5 (20.8%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11 (37.9%)<inline-formula><mml:math id=\"M31\"><mml:msup><mml:mrow/><mml:mo>†</mml:mo></mml:msup></mml:math></inline-formula>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.181</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6 (13.6%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5 (62.5%)<inline-formula><mml:math id=\"M32\"><mml:msup><mml:mrow/><mml:mo>†</mml:mo></mml:msup></mml:math></inline-formula>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.001*</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">SD-OCT, spectral-domain optical coherence tomography; CCT, central corneal thickness; MCT, minimum corneal thickness; CTL, corneal thickness at lesion site; CETL, corneal epithelium thickness at lesion site; <inline-formula><mml:math id=\"M33\"><mml:msup><mml:mrow/><mml:mo>†</mml:mo></mml:msup></mml:math></inline-formula>confirmed by topography\n<inline-formula><mml:math id=\"M34\"><mml:msup><mml:mrow/><mml:mo>*</mml:mo></mml:msup></mml:math></inline-formula>\n<italic>P</italic>-value <inline-formula><mml:math id=\"M35\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05 was considered as statistically significant (Mann–Whitney U test for two independent samples).</td></tr></tbody></table></table-wrap><p>The corneal shape was distorted within the affected zones, showing a hypodense and thickened stroma due to edema. There was also a higher reflectivity at the site of stromal inflammatory infiltrates compared to unaffected areas (Figure 1).</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>(A) Slit-lamp photograph of a cornea with active necrotizing HSK showing stromal inflammatory infiltration and edema. (B) SD-OCT pachymetry map showing a thickened cornea (max = 670 µm) corresponding to the area with stromal edema. (C) A cross-sectional view of the central lesion showing areas of epithelial thickening (max = 76 µm) over inflammatory infiltrates, corresponding to areas of hyperreflectivity, and hypodense areas, corresponding to stromal edema. (D) Color scale of the same lesion showing the inflammatory infiltrates and scarring in warm (orange to yellow) tones.</p></caption><graphic xlink:href=\"jovr-15-279-001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>(A) Slit-lamp photograph of a cornea with inactive nonnecrotizing HSK, showing a diffuse leukoma with no stromal edema. (B) SD-OCT pachymetry showing a thinner cornea (min = 423 µm) in the area of the lesion. (C) A cross-sectional view of the leukoma showing areas of epithelial remodeling and thickening compensation (max = 83 µm) under areas of scarring and stromal compaction (min = 470 µm). (D) Color scale of the lesion showing higher density areas corresponding to fibrosis and scarring on the anterior and medial stroma, fading to yellow and green tones as the lesion becomes less dense.</p></caption><graphic xlink:href=\"jovr-15-279-002\"/></fig><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>(A) A cross-sectional image of a cornea with inactive HSK showing epithelial remodeling (top arrow) over a zone of stromal fibrosis and compaction (bottom arrow) with the MCT = 441 µm. (B) A cross-sectional image of a cornea with active HSK showing marked stromal edema (CTL = 641 µm) and an active inflammatory infiltrate (bottom arrow) underlying an area of epithelial edema.</p></caption><graphic xlink:href=\"jovr-15-279-003\"/></fig></sec><sec sec-type=\"subsection\"><title>SD-OCT Microstructural Analysis for Inactive Keratitis</title><p>The most common SD-OCT morphologic changes seen in corneas with inactive keratitis consisted of stromal scarring (89.6%), epithelial remodeling (72.4%), and stromal thinning (68.9%) (Table 1).</p><p>Similar to the eyes with active HSK, nearly half of the lesions were located in the visual axis, and the other 48.3% were located in the paracentral and peripheral cornea. Additionally, 44.8% of the lesions extended to the mid-stromal depth and had a medium density. Finally, most of the inactive lesions (72.4%) covered an area between 25 and 45% of the ocular surface (Table 2). In general, the SD-OCT analysis of inactive HSK showed thinner corneas with stromal compaction due to fibrosis and scarring. The overlying epithelium was thicker in the affected areas, showing remodeling and thickness compensation. Additionally, there was a higher reflectivity, particularly in areas of dense leukomas (Figure 2).</p></sec><sec sec-type=\"subsection\"><title>SD-OCT Microstructural Differences Between Non-necrotizing and Necrotizing HSK</title><p>There was a clear difference in the morphologic appearance between non-necrotizing and necrotizing keratitis. All corneas with necrotizing HSK showed severe stromal scarring, and the vast majority also had significant stromal inflammatory infiltration and thinning (Table 1). On the other hand, the non-necrotizing corneas showed significantly less inflammatory infiltration (<italic>P</italic> = 0.011).</p><p>Following the SD-OCT cross-sectional analysis, the majority of necrotizing HSK lesions (77.7%) were located centrally, affecting the visual axis, and showed a higher density compared to non-necrotizing lesions (<italic>P</italic> = 0.01) (Table 2). Additionally, 66.7% of the lesions analyzed were located deep into the corneal stroma (<italic>P</italic> = 0.001), and many of them covered a significant area of the corneal surface (<italic>P</italic> = 0.001). By contrast, most non-necrotizing lesions were in the low- to medium-density range, extended between the superficial to medium stromal depths, and affected a smaller area of the cornea than in necrotizing keratitis (Table 2).</p></sec><sec sec-type=\"subsection\"><title>Pachymetry Comparative Analysis </title><p>The comparative analysis of corneal thickness between the active and inactive keratitis groups showed significant differences in the four measured parameters: CCT, MCT, CTL, and CETL (Table 3). Eyes with inactive HSK had significant stromal thinning compared to those with active disease (<italic>P </italic>\n<inline-formula><mml:math id=\"M36\"><mml:mo>≤</mml:mo></mml:math></inline-formula> 0.007). However, the corneal epithelial thickness at the site of the lesion was significantly increased in the inactive cases (<italic>P =</italic> 0.048). On the other hand, the values of MCT (<italic>P =</italic> 0.002) and CETL (<italic>P</italic> = 0.043) were significantly lower in corneas with necrotizing keratitis than in non-necrotizing keratitis. Finally, almost one-third of the eyes showed irregular astigmatism and severe stromal thinning with a major predominance in the necrotizing group (<italic>P</italic>\n<inline-formula><mml:math id=\"M37\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001).</p></sec></sec><sec sec-type=\"section\"><title> DISCUSSION</title><p>Clinically, the corneal pathologic characteristics seen in HSK depend on direct slit-lamp observation of inflammatory stromal infiltration, edema, neovascularization, scarring, and thinning with a history of herpes infection and previously known herpetic epithelial ulceration and corneal hypoesthesia.<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>,<xref rid=\"B23\" ref-type=\"bibr\">23</xref>]</sup>\n</p><p>SD-OCT allows an <italic>in vivo</italic>, accurate, noncontact, and fast examination of the entire corneal microstructure in different inflammatory pathologies, including HSK.<sup>[<xref rid=\"B24\" ref-type=\"bibr\">24</xref>,<xref rid=\"B25\" ref-type=\"bibr\">25</xref>,<xref rid=\"B26\" ref-type=\"bibr\">26</xref>]</sup> To the best of our knowledge, no systematic microstructural analyses have been performed of the corneal pathologic changes seen in HSK using SD-OCT. In the present study, we described these changes in different stages of disease activity and severity. Spectral-domain tomography permits a detailed analysis of the entire cornea in cross-sections of variable orientations as well as en face fragmentation analysis.<sup>[<xref rid=\"B24\" ref-type=\"bibr\">24</xref>]</sup> Most corneas analyzed in the inactive stage of the disease showed epithelial remodeling, consisting of thickening over the scarred stroma that was characterized by fibrosis, thinning, and compaction (Figure 2).</p><p>In contrast, more than two-thirds of the corneas from patients with active keratitis showed significant stromal edema characterized by increased CCT and CTL (Table 3), as well as a hypodense and thickened stromal appearance (Figure 1). In general, patients with the inactive stage of HSK did not show corneal stromal edema; their CCT measurements were in the low to normal range (356–537 µm), and the mean CTL was even lower (439.9 µm), corresponding to areas of stromal scarring and thinning.</p><p>Corneas from the active keratitis group showed a thicker epithelium than average at the site of the lesion (mean CETL = 68.7 µm) and a significantly increased corneal thickness (mean CTL = 644.7 µm), reflecting the presence of stromal inflammatory infiltration and edema.</p><p>Of note was the occurrence of severe corneal thinning in a total of 16 (30.1%) eyes with HSK, with a higher proportion in corneas with necrotizing disease (Table 3). Necrotizing HSK, the most severe form of herpetic keratitis, is characterized by significant leukocyte stromal infiltration and tissue necrosis with consequent thinning of the cornea, which explains the higher percentage of severe corneal thinning and irregular astigmatism seen in these corneas.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B27\" ref-type=\"bibr\">27</xref>]</sup>\n</p><p>SD-OCT measures the intensity of a backscattered optical signal, which represents the reflectivity of the tissue.<sup>[<xref rid=\"B28\" ref-type=\"bibr\">28</xref>]</sup> Since reflectivity varies among different tissues, we can differentiate them by measuring their reflectivities in transverse scans that can be displayed as false-color or grayscale cross-sectional images.<sup>[<xref rid=\"B29\" ref-type=\"bibr\">29</xref>,<xref rid=\"B30\" ref-type=\"bibr\">30</xref>]</sup> The intensity of the backscattered optical signal is represented on a logarithmic scale with varying degrees of brightness. White corresponds to the highest reflection, while dark gray and black correspond to weaker back reflection.<sup>[<xref rid=\"B30\" ref-type=\"bibr\">30</xref>]</sup> SD-OCT software allows toggling between gray- and color scales for each image created. The color scale allows easier detection of subtle variations in the OCT signal to define reflectivity levels. Conversely, the grayscale allows the viewer to visualize contrast more easily.<sup>[<xref rid=\"B31\" ref-type=\"bibr\">31</xref>]</sup> We used the color scale to measure the density of inflammatory infiltrates in eyes with active keratitis and leukomas or scarring in eyes with inactive HSK. In both active and inactive disease, approximately two-thirds of the eyes showed medium to high density lesions; however, corneas with necrotizing stromal keratitis had a significantly higher percentage of high-density lesions (Table 2).</p><p>We found a particular limitation in differentiating between stromal edema and inflammatory infiltration in corneas with active keratitis, where both lesions coexisted within the same stromal area. However, differences in reflectivity, where stromal edema appears as a hypodense area of increased stromal thickness and low reflectivity and stromal infiltrates as areas of increased stromal thickness but with higher reflectivity (yellow-to-orange color), help to facilitate their differentiation.</p><p>Since there are no previous specific reports on SD-OCT findings in this pathologic condition, we find the present study of value to improve our understanding of the <italic>in vivo</italic> pathologic changes occurring at different stages and severity of HSK. As in other corneal pathologies, we consistently found epithelial remodeling, consisting of zonal thickening at sites of fibrosis and scarring in inactive disease (Figure 3A). Epithelial remodeling has also been observed as a way of anterior curvature compensation in advanced keratoconus under areas of significant stromal thinning and ectasia.<sup>[<xref rid=\"B32\" ref-type=\"bibr\">32</xref>]</sup> In contrast, epithelial thickening was present in most eyes during active inflammation (Figure 3B).</p><p>In a clinical setting, corneal SD-OCT could be useful to analyze scarring extension and leukoma depth, hence facilitating surgical decisions regarding partial or total corneal transplantation. Additionally, it may be useful for the detection and grading of stromal edema during recurrent inflammation under challenging situations. During active HSK, leucocyte infiltration induces stromal edema characterized by diffuse haziness, giving a “ground glass” appearance in the area surrounding the inflammatory infiltrate. Subtle stromal edema may hide in the infiltrate or under a dense leukoma. In such circumstances, corneal SD-OCT represents a useful aid for its detection. The potential limitations of the present study include distortions that occur in the SD-OCT scans as a result of the refractive indexes of the tissues. The acquisition of SD-OCT scans was performed along meridional planes to minimize this effect, and all images were de-warped by the SD-OCT system's corneal adaptor module software.<sup>[<xref rid=\"B31\" ref-type=\"bibr\">31</xref>]</sup>\n</p><p>In conclusion, <italic>in vivo</italic> SD-OCT pathologic analysis of corneas with different disease activity and types of inflammation has permitted a better understanding of the pathologic mechanisms adopted by the affected corneas. Additionally, it allowed the evaluation of the grade and extent of tissue damage and repair seen in HSK. Future studies of HSK imaging analysis with SD-OCT should be carried out to monitor disease progression or therapeutic response, including OCT angiography of the cornea to analyze blood perfusion and neovascularization responses. Additionally, the commercial development of ultra-high-definition OCTs for corneal and anterior segment imaging analysis surely would yield more detailed <italic>in vivo</italic> ultrastructure pathologic changes occurring in this sight-threatening disease.</p></sec><sec sec-type=\"section\"><title> Acknowledgements</title><p>The authors are thankful to Susana Imperial-Sauceda for her technical support in performing all corneal SD-OCT tests.</p></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>This study has been partially funded by the Immuneye Foundation.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Liesegang TJ. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"editorial\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864078</article-id><article-id pub-id-type=\"pmc\">PMC7431722</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7466</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Letter to Editor</subject></subj-group></article-categories><title-group><article-title>Guidance for Ophthalmologists and Ophthalmology Centers during the COVID-19 Pandemic</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Rajavi</surname><given-names>Zhale</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Safi</surname><given-names>Sare</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mohammadzadeh</surname><given-names>Maryam</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Ophthalmic Epidemiology Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran</aff><aff id=\"I2\">\n<sup>2</sup>Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Zhale Rajavi, MD. Ophthalmic Research Center,\nResearch Institute for Ophthalmology and Vision\nScience, Shahid Beheshti University of Medical\nSciences, 23 Paidar Fard, Bostan 9, Pasdaran Ave.,\nTehran 16666, Iran.\nE-mail: zhalerajavi@gmail.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>438</fpage><lpage>441</lpage><history><date date-type=\"received\"><day>28</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>22</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Rajavi et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><counts><ref-count count=\"6\"/><page-count count=\"4\"/></counts></article-meta></front><body><p>Dear Editor,</p><p>Coronavirus Disease (COVID-19) caused by the SARS-CoV-2 virus usually manifests with respiratory symptoms including cough and dyspnea associated with fever and diarrhea, while it rarely presents with conjunctivitis. Ophthalmologists are at high risk for being infected or transmitting the virus due to the following reasons: examining patients with conjunctivitis or asymptomatic carriers from a short distance (usually <inline-formula><mml:math id=\"M1\"><mml:mo><</mml:mo></mml:math></inline-formula> 20 cm), the long duration of eye examinations/procedures (<inline-formula><mml:math id=\"M2\"><mml:mo>></mml:mo></mml:math></inline-formula> 15 min), touching patients' eyelids during slit-lamp examination, and the possibility of viral shedding and transmission in contact with ocular secretions. During the pandemic peak of COVID-19 in March 2020, a guidance was prepared by the Knowledge Management Unit at the Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran for ophthalmologists and eye care centers.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> We also discuss on necessary precautions for the post-peak period starting from May 2020 and during the partial recovery phase of the COVID-19 pandemic.</p><sec sec-type=\"section\"><title> Characteristics </title><p>\n<inline-formula><mml:math id=\"M3\"><mml:mo>•</mml:mo></mml:math></inline-formula>Person to person transmission is caused by respiratory droplets produced by cough and sneezes of the patient. Furthermore, the virus can spread when someone touches infected surfaces and then touches their nose, mouth, and eyes.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M4\"><mml:mo>•</mml:mo></mml:math></inline-formula>Symptoms can manifest early (in 2 days) or late (within 14 days) after exposure to the virus. Since the incubation period is between 5 and 7 days, the quarantine period is considered to be at least 14 days.</p><p>\n<inline-formula><mml:math id=\"M5\"><mml:mo>•</mml:mo></mml:math></inline-formula>Even a carrier without any respiratory symptoms can transmit the infection.</p><p>\n<inline-formula><mml:math id=\"M6\"><mml:mo>•</mml:mo></mml:math></inline-formula>The virus could survive for 24 hours on cardboard surfaces and credit cards, while it lasts for two–three days on plastic and metal surfaces.</p><p>\n<inline-formula><mml:math id=\"M7\"><mml:mo>•</mml:mo></mml:math></inline-formula>No vaccines have been produced against the virus so far and research projects in this field are in process.</p><p>\n<inline-formula><mml:math id=\"M8\"><mml:mo>•</mml:mo></mml:math></inline-formula>Currently, there is no specific drug for prophylaxis and treatment of this virus.</p></sec><sec sec-type=\"section\"><title> Risk factors</title><p>The risk of COVID-19 infection is higher in persons with the following characteristics:</p><p>\n<inline-formula><mml:math id=\"M9\"><mml:mo>•</mml:mo></mml:math></inline-formula>Age <inline-formula><mml:math id=\"M10\"><mml:mo>></mml:mo></mml:math></inline-formula> 55 years</p><p>\n<inline-formula><mml:math id=\"M11\"><mml:mo>•</mml:mo></mml:math></inline-formula>History of respiratory disease</p><p>\n<inline-formula><mml:math id=\"M12\"><mml:mo>•</mml:mo></mml:math></inline-formula>History of renal disease</p><p>\n<inline-formula><mml:math id=\"M13\"><mml:mo>•</mml:mo></mml:math></inline-formula>Diabetes (HbA1C <inline-formula><mml:math id=\"M14\"><mml:mo>></mml:mo></mml:math></inline-formula> 7.6%)</p><p>\n<inline-formula><mml:math id=\"M15\"><mml:mo>•</mml:mo></mml:math></inline-formula>History of hypertension</p><p>\n<inline-formula><mml:math id=\"M16\"><mml:mo>•</mml:mo></mml:math></inline-formula>History of cardiopulmonary disease</p><p>\n<inline-formula><mml:math id=\"M17\"><mml:mo>•</mml:mo></mml:math></inline-formula>History of organ transplant and use of immunosuppressive drugs</p></sec><sec sec-type=\"section\"><title> General precautions</title><p>It is very important to follow routine health instructions against this virus such as washing hands frequently, disinfecting surfaces, and using personal protection equipment (PPE).<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>][<xref rid=\"B3\" ref-type=\"bibr\">3</xref>][<xref rid=\"B4\" ref-type=\"bibr\">4</xref>][<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p></sec><sec sec-type=\"section\"><title> Recommendations for ophthalmologists in the epidemic peak of March 2020</title><p>\n<inline-formula><mml:math id=\"M18\"><mml:mo>•</mml:mo></mml:math></inline-formula>Cancel previously scheduled appointments and avoid scheduling new appointments for future months.</p><p>\n<inline-formula><mml:math id=\"M19\"><mml:mo>•</mml:mo></mml:math></inline-formula>Provide facilities for telemedicine communications with patients for further consultations, answering their questions, and dose adjustment of their medications.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M20\"><mml:mo>•</mml:mo></mml:math></inline-formula>In case of unavoidable in-person appointments, ask the patient during a phone contact about symptoms such as cough, sneezing, fever, dyspnea, and traveling to high-risk regions. If their answer is yes, avoid visiting the patient at least for three weeks.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>][<xref rid=\"B3\" ref-type=\"bibr\">3</xref>][<xref rid=\"B4\" ref-type=\"bibr\">4</xref>][<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M21\"><mml:mo>•</mml:mo></mml:math></inline-formula>The patients should be examined alone (without companions) and if needed only one person can accompany elderly patients and children.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M22\"><mml:mo>•</mml:mo></mml:math></inline-formula>Avoid crowding in the waiting room while maintaining at least 3 m distance between patients. If possible, encourage patients to wait for the appointment outside the facilities (i.e., in their cars) and notify them when they should return to the office.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M23\"><mml:mo>•</mml:mo></mml:math></inline-formula>It is recommended to screen patients and their companions for fever and respiratory symptoms and ask them to wear masks and wash their hands before entering the examination room. Suspicious cases should be referred to relevant healthcare centers.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M24\"><mml:mo>•</mml:mo></mml:math></inline-formula>Limit ophthalmic examinations to urgent cases such as retinal detachment, trauma, chemical contact, and severe ocular infections. It is recommended to perform the examination faster and also avoid unnecessary ones.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M25\"><mml:mo>•</mml:mo></mml:math></inline-formula>It is recommended to postpone non-urgent examinations and elective surgeries.</p><p>\n<inline-formula><mml:math id=\"M26\"><mml:mo>•</mml:mo></mml:math></inline-formula>It is necessary for the ophthalmologists to wear masks during the examination and it is also essential for doctors and health caregivers to wash their hands after contact with every patient.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>][<xref rid=\"B3\" ref-type=\"bibr\">3</xref>][<xref rid=\"B4\" ref-type=\"bibr\">4</xref>][<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M27\"><mml:mo>•</mml:mo></mml:math></inline-formula>In order to prevent exposure to patients' respiratory droplets, it is recommended to use slit-lamp shield.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M28\"><mml:mo>•</mml:mo></mml:math></inline-formula>Avoid talking with patients during the examination.</p><p>\n<inline-formula><mml:math id=\"M29\"><mml:mo>•</mml:mo></mml:math></inline-formula>To prevent the patient to patient transmission, single-dose drops are preferred.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M30\"><mml:mo>•</mml:mo></mml:math></inline-formula>Use applicators to examine the eyelids.</p><p>\n<inline-formula><mml:math id=\"M31\"><mml:mo>•</mml:mo></mml:math></inline-formula>If possible, use non-contact tonometer to measure the intraocular pressure.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> If not possible, disinfect the tip of Goldmann applanation as follows. First of all, wash the tonometer with water and soap, then allow it to dry. Afterward, put almost 2 mm of its tip in the bleach-based solution with 5% concentration. Isopropyl alcohol and ethyl alcohol are also frequently used; however, they are not as effective as the aforementioned method.</p><p>\n<inline-formula><mml:math id=\"M32\"><mml:mo>•</mml:mo></mml:math></inline-formula>It is recommended to disinfect the examination room, all equipment including slit-lamp and its accessories after every visit.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M33\"><mml:mo>•</mml:mo></mml:math></inline-formula>Use disposable gloves when sanitizing surfaces and equipment.</p><p>\n<inline-formula><mml:math id=\"M34\"><mml:mo>•</mml:mo></mml:math></inline-formula>Use bleach-based disinfectants (with a concentration of one spoon in a gallon of water) for disinfecting surfaces and use alcohol 70% for instruments.</p><p>\n<inline-formula><mml:math id=\"M35\"><mml:mo>•</mml:mo></mml:math></inline-formula>If possible, use telemedicine communication such as video or telephone consultations.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M36\"><mml:mo>•</mml:mo></mml:math></inline-formula>When examining confirmed or suspected cases, ophthalmologists should wear N95 face masks, goggles or face-protective shield, gloves, gown, and disposable overshoes.</p><p>\n<inline-formula><mml:math id=\"M37\"><mml:mo>•</mml:mo></mml:math></inline-formula>If any of the hospitalized patients need urgent diagnostic or therapeutic measures, in order to avoid patient transport, it is recommended to perform necessary procedures at the host hospital. If further examinations or urgent surgery is needed, refer the patient to an equipped ophthalmic center.</p></sec><sec sec-type=\"section\"><title> Recommendations for Ophthalmic Centres in the epidemic peak in March 2020</title><p>\n<inline-formula><mml:math id=\"M38\"><mml:mo>•</mml:mo></mml:math></inline-formula>It is recommended that managers assure their healthcare workers that all clients are going to be screened on arrival.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M39\"><mml:mo>•</mml:mo></mml:math></inline-formula>Managers should monitor cancelation of routine examinations and elective procedures. Also, they should prevent companions from entering the center and keep the waiting rooms uncrowded. If necessary, each patient may only have one companion who should be screened in a similar manner as patients.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M40\"><mml:mo>•</mml:mo></mml:math></inline-formula>Provide facilities for telemedicine consultations through phone calls, video conferences, and social networks.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M41\"><mml:mo>•</mml:mo></mml:math></inline-formula>Healthcare personnel working hours should be scheduled to minimize work fatigue.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M42\"><mml:mo>•</mml:mo></mml:math></inline-formula>Educate personnel to distinguish and isolate high-risk patients.</p><p>\n<inline-formula><mml:math id=\"M43\"><mml:mo>•</mml:mo></mml:math></inline-formula>Educate and monitor the process of disinfection of ophthalmic instruments, surfaces, chairs, and elevator handles regularly.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M44\"><mml:mo>•</mml:mo></mml:math></inline-formula>Give leave to non-essential personnel in each working shift.</p><p>\n<inline-formula><mml:math id=\"M45\"><mml:mo>•</mml:mo></mml:math></inline-formula>Provide enough disinfectants including soap, tissue paper, masks, disposable gloves, 70% alcohol, disinfectant gel, and thermometers for patients and healthcare workers.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>][<xref rid=\"B3\" ref-type=\"bibr\">3</xref>][<xref rid=\"B4\" ref-type=\"bibr\">4</xref>][<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M46\"><mml:mo>•</mml:mo></mml:math></inline-formula>Supply slit-lamp shields, goggles, and counter shields.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup>\n</p><p>\n<inline-formula><mml:math id=\"M47\"><mml:mo>•</mml:mo></mml:math></inline-formula>Administrate social distancing while in self-service dining rooms and handover foods in packages.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p></sec><sec sec-type=\"section\"><title> Recommendations for ophthalmologists and ophthalmic centres during the post-peak period </title><p>All practitioners should follow these recommendations until Food and Drug Administration (FDA)-approved treatments and/or vaccines for COVID-19 are available:</p><p>\n<inline-formula><mml:math id=\"M48\"><mml:mo>•</mml:mo></mml:math></inline-formula> Respect social distancing.</p><p>\n<inline-formula><mml:math id=\"M49\"><mml:mo>•</mml:mo></mml:math></inline-formula> Both patients and healthcare workers should utilize face masks.</p><p>\n<inline-formula><mml:math id=\"M50\"><mml:mo>•</mml:mo></mml:math></inline-formula> Maintain a lower in-office appointment numbers than that in the pre-COVID-19 period.</p><p>\n<inline-formula><mml:math id=\"M51\"><mml:mo>•</mml:mo></mml:math></inline-formula>Lengthen the turnover time in the operating room.</p><p>\n<inline-formula><mml:math id=\"M52\"><mml:mo>•</mml:mo></mml:math></inline-formula> Consider cataract surgery as a semi-urgent surgery, not elective, when the patient cannot drive or work or has a risk of falling.</p><p>\n<inline-formula><mml:math id=\"M53\"><mml:mo>•</mml:mo></mml:math></inline-formula>It is recommended to test ophthalmologists, health caregivers, and patients prior to elective visits and surgeries. The tests and their characteristics are:</p><p>RT-PCR which is usually done on nasopharyngeal swab specimens can be positive even after 35 days since the onset of symptoms.</p><p>The specificity of the SARS-CoV-2 antibody test is 99.8% and has no cross-reactivity to other coronaviruses. A positive serology test shows recent infection. However, the virus can shed for at least five weeks from the onset of the infection. The duration and degree of protection of the IgG antibody response from reinfection are unknown. In regions where the prevalence of COVID-19 is low, positive serologic tests, without prior COVID-19 (RT-PCR) positive test, is more probable to be an artifact or the testing error rather than true infections.</p><p>A rapid antigen detection test is available but is more probable to report false-negative results and needs a fluorescent immunoassay analyzer to be acceptable.</p><p>\n<inline-formula><mml:math id=\"M54\"><mml:mo>•</mml:mo></mml:math></inline-formula>In the case of in-office procedures that necessitate close interaction between the surgeon and patients, wearing surgical masks for both patients and surgeons is recommended. Surgeons are also encouraged to wear eye protection and N95 masks.</p><p>\n<inline-formula><mml:math id=\"M55\"><mml:mo>•</mml:mo></mml:math></inline-formula>During procedures needing general anesthesia (GA), caregivers without N95 mask should remain out of the operating room throughout intubation/extubation. Furthermore, for procedures with monitored anesthesia/conscious sedation, the patient should wear a surgical mask. Due to prolonged physical proximity between the ophthalmologist and the patient during surgery, it is recommended for the surgeon to wear an N95 mask.</p><p>As time goes by, ophthalmologists will be required to perform routine in-office procedures and examine patients who have recovered or are recovering from COVID-19. Due to prolonged viral shedding (even reported for up to 37 days), it is recommended to repeat RT-PCR testing for patients who are being scheduled for operation <inline-formula><mml:math id=\"M56\"><mml:mo><</mml:mo></mml:math></inline-formula> 6 weeks after their diagnosis (except for emergency cases).</p><p>If the repeat test is positive, the patient should wear a surgical mask and the surgeon should wear an N-95 mask, gown, and eye protection.</p></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>Nil.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ack><title> Acknowledgements</title><p>We appreciate the Scientific Committee of the Iranian Society of Ophthalmology for their valuable comments and advice on this guidance.</p></ack><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Sallet G. Update on COVID-19 situation in Belgium from Dr. Guy Sallet [Internet]. Euro Times; 2020 [accessed March 24, 2020]. Available from: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.eurotimes.org/update-on-covid-19-situation-in-belgium-from-dr-guy-sallet/\">https://www.eurotimes.org/update-on-covid-19-situation-in-belgium-from-dr-guy-sallet/</ext-link>.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">The College of Optometrists. Coronavirus (COVID-19) pandemic: guidance for optometrists [Internet]. The College of Optometrists; 2020 [accessed March 24, 2020]. Available from: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.college-optometrists.org/the-college/media-hub/news-listing/coronavirus-covid-19-guidance-for-optometrists.html\">https://www.college-optometrists.org/the-college/media-hub/news-listing/coronavirus-covid-19-guidance-for-optometrists.html</ext-link>.</mixed-citation></ref><ref id=\"B3\"><label>3</label><mixed-citation publication-type=\"other\">CDC, WHO. Important coronavirus updates for ophthalmologists [Internet]. American Academy of Ophthalmology; 2020 [accessed March 24, 2020]. Available from: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.aao.org/headline/alert-important-coronavirus-context\">https://www.aao.org/headline/alert-important-coronavirus-context</ext-link>.</mixed-citation></ref><ref id=\"B4\"><label>4</label><mixed-citation publication-type=\"other\">COS PRC. COS and ACUPO Guidelines for ophthalmic care during COVID-19 pandemic [Internet]. COS PRC; 2020 [accessed March 20, 2020]. Available from: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.cosprc.ca/resource/guidelines-for-ophthalmic-care/\">https://www.cosprc.ca/resource/guidelines-for-ophthalmic-care/</ext-link>.</mixed-citation></ref><ref id=\"B5\"><label>5</label><mixed-citation publication-type=\"other\">Coronavirus RCOphth– summary of key actions [Internet]; 2020 [accessed March 20, 2020]. Available from: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.rcophth.ac.uk/wp-content/uploads/2020/03/Coronavirus-RCOphth-key-points-March-19th.pdf\">https://www.rcophth.ac.uk/wp-content/uploads/2020/03/Coronavirus-RCOphth-key-points-March-19th.pdf</ext-link>.</mixed-citation></ref><ref id=\"B6\"><label>6</label><mixed-citation publication-type=\"other\">Nuijts RMMA. ESCRS update on COVID-19 [Internet]. Euro Times; 2020 [accessed March 19, 2020]. Available from: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.eurotimes.org/a-message-from-the-escrs-president-on-covid-19/.\">https://www.eurotimes.org/a-message-from-the-escrs-president-on-covid-19/.</ext-link> Accessed: March 19, 2020.</mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864064</article-id><article-id pub-id-type=\"pmc\">PMC7431723</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7452</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Long-term Outcomes of Treat and Extend Regimen of Anti-vascular Endothelial Growth Factor in Neovascular Age-related Macular Degeneration</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Lee</surname><given-names>Andy</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Garg</surname><given-names>Pooja G</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Lyon</surname><given-names>Alice T</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Mirza</surname><given-names>Rukhsana</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Gill</surname><given-names>Manjot K</given-names></name><degrees>MD</degrees></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Department of Ophthalmology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Manjot K Gill, MD. Department of Ophthalmology,\nFeinberg School of Medicine, Northwestern University,\n645 North Michigan Ave., Suite 440. Chicago, IL 60611,\nUSA.\nEmail: mgill@nm.org\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>331</fpage><lpage>340</lpage><history><date date-type=\"received\"><day>09</day><month>10</month><year>2019</year></date><date date-type=\"accepted\"><day>21</day><month>3</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Lee et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><sec><title>Purpose</title><p>This study describes the long-term visual and anatomic outcomes of anti-vascular endothelial growth factor (VEGF) treatment using a treat and extend dosing regimen.</p></sec><sec><title>Methods</title><p>This cross-sectional cohort study consisted of 224 treatment-naïve eyes with neovascular age-related macular degeneration (NV-AMD) from 202 patients that were treated with anti-VEGF agents bevacizumab, ranibizumab, and aflibercept using a treat and extend (TAE) regimen by four physician investigators in a large urban referral center from 2008 to 2015. Subjects were evaluated for visual acuity, injection frequency, and optical coherence tomography (OCT).</p></sec><sec><title>Results</title><p>Over a seven-year follow-up period (mean 3.4 years), an average 20.2 <inline-formula><mml:math id=\"M1\"><mml:mo>±</mml:mo></mml:math></inline-formula> 14.7 injections were administered with 8.4 injections in the first year and 5.5 injections by the seventh year of remaining eyes undergoing treatment. Visual acuity was 0.70 logMAR (20/100 Snellen) at the first visit and 0.67 logMAR (20/93 Snellen) at the final visit, with 74% of eyes maintaining or gaining more than 2 lines of vision. Long-term, 45.1% of eyes achieved 20/50 or better, while 27.1% were 20/200 or worse. Of the treated patients, 61.2% received monotherapy with no difference in visual acuity outcomes or number of injections between the agents used. OCT analysis showed decreased fluid from initial to final follow-up visit: 70.1–15.6% with sub-retinal fluid (SRF) and 47.3–18.8% with intra-retinal fluid (IRF) with no difference between the agents were used.</p></sec><sec><title>Conclusion</title><p>This study demonstrates that most patients (74%) improve or maintain visual acuity long-term using a TAE model with a significant portion (45.1%) achieving 20/50 or better visual acuity with sustained treatment.</p></sec></abstract><kwd-group><kwd>Age-related Macular Degeneration (AMD)</kwd><kwd> Intraocular Drugs</kwd><kwd> Visual Acuity</kwd></kwd-group><counts><fig-count count=\"4\"/><table-count count=\"2\"/><ref-count count=\"24\"/><page-count count=\"10\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> INTRODUCTION</title><p>Neovascular age-related macular degeneration (NV-AMD) is the leading cause of vision loss in individuals aged 50 years or older. Over the past decade, treatment has evolved to control subfoveal choroidal neovascularization (CNV) growth with intravitreal drug delivery directed toward inhibition of vascular endothelial growth factor (VEGF). Specifically, the MARINA and ANCHOR studies were amongst the first to demonstrate the effects of targeting angiogenesis by blocking VEGF-A with ranibizumab, a recombinant humanized monoclonal antibody fragment (Fab). These studies clearly showed that monthly treatment was beneficial in preventing vision loss and allowing for visual gain compared to sham and photodynamic therapy (PDT), respectively, over a two-year period.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup> The VIEW 1 and 2 trials demonstrated the efficacy of aflibercept, a soluble decoy receptor fusion protein with a higher affinity to VEGF-A and VEGF-B as well as placental growth factor (PIGF) with decreased treatment burden allowing improvement or maintenance of vision over two years.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> However, monthly or bimonthly injections along with monthly follow-up is challenging for patients to maintain in clinical practice.</p><p>Due to treatment burden, pro re nata (PRN) treatment was studied to examine the effects of monthly follow-up with an individualized retreatment regimen. CATT and IVAN trials demonstrated equivalent efficacy between ranibizumab versus bevacizumab; however, there was an overall less favorable outcome in the PRN arms compared to monthly dosing with respect to final visual acuity.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>,<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup>\n</p><p>In 2009, Freund and colleagues were the first to describe the “treat-and-extend (TAE)” regimen with treatment of Type 3 CNV lesions in a small cohort over three years.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> With use of ranibizumab and/or bevacizumab, they showed an overall improvement of vision from 20/80 to 20/40 with an average of 6–7 injections per year. Since then, several other retrospective studies have proposed using a TAE approach as an alternate to monthly or PRN dosing to reduce treatment burden while maintaining or improving visual outcome.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>,<xref rid=\"B11\" ref-type=\"bibr\">11</xref>,<xref rid=\"B12\" ref-type=\"bibr\">12</xref>]</sup>\n</p><p>Despite the multitude of trials demonstrating the safety and efficacy of anti-VEGF drug therapy, there have been limited studies describing the long-term follow-up of anti-VEGF treatment. SEVEN-UP and CATT were among the first studies to describe the long-term outcomes with either monthly or PRN dosing.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>,<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup> Of the TAE trials, the longest to date was by Mrejen et al over a six-year period with 185 patients and a retention rate of 62.9%.<sup>[<xref rid=\"B12\" ref-type=\"bibr\">12</xref>]</sup> Their study showed an improvement or maintenance in visual acuity with an average of 8.3 injections per year. They demonstrated that a greater number of injections was an independent predictor of better visual outcome. Other studies have compared TAE to PRN revealing a worse visual outcome with a smaller number of injections with the PRN group.<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>,<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup> Specifically, Calvo et al showed that over a three-year follow-up period, 42.4% in the TAE dosing group versus 24.1% in the PRN dosing group gained at least three lines of vision. Over the study period, the TAE group was treated with an average of 20.31 injections, while the PRN group was treated with an average of 18.41.<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>]</sup> TAE is a practical option to reducing the number of injections and office visits as compared to a monthly and PRN regimen.</p><p>Our current study reports a seven-year follow-up period of the long-term outcomes as measured by visual acuity and OCT imaging of the treatment-naïve NV-AMD patients using a TAE model.</p></sec><sec sec-type=\"section\"><title> METHODS</title><p>The Institutional Review Board of Northwestern University Feinberg School of Medicine approved this retrospective cohort study at a large urban tertiary medical center. Study data was obtained through the Northwestern Medicine Enterprise Data Warehouse (NMEDW) and through direct chart review. Our study population consisted of treatment-naïve patients of four retina specialists receiving intravitreal anti-VEGF with the diagnosis of neovascular AMD (ICD-9 code 362.52) from March 2008 to October 2015. Other inclusion criteria were: age more than 50 years, visual acuity of hand motion (HM) or better at baseline, and a follow-up duration of at least one year. All four physicians used a TAE protocol consisting of initial intensive monthly anti-VEGF injections until there was no evidence of exudation on OCT followed by extension of treatment interval by two weeks up until 12 weeks. If there was a mild recurrence of subretinal fluid (SRF), intraretinal fluid (IRF), or a new macular hemorrhage, then the interval was reduced by one–two weeks until the macula was dry or hemorrhage stabilized. In the case of more severe recurrences, monthly treatment was reinitiated.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>]</sup> The interval was not increased in the presence of persistent but stable fluid, however, if a pigment epithelial detachment (PED) persisted in the absence of SRF or IRF, then the interval was extended. Patient demographics, type of anti-VEGF agent used (bevacizumab, ranibizumab, or aflibercept), number and frequency of injections, best-corrected visual acuities (BCVA), and intraocular pressure (IOP) were obtained at each office visit from the EDW database. In this article, single-agent monotherapy is defined as treatment with only one type of anti-VEGF agent over the entire treatment course, while multi-agent therapy is defined as treatment with multiple types of anti-VEGF agents but not during the same office visit.</p><p>OCT images of the affected eye were obtained from direct chart review at baseline and at the last follow-up visit. OCT images of each affected eye at baseline and the last follow-up visit were directly reviewed for the presence or absence of SRF and IRF. BCVA and IOP were extracted for each affected eye at the start of treatment and at time-points of six months, one year, and every year thereafter until the last office visit. Measurements from the office visit whose date was closest to the specific time-point were selected but was required to be within three months of the specific time-point to be included. Visual acuity values were converted from Snellen to logMAR to allow the paired <italic>t</italic>-test comparisons. Visual acuity values were also categorized as 20/50 or better, between 20/50 and 20/200, and 20/200 or worse for further interpretation. The number and types of injections were also tallied for each affected eye.</p><p>Subsequent numerical and statistical analyses were performed in Microsoft Excel 2016 (Microsoft Corporation, Redmond, WA) and GraphPad Prism 7 (GraphPad Software, San Diego, CA). The paired Student <italic>T</italic>-test was performed to compare visual acuities and intraocular pressure at the first and last office visits. Pearson's correlation coefficient was calculated to test for the linearity of changes in visual acuity over time. Statistical analysis was also performed for visual acuity categories using contingency tables with Fisher's exact test and the OCT data was analyzed with McNemar's test. Subgroup analysis was conducted on eyes treated with single-agent monotherapy to examine the visual acuity and OCT outcomes for each drug.</p></sec><sec sec-type=\"section\"><title> RESULTS</title><p>In total, 224 treatment-naïve eyes of 202 patients were analyzed with an average follow-up period of 3.4 years (range, 1.0–7.6 years). The majority (80%) of patients in this study were between 70 and 89 years of age at initial presentation. Of the 224 eyes, 137 (61.2%) were treated with only one type of anti-VEGF agent: ranibizumab (71, 51.8%), aflibercept (47, 34.3%), or bevacizumab (19, 13.9%). Visual acuity at baseline was 20/100 in Snellen and did not differ significantly between the treatment groups (F = 1.33, <italic>P</italic> = 0.27). Most eyes had either SRF (70%) or IRF (47%) present on OCT imaging at baseline (Table 1).</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Patient baseline characteristics</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"7\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" colspan=\"3\" rowspan=\"1\"><bold>Monotherapy (</bold>\n<italic><bold>N</bold></italic>\n<bold> = 137)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Multi-drug therapy (</bold>\n<italic><bold>N</bold></italic>\n<bold> = 87)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Total (</bold>\n<italic><bold>N</bold></italic>\n<bold> = 224)</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Bevacizumab (<italic>N</italic> = 19)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ranibizumab (<italic>N</italic> = 71)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Aflibercept (<italic>N</italic> = 47)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" colspan=\"7\" rowspan=\"1\">\n<bold>Eye – no (%)</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">OD</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7(37)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33 (47)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21 (45)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">48 (55)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">109 (49)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">OS</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12(63)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">38 (54)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26 (55)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">39 (45)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">115 (51)</td></tr><tr><td align=\"center\" colspan=\"7\" rowspan=\"1\">\n<bold>Gender – no (%)</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">F</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16(84)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">49 (69)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">38 (81)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">58 (67)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">161 (72)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3(16)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22 (31)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9 (19)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">29 (33)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">63 (28)</td></tr><tr><td align=\"center\" colspan=\"7\" rowspan=\"1\">\n<bold>Race – no (%)</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Caucasian</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12(63)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">52 (73)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">40 (85)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">72 (83)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">176 (79)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">African American</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2(11)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5 (6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11 (5)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Hispanic</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3(16)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 (2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6 (3)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Other/Unknown</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2(11)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (20)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (15)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8 (9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">31 (14)</td></tr><tr><td align=\"center\" colspan=\"7\" rowspan=\"1\">\n<bold>Age</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Mean</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">78.1 <inline-formula><mml:math id=\"M2\"><mml:mo>±</mml:mo></mml:math></inline-formula> 12.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">82.1 <inline-formula><mml:math id=\"M3\"><mml:mo>±</mml:mo></mml:math></inline-formula> 6.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">83.3 <inline-formula><mml:math id=\"M4\"><mml:mo>±</mml:mo></mml:math></inline-formula> 6.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">78.2 <inline-formula><mml:math id=\"M5\"><mml:mo>±</mml:mo></mml:math></inline-formula> 8.7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">80.5 <inline-formula><mml:math id=\"M6\"><mml:mo>±</mml:mo></mml:math></inline-formula> 8.3</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">50–69 -no. (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (21)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13 (15)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21 (9)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">70–89 -no. (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12 (63)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">62 (87)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">40 (85)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">66 (76)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">180 (80)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">90+ -no. (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 (16)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (10)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5 (11)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8 (9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23 (10)</td></tr><tr><td align=\"center\" colspan=\"7\" rowspan=\"1\">\n<bold>Visual Acuity (Snellen)</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Mean</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20/94</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20/124</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20/91</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20/89</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">20/100</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">20/50 or Better - no. (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (37)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19 (27)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20 (43)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32 (37)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">78 (35)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Between 20/50 and 20/200 - no. (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (37)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29 (41)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17 (36)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33 (38)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">86 (38)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Worse than 20/200 - no. (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5 (26)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23 (32)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 (21)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">22 (25)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">60 (27)</td></tr><tr><td align=\"center\" colspan=\"7\" rowspan=\"1\">\n<bold>OCT Findings</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">SRF-no. (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (74)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">42 (59)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30 (64)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">73 (84)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">159 (71)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">IRF-no. (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8 (42)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">43 (61)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27 (57)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">28 (32)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">106 (47)</td></tr><tr><td align=\"left\" colspan=\"7\" rowspan=\"1\">OCT, optical coherence tomography; SRF, subretinal fluid; IRF, intraretinal fluid</td></tr></tbody></table></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>OCT* characteristics of single-agent vs multi-agent anti-VEGF therapy</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"7\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Anti-VEGF Drug for Single-agent Monotherapy</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold># SRF + Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold># SRF + Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold/>\n<italic><bold>P</bold></italic>\n<bold>-value</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold># IRF + Pre-treatment</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold># IRF + Post-treatment</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold/>\n<italic><bold>P</bold></italic>\n<bold>-value</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Bevacizumab (</bold>\n<italic><bold>N</bold></italic>\n<bold> = 19)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>\n<inline-formula><mml:math id=\"M7\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.01</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic> = 0.077</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Ranibizumab (</bold>\n<italic><bold>N</bold></italic>\n<bold> = 71)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">42</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>\n<inline-formula><mml:math id=\"M8\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>\n<inline-formula><mml:math id=\"M9\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Aflibercept (</bold>\n<italic><bold>N</bold></italic>\n<bold> = 47)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>\n<inline-formula><mml:math id=\"M10\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>\n<inline-formula><mml:math id=\"M11\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Multi-agent Therapy</bold>\n<bold>(</bold>\n<italic><bold>N</bold></italic>\n<bold> = 87)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">73</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>\n<inline-formula><mml:math id=\"M12\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>\n<inline-formula><mml:math id=\"M13\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Total Cohort</bold>\n<bold>(</bold>\n<italic><bold>N</bold></italic>\n<bold> = 224)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">159</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>\n<inline-formula><mml:math id=\"M14\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">106</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">42</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>P</italic>\n<inline-formula><mml:math id=\"M15\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"left\" colspan=\"7\" rowspan=\"1\">OCT, optical coherence tomography; SRF, subretinal fluid; IRF, intraretinal fluid; VEGF, vascular endothelial growth factor</td></tr></tbody></table></table-wrap><p>The average visual acuity at baseline of 0.698 logMAR (20/100 Snellen equivalent) remained stable at the final follow-up visit at 0.666 logMAR (20/93 Snellen) (<italic>P</italic> = 0.30; Figure 1a). A significant portion of eyes (40%) maintained their visual acuities within two Snellen lines, while 34% of eyes gained more than two lines and 25% lost more than two lines. The percentage of eyes with visual acuities of 20/50 or better increased significantly from 34.8% at baseline to 45.1% by the last follow-up visit (Fischer's exact test, <italic>P</italic> = 0.037; Figure 1b). There was no significant difference between baseline IOP (14.8 <inline-formula><mml:math id=\"M16\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.3) and IOP at the last follow-up visit (15.2 <inline-formula><mml:math id=\"M17\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.6) (<italic>P</italic> = 0.052).</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Comparison of mean visual acuity between baseline and last visit. Figure 1A shows boxplot comparisons between baseline and final visual acuities. Figure 1B shows the percentage of patients in each visual acuity category by the last follow-up visit compared to baseline.</p></caption><graphic xlink:href=\"jovr-15-331-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Cumulative gain in logMAR over treatment course (Mean <inline-formula><mml:math id=\"M18\"><mml:mo>±</mml:mo></mml:math></inline-formula> SE). Visual acuities recorded during patients' treatment visits were compared with the visual acuity at baseline. Only those patients actively continuing to receive injections were included in this figure; patients who discontinued injections after a specific time were not included in subsequent time points in this graph.</p></caption><graphic xlink:href=\"jovr-15-331-g002\"/></fig><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>Number of injections over time. The figure shows the average annual number of injections administered over time.</p></caption><graphic xlink:href=\"jovr-15-331-g003\"/></fig><fig id=\"F4\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><p>Comparison of OCT finding between baseline and last visit. The figure shows the number of eyes with the presence of SRF and IRF pre- and post-treatment.</p></caption><graphic xlink:href=\"jovr-15-331-g004\"/></fig><p>Visual acuities of patients receiving ongoing injections recorded at six months, one year, and annually thereafter showed an overall steady gain that peaks near the end of the third year, with slight reductions thereafter (Figure 2). The smaller sample size in these groups impedes any individual subgroup analysis of the treatment type.</p><p>The baseline visual acuity at initial presentation was tested against the change in visual acuity along with demographic factors of sex and age as possible predictors of patient's response to treatment. Baseline visual acuities exhibited a weak linear correlation with the cumulative change in visual acuities at all time-points (Pearson's coefficient r averaged over timepoints = –0.45, <italic>P</italic>\n<inline-formula><mml:math id=\"M19\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05). Age was not found to be linearly correlated with the change in visual acuity at the last follow-up (Pearson's r = 0.13, p = 0.33). Similarly, patients' sex and also race (Caucasian vs non-Caucasian) were not correlated with the treatment response (t = –1.13, <italic>P</italic> = 0.26 and t = –0.6, <italic>P</italic> = 0.55, respectively).</p><p>Over the course of the study, 224 eyes received an average of 20.3 <inline-formula><mml:math id=\"M20\"><mml:mo>±</mml:mo></mml:math></inline-formula> 14.7 injections (range, 2–95) during a mean of 3.4 years of follow-up (range, 1.0–7.6), for a total of 4,543 injections. Of the 224 eyes, 137 (61.2%) were treated with a single agent for the duration of their treatment [71 (51.8%) with ranibizumab, 47 (34.3%) with aflibercept, and 19 (13.9%) with bevacizumab] while 87 (38.8%) eyes were treated with more than one agent type. For those patients receiving single-agent therapy, the number of total injections did not differ significantly based on the agent used (14.8 for bevacizumab vs 14.7 for ranibizumab vs 13.0 for aflibercept, <italic>P</italic> = 0.54) over the course of treatment, although the average duration of treatment in weeks varied significantly (35.5 for bevacizumab vs 28.6 for ranibizumab vs 21.7 for aflibercept, <italic>P</italic> = 0.014). The number of injections that patients received differed over time (Figure 3). Eyes in the first year of treatment received an average of 8.4 injections that decreased on average by 0.3 injections per year to 5.5 injections by the seventh year (R<inline-formula><mml:math id=\"M21\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula> = 0.68). Moreover, eyes that gained more than two lines received significantly more injections with an average number of 24.1 <inline-formula><mml:math id=\"M22\"><mml:mo>±</mml:mo></mml:math></inline-formula> 15.3 injections, while eyes that maintained within two lines or lost more than two lines received 18.1 <inline-formula><mml:math id=\"M23\"><mml:mo>±</mml:mo></mml:math></inline-formula> 13.3 injections and 18.6 <inline-formula><mml:math id=\"M24\"><mml:mo>±</mml:mo></mml:math></inline-formula> 15.2 injections, respectively (<italic>P</italic>\n<inline-formula><mml:math id=\"M25\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05). It was also found that eyes with visual acuities of 20/200 or better at last follow-up tended to receive treatment over a longer period (average 3.1 years vs 2.2 years, <italic>P</italic>\n<inline-formula><mml:math id=\"M26\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.01) and received a greater number of injections (average 23 vs 14, <italic>P</italic>\n<inline-formula><mml:math id=\"M27\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001) compared to eyes with visual acuities of 20/200 or worse at last follow-up.</p><p>In addition to visual acuity analysis, OCT images were compared at baseline and at the last follow-up visit. Out of the 224 eyes, 159 eyes had SRF at baseline compared with 35 eyes by the date of last follow-up (<italic>P</italic>\n<inline-formula><mml:math id=\"M28\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.0001; Figure 4). Similarly, 106 eyes had IRF at baseline compared with 42 eyes by the date of last follow-up (<italic>P</italic>\n<inline-formula><mml:math id=\"M29\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.0001). When aggregated, 208 eyes had some type of fluid at baseline, compared with 69 eyes by the date of last follow-up (<italic>P</italic>\n<inline-formula><mml:math id=\"M30\"><mml:mo><</mml:mo></mml:math></inline-formula>0.0001). Subset analysis of eyes receiving single-agent anti-VEGF therapy did not reveal any differences in OCT outcomes and mirrored the trends seen in the overall group. All treatment groups showed a statistically significant decrease in the presence of fluid over the course of the treatment except for the presence of IRF in the bevacizumab group (Table 2), although this may be attributed to the smaller sample size of this subgroup (<italic>n</italic> = 19).</p></sec><sec sec-type=\"section\"><title> DISCUSSION</title><p>We report up to a seven-year (average, 3.4 years) follow-up period of treatment-naïve NV-AMD patients undergoing anti-VEGF therapy using a TAE model. All four investigators in our study used the consensus recommendations of the TAE regimen following monthly injections until the macula was dry based on OCT, then extending the interval between treatments by two weeks to a maximum of twelve weeks. If fluid recurred, then the interval would be shortened. Using this approach, the patients' treatment is individually tailored to its response. The TAE regimen offers an alternate and preferred treatment practice due to reduced burden for office visits compared to monthly and OCT-guided dosing regimens.</p><p>Prior to the development of TAE regimen, long-term outcomes of monthly and PRN anti-VEGF treatments have been described in several other studies, most notably in the SEVEN-UP and 5-year CATT study.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>,<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup> The SEVEN-UP study reported on ranibizumab-treated patients after an average of 7.3 years from the time of first injection with patients receiving monthly injections in the first two years followed by PRN treatment over the subsequent years. Patients received an average of 6.8 total injections over a mean 3.4 year interval. The subgroup that received more frequent injections yielded a better result in visual acuity gains. In their study, 23% attained a visual acuity of 20/40 or better whereas 37% were 20/200 or worse. There was an overall mean decline of 8.2 letters over the course of follow-up.<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup>\n</p><p>In the five-year CATT study, patients were followed an average of 5.5 years from the time of first injection. Ranibizumab- or bevacizumab-treated patients were stratified into monthly or PRN arms in the first year with the monthly arm stratified again into monthly or PRN treatment in the second year. In the subsequent three years, a variety of treatment drug combinations and regimens were used with patients receiving an average of 15.4 injections over three years. In their study, 49.6% attained visual acuity of 20/40 or better whereas 20% were 20/200 or worse. There was a mean overall decline of 3.3 letters.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>]</sup>\n</p><p>Since the initial description of the TAE regimen, several studies have reproduced results favoring maintenance or improvement of BCVA similar to monthly dosing while reducing injection frequency and treatment burden. Our study is comparable to others that describe outcomes using a TAE regimen.<sup>[<xref rid=\"B18\" ref-type=\"bibr\">18</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>,<xref rid=\"B20\" ref-type=\"bibr\">20</xref>,<xref rid=\"B21\" ref-type=\"bibr\">21</xref>,<xref rid=\"B22\" ref-type=\"bibr\">22</xref>,<xref rid=\"B23\" ref-type=\"bibr\">23</xref>]</sup> Similar to our study, BCVA in these studies was either maintained or improved throughout treatment with 30–34% of patients on average improving by 2–3 lines and 94–97.5% patients losing less than 2–3 lines. The number of injections in the first year averaged a total of 7.6–8.6, which is comparable to our mean of 8.4. Most of these studies, however, only reported on outcomes over a two-year follow-up while our study looks at outcomes over a longer treatment period. Of note, in our study, the average number of injections decreased to 5.5 during the seventh year while maintaining BCVA.</p><p>A study by Mrejen et al with a longer follow-up period of six years (average 3.5 years) compared to the aforementioned studies<sup>[<xref rid=\"B18\" ref-type=\"bibr\">18</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>,<xref rid=\"B20\" ref-type=\"bibr\">20</xref>,<xref rid=\"B21\" ref-type=\"bibr\">21</xref>,<xref rid=\"B22\" ref-type=\"bibr\">22</xref>,<xref rid=\"B23\" ref-type=\"bibr\">23</xref>]</sup> demonstrated similar favorable results.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>,<xref rid=\"B11\" ref-type=\"bibr\">11</xref>,<xref rid=\"B12\" ref-type=\"bibr\">12</xref>]</sup> In their study, BCVA peaked at 18 months with a steady decline afterward. On average, patients received 28.5 injections over the study period with 8.3 injections per year and a mean interval of 6.6 weeks between injections. The majority of their patients (64.3%) were treated with injection of a single agent of which 59% of them were ranibizumab alone, 4.3% were bevacizumab alone, and 1% was aflibercept alone. Their multivariant analysis showed a greater number of injections as an independent predictor of better visual outcomes. On the other hand, older age of starting injections, hypertension, and anticoagulation were correlated with poorer visual outcomes.</p><p>In the current study, we utilized a TAE approach in which patients were followed for an average of 3.4 years (range, 1.0–7.6) receiving an injection regimen with an average of 20.3 <inline-formula><mml:math id=\"M31\"><mml:mo>±</mml:mo></mml:math></inline-formula> 14.7 total injections with 8.4 injections in the first year and 5.5 injections by the seventh year of follow-up. The majority of patients (61.1%) were treated with a single anti-VEGF agent for the duration of their treatment, of which 51.8% were ranibizumab alone, 34.3% were aflibercept alone, and 13.9% were bevacizumab alone. Having more single-agent data analysis allows us to validate similarities of BCVA outcomes regardless of the drug type. BCVA peaked after three years of treatment with a slow decline thereafter. Baseline visual acuity was weakly shown to be the only significant predictor of change in visual acuity. There were no significant differences between drug type and visual acuity effect, number of injections needed, or OCT outcomes.</p><p>Eyes with a final visual acuity of 20/50 or better increased from 34.8% at the beginning of the study to 45.1% at the latest follow-up (<italic>P</italic> = 0.037), while eyes with 20/200 or worse remained stable at 26.8% at baseline compared to 27.7% at last follow-up (<italic>p</italic> = 0.92). In contrast, in the five-year CATT study, eyes with 20/200 or worse increased significantly from 6% at baseline to 20% at the last follow-up<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>]</sup> even though in our study there were more patients with baseline vision of 20/200 or worse. At the end of the SEVEN-UP study, 37% of patients were reported to have visual acuity of 20/200 or worse. Overall, 74% of patients in our study maintained or gained at least two Snellen lines of visual acuity, compared with the SEVEN-UP trial where only 55% of eyes maintained or improved their vision.<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup> At the end of our study, eyes that improved by at least two lines received an average of 24 injections, while all other eyes received an average of 18 injections. Similarly, in the SEVEN-UP study, eyes receiving a greater number of injections (11 vs 6.8) gained 3.9 letters overall and were more likely to show improvement in vision.<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup>\n</p><p>The better visual outcomes in our population compared to the five-year CATT study may be explained by the OCT analysis. At the last follow-up visit, 16% of eyes in our study had SRF and 19% of eyes had IRF. Those without SRF or IRF had either a PED or were without any fluid. In comparison, at the end of five years in the CATT study, 38% had SRF and 61% had IRF.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>]</sup> Eyes with residual IRF yield worse visual outcomes compared with eyes with residual SRF or absence of fluid.<sup>[<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup> More frequent treatments may have an impact on the amount of fluid on OCT to allow for maintenance or gain in visual acuity. However, other factors such as geographic atrophy also contribute to the final visual acuity. Though not studied in our population, the CATT study demonstrated that 24% of eyes with monthly dosing showed geographic atrophy compared to 15% in the PRN group. Similarly, in the IVAN trial, 34% versus 26% showed progressive atrophy in the monthly versus PRN groups, respectively.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>,<xref rid=\"B13\" ref-type=\"bibr\">13</xref>,<xref rid=\"B17\" ref-type=\"bibr\">17</xref>]</sup>\n</p><p>Our study demonstrates that a TAE model allows for a frequent albeit lower treatment burden as compared to monthly dosing with reduction in fluid on OCT. This may theoretically lower the rate of geographic atrophy while maintaining similar gains in visual potential compared with a monthly dosing regimen.</p><p>There are several limitations in our study most notably its retrospective nature. With data being compiled via electronic database, records may be incomplete and there may be innate errors in how the data was recorded. Patients began treatment at different times between 2008 and 2015 and there may be differences both in medical technology and in practice patterns amongst providers. There is no monthly regimen treatment arm to compare its efficacy with our TAE model. Due to the method of data collection, there were fewer patients with more than four to five years of treatment available for analysis thereby limiting sample size and comparisons across different anti-VEGF agents. Some patients have undergone cataract surgery during treatment period, which can confound BCVA amongst patients. Furthermore, more in-depth studies are needed to analyze the impact of residual fluid type on visual acuity.</p><p>In conclusion, our retrospective uncontrolled review of a large urban cohort of NV-AMD reveals favorable long-term visual and anatomic results of anti-VEGF therapy using a TAE regimen. Our study demonstrates that visual acuity seems to improve with more frequent injections over a longer period of time. The majority of patients (74%) maintained or improved vision with 45% of patients achieving VA of 20/50 or better at their last follow-up over a seven-year period. Our study supports the use of a TAE treatment paradigm to reduce both office visits and treatment burden while still achieving positive functional and anatomic results.</p></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>This study was supported in part by an unrestricted grant from Research to Prevent Blindness and by the Northwestern Medicine Enterprise Data Warehouse. The sponsor or funding organization had no role in the design or conduct of this research.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Rosenfeld PJ, Brown DM, Heier JS, Boyer DS, Kaiser PK, Chung CY, et al. Ranibizumab for neovascular age-related macular degeneration. <italic>New Engl J Med</italic> 2006;355:1419–1431.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">Lalwani GA, Rosenfeld PJ, Fung AE, Dubovy SR, Michels S, Feuer W, et al. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864073</article-id><article-id pub-id-type=\"pmc\">PMC7431724</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7461</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Case Report</subject></subj-group></article-categories><title-group><article-title>Focal Choroidal Excavation in a Case of Choroidal Osteoma Associated with Choroidal Neovascularization</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Azimizadeh</surname><given-names>Mahdieh</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hosseini</surname><given-names>Seyedeh Maryam</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Babaei</surname><given-names>Esmaeil</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Eye Research Center, Mashhad University of Medical Sciences, Mashhad, Iran</aff><aff id=\"I2\">\n<sup>2</sup>Retina Research Center, Mashhad University of Medical Sciences, Mashhad, Iran</aff><aff id=\"I3\">\n<sup>3</sup>Eye Research Center, Yazd University of Medical Sciences, Yazd, Iran</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Esmaeil Babaei, MD. Retina Research Center, Khatam\nAl Anbia Eye Hospital, Mashhad, Aboutaleb Junction,\nGharanei BLVD, Mashahad University of Medical\nSciences, Mashhad 917789, Iran.\nEmail: esmaeil.babaei@yahoo.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>419</fpage><lpage>423</lpage><history><date date-type=\"received\"><day>10</day><month>12</month><year>2018</year></date><date date-type=\"accepted\"><day>29</day><month>2</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Azimizadeh et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><sec><title>Purpose</title><p>To report a case of choroidal osteoma associated with reactivation of choroidal neovascularization (CNV) and development of focal choroidal excavation (FCE).</p></sec><sec><title>Case Report</title><p>A 34-year-old woman with choroidal osteoma complicated by CNV in the right eye for two years presented with deterioration of visual acuity in her right eye. A small retinal hemorrhage accompanied by subretinal fluid (SRF) was seen in the macular area of the right eye. Optical coherence tomography (OCT) showed that the inner retina was intact, and the outer retinal layers had outward displacement. SRF and a wedge-shaped choroidal depression were also seen. This choroidal excavation was not present on previous OCT images. The integrity of the inner retinal layers was maintained, and an optically clear space was present between the neurosensory retina and the retinal pigment epithelium.</p></sec><sec><title>Conclusion</title><p>Choroidal osteoma can be complicated by CNV and FCE could occur as a consequence. Again, FCE can lead to CNV development. This cascade can deteriorate vision and sometime lead to permanent visual loss.</p></sec></abstract><kwd-group><kwd>Bevacizumab</kwd><kwd> Choroidal Excavation</kwd><kwd> Choroidal Neovascularization</kwd><kwd> Choroidal Osteoma</kwd><kwd> Multifocal</kwd></kwd-group><counts><fig-count count=\"4\"/><ref-count count=\"15\"/><page-count count=\"5\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> INTRODUCTION</title><p>Choroidal osteoma is a rare, ossifying, benign tumor of the choroid. It occurs predominantly in young healthy women in the second and third decades of life and is unilateral in 80% of cases.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> Although it has been recognized as a benign tumor, it can grow in 51% of patients by 10 years and can be complicated by enlargement, decalcification, and choroidal neovascularization (CNV).<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup>\n</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Color fundus photograph of the right eye.</p></caption><graphic xlink:href=\"jovr-15-419-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>SD- OCT of the right eye: (A) Spectral-domain OCT (SD-OCT) of the right eye performed in 2014 revealed subretinal fluid (SRF) associated with choroidal osteoma complicated by choroidal neovascularization (CNV). (B) OCT of the right eye (2016) clearly shows focal choroidal excavation and SRF due to CNV development. (C) OCT of the right eye clearly shows FCE and complete resolution of SRF after two consecutive intravitreal bevacizumab injections.</p></caption><graphic xlink:href=\"jovr-15-419-g002\"/></fig><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>Fluorescein angiography (FAG) showing choroidal neovascularization in the right eye.</p></caption><graphic xlink:href=\"jovr-15-419-g003\"/></fig><fig id=\"F4\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><p>OCTA images of the right eyes illustrate high-flow vessels above the RPE in the outer retina as well as in the choriocapillaris.</p></caption><graphic xlink:href=\"jovr-15-419-g004\"/></fig><p>Focal choroidal excavation (FCE) refers to focal depression of the choroid that has been recently described thanks to the development of spectral-domain optical coherence tomography (SD-OCT). It may be solitary or occur in association with other chorioretinal disorders such as central serous chorioretinopathy (CSC), CNV, and polypoidal choroidal vasculopathy (PCV).<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup> It has also been reported in eyes with choroidal osteoma associated with CNV.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup>\n</p><p>Here, we report an interesting case involving a 34-year-old woman with bilateral choroidal osteoma complicated by CNV in the right eye, with development of FCE following reactivation of the CNV.</p></sec><sec sec-type=\"section\"><title> CASE REPORT</title><p>A 34-year-old healthy woman with bilateral choroidal osteoma complicated by CNV in the right eye for the two years presented with acute deterioration of visual acuity (VA) in the right eye. The findings of multimodal imaging for this case were reported and published in 2015, and there was no FCE in the right eye at that time.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>Best corrected visual acuity (BCVA) of the right eye had decreased from 20/40 to 20/100, while that of the left eye was 20/20. Fundus examination of both eyes showed a yellow–orange lesion with sharp borders at the level of the choroid (Figure 1). A small retinal hemorrhage accompanied by subretinal fluid (SRF) was seen in the macular area of the right eye. Enhanced depth imaging OCT (EDI-OCT) (Spectralis HRA + OCT, Heidelberg Engineering, Heidelberg, Germany) showed that the inner retina was intact, although the outer retinal layers had outward displacement due to active CNV along with SRF and a wedge-shaped choroidal depression in the right eye. The choroidal excavation involved the retinal pigment epithelium (RPE) and outer retinal layers up to the outer plexiform layer. The integrity of the inner retinal layers was maintained, and an optically clear space was present between the neurosensory retina and RPE. EDI-OCT showed thinning of the choriocapillaris and large choroidal vessels and a sponge-like choroidal lesion (Figure 2). Fluorescein angiography (FAG) demonstrated mild hyperfluorescence in the choroidal osteoma due to window defect, as well as juxtafoveal hyperfluorescence corresponding to active CNV in the right eye (Figure 3), which was better delineated on OCT angiography (Figure 4).</p><p>Thus, the diagnosis was reactivation of CNV associated with the development of FCE in the right eye in a known case of choroidal osteoma. The patient received two consecutive intravitreal bevacizumab (Avastin, Genentech Inc., San Francisco, CA) injections, which resulted in improvement of BCVA to 20/40. OCT showed disappearance of SRF and resolution of the disturbances in the outer retinal layers. FCE was more clearly visible and associated with severe choroidal thinning after quiescence of the CNV (Figure 2c). BCVA was maintained in both eyes during a follow-up period of one year.</p></sec><sec sec-type=\"section\"><title> DISCUSSION</title><p>We report development of FCE following reactivation of CNV after two years of inactivity in an eye with choroidal osteoma.</p><p>Initially, FCE was defined as choroidal excavation in the submacular area without posterior staphyloma, any positive history of trauma, or any chorioretinal disease; however, some chorioretinal disorders such as CSC, PCV, CNV, and choroidal osteoma have been reported in association with FCE.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup> Although the inner retina is commonly intact in eyes with FCE, the outer retinal layers, RPE, choriocapillaris, and choroid can be involved.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup>\n</p><p>FCE can be detected by SD-OCT. However, in cases with bilateral involvement or multiple lesions in one eye, foveal SD-OCT which is confined to the posterior pole may not detect all lesions. Therefore, multiple imaging modalities such as fundus photography, fundus autofluorescence, indocyanine green angiography, and FAG are considered helpful.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup>\n</p><p>In the present case, FCE developed adjacent to a previous CNV scar and the decalcified part of the tumor as seen on previous OCT images, and it was detected two years after CNV occurrence. Thus, we can conclude that FCE was a consequence of the CNV scar and/or tumor decalcification that resulted in tissue loss, contraction of fibrosis, and outward displacement and excavation of the outer retina and choroid. On the other hand, FCE may have been involved in reactivation of CNV,<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> as choroidal excavation has been reported to be associated with the development or reactivation of CNV.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>]</sup> There are multiple options for treating CNV in eyes with choroidal osteoma,<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>,<xref rid=\"B12\" ref-type=\"bibr\">12</xref>,<xref rid=\"B13\" ref-type=\"bibr\">13</xref>,<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup> although the best response has been achieved by intravitreal injection of anti-vascular endothelial growth factor agents such as bevacizumab.<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>]</sup>\n</p><p>This is a documented development of FCE in an eye with CNV associated with choroidal osteoma. FCE could occur as a consequence of CNV, tumor decalcification, or both. Close follow-up and prompt treatment are necessary to achieve the best results.</p></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>Nil.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Erol MK, Coban DT, Ceran BB, Bulut M. Enhanced depth imaging optical coherence tomography and fundus autofluorescence findings in bilateral choroidal osteoma: a case report. <italic>Arq Bras Oftalmol</italic> 2013; 76:189-91.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">Shields CL, Perez B, Materin MA, Mehta S, Shields JA. Optical coherence tomography of choroidal osteoma in 22 cases: evidence for photoreceptor atrophy over the decalcified portion of the tumor. <italic>Ophthalmology</italic> 2007; 114:e53-8</mixed-citation></ref><ref id=\"B3\"><label>3</label><mixed-citation publication-type=\"other\">Cebeci Z, Bayraktar Ş, Oray M, K<inline-formula><mml:math id=\"M1\"><mml:mo>ı</mml:mo></mml:math></inline-formula>r N. Focal choroidal excavation. <italic>Turk J Ophthalmol</italic> 2016; 46:296.</mixed-citation></ref><ref id=\"B4\"><label>4</label><mixed-citation publication-type=\"other\">Pierro L, Marchese A, Gagliardi M, Introini U, Parodi MB, Casalino G, et al. Choroidal excavation in choroidal osteoma complicated by choroidal neovascularization. <italic>Eye (Lond)</italic>. 2017;31:1740-1743.</mixed-citation></ref><ref id=\"B5\"><label>5</label><mixed-citation publication-type=\"other\">Wakabayashi Y, Nishimura A, Higashide T, Ijiri S, Sugiyama K. 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Focal choroidal excavation: a preliminary interpreta-tion based on clinic and review. <italic>Int J Ophthalmol</italic> 2015;8:513-21.</mixed-citation></ref><ref id=\"B9\"><label>9</label><mixed-citation publication-type=\"other\">Chung H, Byeon SH, Freund KB. Focal choroidal excavation and its association with pachychoroid spectrum disorders.\n<italic>Retina</italic> 2017;37:199-221.</mixed-citation></ref><ref id=\"B10\"><label>10</label><mixed-citation publication-type=\"other\">Xu H, Zeng F, Shi D, Sun X, Chen X, Bai Y. Focal choroidal excavation complicated by choroidal neovascularization. <italic>Ophthalmology</italic> 2014;121:246-250.</mixed-citation></ref><ref id=\"B11\"><label>11</label><mixed-citation publication-type=\"other\">Mansour AM, Arevalo JF, Al Kahtani E, Zegarra H, Abboud E, Anand R, et al. Role of intravitreal antivascular endothelial growth factor injections for choroidal neovascularization due to choroidal osteoma. <italic>J Ophthalmol</italic> 2014;2014:210458.</mixed-citation></ref><ref id=\"B12\"><label>12</label><mixed-citation publication-type=\"other\">Shields CL, Sun H, Demirci H, Shields JA. Factors predictive of tumor growth, tumor decalcification, choroidal neovascularization, and visual outcome in 74 eyes with choroidal osteoma. <italic>Arch Ophthalmol</italic> 2005;123:1658-66.</mixed-citation></ref><ref id=\"B13\"><label>13</label><mixed-citation publication-type=\"other\">Browning D. J, Choroidal osteoma: observations from a community setting, <italic>Ophthalmology</italic> 2003;110:1327-34.</mixed-citation></ref><ref id=\"B14\"><label>14</label><mixed-citation publication-type=\"other\">Shields CL, Perez B, Materin MA, Mehta S, Shields JA. Optical coherence tomography of choroidal osteoma in 22 cases: evidence for photoreceptor atrophy over the decalcified portion of the tumor. <italic>Ophthalmology</italic> 2007;114:e53-8.</mixed-citation></ref><ref id=\"B15\"><label>15</label><mixed-citation publication-type=\"other\">Ahmadieh H, Vafi N. Dramatic response of choroidal neovascularization associated with choroidal osteoma to the intravitreal injection of bevacizumab (Avastin). <italic>Graefes Arch Clin Exp Ophthalmol</italic> 2007;245:1731-3.</mixed-citation></ref></ref-list></back></article>\n"
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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864059</article-id><article-id pub-id-type=\"pmc\">PMC7431725</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7447</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Effects of Topical Ozone Application on Outcomes after Accelerated Corneal Collagen Cross-linking: An Experimental Study</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Dogan</surname><given-names>Aysun Sanal</given-names></name><degrees>MD, FEBO, FICO</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Gurda</surname><given-names>Canan</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Caliskan</surname><given-names>Sinan</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Onder</surname><given-names>Evrim</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kaymaz</surname><given-names>Figen</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Bilgic</surname><given-names>Elif</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Department of Ophthalmology, Saglik Bilimleri University, Diskapi Yildirim Beyazit Training and Research Hospital, Ankara, Turkey</aff><aff id=\"I2\">\n<sup>2</sup>Department of Pathology, Saglik Bilimleri University, Diskapi Yildirim Beyazit Training and Research Hospital, Ankara, Turkey</aff><aff id=\"I3\">\n<sup>3</sup>Department of Histology and Embryology, Hacettepe University, School of Medicine, Ankara, Turkey</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Aysun Sanal Dogan, MD, FEBO, FICO. Diskapi Yildirim\nBeyazit Egitim ve Arastirma Hastanesi, Sehit Omer\nHalisdemir Cad. Diskapi 06130, Ankara, Turkey.\nE-mail: asanaldogan@gmail.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>289</fpage><lpage>298</lpage><history><date date-type=\"received\"><day>02</day><month>10</month><year>2019</year></date><date date-type=\"accepted\"><day>03</day><month>4</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Dogan et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><sec><title>Purpose</title><p> Ozone is a trioxygen molecule that spontaneously degrades into oxygen and oxygen free radicals. This study was designed to assess the effects of topical ozone application on outcomes after corneal collagen cross-linking (CXL).</p></sec><sec><title>Methods</title><p> Enucleated fresh cadaver yearling sheep eyes (<italic>n</italic> = 28) were divided into five groups: control (C, <italic>n</italic> = 6), sham (S, <italic>n</italic> = 6), ozone only (Z, <italic>n</italic> = 6), CXL only (X, <italic>n</italic> = 5), and Ozone + CXL (ZX, <italic>n</italic> = 5). In all groups, except C, the epithelial layer was removed. In group Z, 20 μg/mL liquid ozone was topically applied. In group X, CXL was performed in the accelerated pulse mode. In group ZX, both CXL and ozone were applied. Post-interventional oxygen levels were determined and corneal confocal microscopy and optical coherence tomography were performed. Corneas were evaluated using light and electron microscopy.</p></sec><sec><title>Results</title><p>Pre-interventional central corneal thickness (CCT) was highest in the control group and considerably similar in the remaining groups (<italic>P</italic> = 0.006). Pre- and post-interventional CCT were significantly different in the ozonated groups (Z and ZX) (<italic>P</italic> = 0.028; <italic>P</italic> = 0.043). Demarcation line depths were similar in groups Z, X, and ZX (<italic>P</italic> = 0.343). Increased stromal tissue reflectivity was observed in groups Z, X, and ZX. Oxygen levels were higher in the ozonated groups (Z and ZX) (<italic>P</italic> = 0.006), and caspase activity was higher in the CXL groups (X and ZX) (<italic>P</italic> = 0.028) as compared to the other groups. Group ZX showed tighter, more regular, and parallel fibrils.</p></sec><sec><title>Conclusion</title><p>Ozone increases corneal stromal oxygenation which can probably augment the effect of CXL. Future studies should investigate the safety and feasibility of ozone application during CXL.</p></sec></abstract><kwd-group><kwd>Ozone</kwd><kwd> Corneal Collagen Cross-linking</kwd><kwd> Corneal Confocal Microscopy</kwd><kwd> Corneal Oxygen</kwd><kwd> Experimental</kwd></kwd-group><counts><fig-count count=\"18\"/><table-count count=\"3\"/><ref-count count=\"29\"/><page-count count=\"10\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> INTRODUCTION</title><p>The cornea contains outer epithelial cell layer, middle stromal layer, and inner endothelial cell layer. The stromal layer comprises the major volume of the tissue and is composed of keratocytes and extracellular matrix (ECM) including collagen lamella. The shape and structure of the corneal stroma are primarily responsible for the transparency, strength, and contour of the cornea.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup>\n</p><p>Keratoconus (KC) is a non-inflammatory ectatic disorder of the cornea, characterized by progressively bulging and thinning in the central or paracentral area of the cornea.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> Ultrastructural changes of stromal matrix include increased corneal stromal protein degradation, decreased collagen lamella density, and disorganization of the stromal matrix, resulting in biochemical instability and weakening.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup> Contemporary treatment has been undertaken with the advent of corneal collagen cross-linking (CXL), demonstrating promising results in terms of halting disease progression or even, to a limited extent, reversing the course of KC.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup>\n</p><p>The application of riboflavin and ultraviolet A during the CXL procedure results in the formation of oxygen radicals which crosslink in the adjacent collagen fibrils. This cross-linking increases the biomechanical strength by modifying the organization of the corneal lamellar structure, the diameter of the corneal stromal collagen, and the spacing between fibrils and proteoglycans.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> It has been shown that tissue oxygen is the key element for this reaction to occur.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>Within the last five years, a growing number of studies have been performed to facilitate the procedure without decreasing the efficacy. These studies have focused on the four basic variables of this procedure.</p><p>1 Delivery of riboflavin to cornea: Chemical de-epithelization, the “epi-on” (without de-epithelization) and intraoperative contact lens using techniques to decrease the operative complications and postoperative pain, and “iontophoresis technique” to increase efficacy have been described.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup>\n</p><p>2 Photosensitizing agent: Reportedly, studies have evaluated dosage modification and duration of riboflavin application; additionally, alternatives to this original photosensitizing agent have been investigated.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup>\n</p><p>3 Duration of exposure to UVA: The reaction is initiated by UVA. In 2014, “accelerated CXL” was described and aimed to shorten the duration by increasing the UVA irradiance, without changing the total energy.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>]</sup>\n</p><p>4 Oxygen: The photochemical reaction requires oxygen. Following the realization that environmental oxygen is rapidly depleted within seconds and rises to normal limits after 3 min, it was proposed that the “pulse CXL” method allows tissue reoxygenation.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>,<xref rid=\"B11\" ref-type=\"bibr\">11</xref>,<xref rid=\"B12\" ref-type=\"bibr\">12</xref>]</sup>\n</p><p>The effect of cross-linking on collagen was first investigated using the crystalline lens.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>]</sup> The lens and lenticular collagen were the first subjects of these studies, which demonstrated the interaction among singlet oxygen, ozone, and applied riboflavin that resulted in free radical-induced cross-linking of the lenticular fibrils.<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>,<xref rid=\"B15\" ref-type=\"bibr\">15</xref>,<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup> These aforementioned studies put forward the possible use of ozone in CXL.</p><p>Ozone is an unstable trioxygen molecule. Its breakdown to oxygen gives rise to oxygen free radicals, which are highly reactive and powerful oxidizing agents.<sup>[<xref rid=\"B17\" ref-type=\"bibr\">17</xref>]</sup> As a result, it serves as an oxygen supply. Therefore, by itself or in conjunction with riboflavin and UVA, it has the potential to augment the cross-linking effect. We hypothesized that ozone can be used as an adjuvant to the cross-linking reaction, as an oxygen generator, or as a cross-linking agent in the cornea. This study was designed to assess the effect of topical ozone application on the outcomes of the CXL procedure.</p></sec><sec sec-type=\"section\"><title> METHODS</title><sec sec-type=\"subsection\"><title>Study Design</title><p>Enucleated fresh cadaver yearling sheep eyes (<italic>n</italic> = 28) were obtained from a local slaughterhouse and the full experimental procedure was performed within 12 hours. The eyes were divided into five groups: control (C, <italic>n</italic> = 6), sham (S, <italic>n</italic> = 6), ozone only (Z, <italic>n</italic> = 6), CXL only (X, <italic>n</italic> = 5), and Ozone + CXL (ZX, <italic>n</italic> = 5).</p><p>During the experiment, the eyes were handled from the equator using gauze sponges. Group C was not touched or handled, and the epithelial layer was mechanically removed for all other groups. In group S, only the epithelial layer was mechanically removed. In group Z, 20 μg/mL ozonated water was topically applied to the de-epithelized cornea. In group X, CXL was performed using a total energy of 5,4 J/cm<inline-formula><mml:math id=\"M1\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula> in the accelerated pulse mode.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> In group ZX, ozone was first applied, followed by CXL treatment. Pre- and post-interventional anterior segment optical coherence tomography (AS-OCT) and corneal confocal microscopy (CCM) were performed. Immediate post-interventional corneal stromal oxygen levels were measured. After the procedure, each cornea was dissected from the globe and specimens were obtained to perform light and electron microscopic evaluations.</p></sec><sec sec-type=\"subsection\"><title>Ozone Application</title><p>Liquid ozone (20 μm/mL) was obtained using an ozone generator (Refresh, Refreshozon Medical, Ankara, Turkey) in distilled water in a separate room. Two milliliters of the ozonated water was applied to the cornea for 2 min using a silica glass apparatus, with a corneal 8 mm aperture.</p></sec><sec sec-type=\"subsection\"><title>Cross-linking</title><p>After removal of the central 8mm portion of the epithelium using a No. 15 knife, 0.1% riboflavin (vitamin B2) with hydroxypropyl methylcellulose (VibeXRapid, Avedro) was applied drop-wise to the cornea at 2 min intervals for 10 min to achieve corneal penetration. Anterior segment diffusion was controlled using handheld biomicroscope. A UVA (370 nm) generator designed for corneal cross-linking was used (KXL System, Avedro Inc., Waltham, MS, USA). First, guiding lights were set to focus the UVA onto the cornea perfectly. The parameters were set as power: 15 mW/cm<inline-formula><mml:math id=\"M2\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula>, a total energy of 5.4 J/cm<inline-formula><mml:math id=\"M3\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula> for each eye, with a pulse of 1.5-sec on and 1.5-sec off mode.</p></sec><sec sec-type=\"subsection\"><title>Oxygen Measurement</title><p>Post-interventional oxygen measurements were performed at room temperature (21ºC). The operator was masked to the groups. An oxygen sensor microprobe (PreSens, Regensburg, Germany), with a tapering end <inline-formula><mml:math id=\"M4\"><mml:mo><</mml:mo></mml:math></inline-formula> 50 μm in diameter, was placed from the epithelized cornea, passing 2 mm into the de-epithelized cornea, with a parallel angle at the half-thickness of the cornea under the biomicroscope. The mean of three consecutive measurements was used for statistical analyses.</p></sec><sec sec-type=\"subsection\"><title>Anterior Segment Optical Coherence Tomography (AS-OCT) </title><p>Corneas were imaged using optical coherence tomography (OCT, RTVue-XR, Optovue Inc., Fremont, CA). The non-contact anterior segment attachment lens (CAM-L), corneal line mode, and automated image analysis system were used. The sections were set to the central cornea. In particular, central corneal thickness (CCT) was measured in micrometers and the depth of demarcation lines, which are the results of different reflectivity, were investigated. The reflective line between the cross-linked and untreated areas was determined and marked.</p></sec><sec sec-type=\"subsection\"><title>Corneal Confocal Microscopy (CCM)</title><p>Laser scanning CCM (HRT III-RCM, Heidelberg Engineering, Dossenheim, Germany) was used to evaluate the cellular structures of the corneas. A transparent ophthalmic gel (Viscotears Ophthalmic Gel, Alcon) was filled into the confocal cap attached to the objective lens and on its external surface. The eyes were handled with a sponge from the equator, providing the central corneal touch to the cap. The captured images were analyzed by a clinician (ASD) experienced in CCM.</p></sec><sec sec-type=\"subsection\"><title>Light Microscopy</title><p>Caspase-3 staining was performed to detect keratocyte apoptosis. Paraffin-embedded tissue blocks were sectioned into 4-5µm thick slides. The slides were deparaffinized, rehydrated, and washed in phosphate-buffered saline (PBS). After treatment with 3% hydrogen peroxide in aqueous solution, the sections were blocked with PBS-6% non-fat dry milk for 1 hour at room temperature. The slides were then incubated at 4ºC overnight with the primary antibody for cleaved caspase-3 (CPP32) (Thermo Scientific). After washing with PBS, the slides were treated with a solution of diaminobenzidine (DAB). Finally, a counterstain with hematoxylin was performed and the slides were allowed to dry. The immunohistochemical (IHC) evaluation was performed to determine the nuclei of stromal cells, and positivity was scored as percentages. Caspase activity was evaluated in stromal cells. In each section, the area with the highest density of nuclear positivity was selected and 100 cells were counted. The ratio of positively stained nuclei to 100 (the percentage value) was accepted as the caspase activity for each given case.</p></sec><sec sec-type=\"subsection\"><title>Electron Microscopy</title><p>Tissue samples were carefully dissected and 1 mm<inline-formula><mml:math id=\"M5\"><mml:msup><mml:mrow/><mml:mn>3</mml:mn></mml:msup></mml:math></inline-formula>-sized samples were fixed overnight in 2.5% glutaraldehyde in PBS. Next, the tissue samples were washed with PBS and fixed with 1% osmium tetroxide solution. After washing with PBS, the tissue samples were dehydrated with a graded alcohol series. An Araldite/Epon812 mixture (Cat no.: 13940, EMS, Hatfield, PA, USA) was used to embed the propylene-oxide-treated tissue. The tissue blocks obtained were maintained at 60ºC for two days to complete polymerization. Thin sections were obtained using the Leica Ultracut R, and contrast double-stained using uranyl acetate and lead citrate (Leica EM AC20). Next, examinations were performed by using transmission electron microscope (TEM; JEM 1400, Jeol, Japan) with an attached digital CCD camera (Gatan Inc., Pleasanton, CA, USA). To compare the differences among the groups, we obtained four blocks from each group, and then examined four sections from each block. Finally, collagen fibril diameters and ECM distances from four non-overlapping areas were calculated at the magnification of 100k.</p></sec></sec><sec sec-type=\"section\"><title> RESULTS</title><p>Tissue oxygen levels were higher in the ozonated groups (Z and ZX) than in the other groups (<italic>P</italic> = 0.006, Kruskal–Wallis test). Pre-interventional CCT measures were higher in the control group (C) as compared to the other groups (<italic>P</italic> = 0.006); no significant difference, however, was found among the remaining groups in the baseline CCT. Post-interventional CCT differed significantly among the groups (<italic>P</italic> = 0.001). Compared to the baseline values, the post-interventional CCT decreased significantly in groups Z and ZX (Group Z: <italic>P</italic> = 0.028, Group X: <italic>P</italic> = 0.768, Group ZX: <italic>P</italic> = 0.043; Table 1). Similar demarcation lines were observed in the interventional groups, although group ZX showed a generalized increase in corneal reflectivity (Figure 1).</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Between and within groups comparison of the examined parameters.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"7\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Group C</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Group S</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Group Z</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Group X</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Group ZX</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>p<inline-formula><mml:math id=\"M6\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula></bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Caspase activity, %, median (range)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.5 (1–4)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (1–3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.5(1–5)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>3 (3–5)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>4 (3–6)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.028*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Oxygen saturation, %, median (range)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.7 (14.5–22.2)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16.7 (11.9–22.3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>21.7 (20.4–26.0)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16.4 (16.0–18.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>22.2 (21.3–23.3)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.006**</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">CCT, preop, mcm, median (range)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>673.5 (664–768)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">587.5 (545–639)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">637 (528–653)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">609 (571–655)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">609 (545–652)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.006***</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">CCT, postop, mcm, median (range)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">673.5 (664–768)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">587.5 (545–639)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">569 (506–635)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">622 (555–657)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">541 (532–558)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.001****</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Demarcation, mcm, median (range)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">203.5 (185–257)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">190 (185–245)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">323 (150–360)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.343</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">Group C: control, Group S: sham, Group Z: ozonated only, Group X: cross-linked only, Group ZX: ozonated and crossl-linked, CCT: central corneal thickness, a: Kruskal–Wallis test\n<bold>Between groups comparisons:</bold>\n*Group C vs ZX, <italic>p</italic> = 0.0025; Group S vs X, <italic>p</italic> = 0.021; Group S vs ZX, <italic>p</italic> = 0.009; Mann–Whitney U-test for each\n**Group C vs Z, <italic>p</italic> = 0.037; Group C vs ZX, <italic>p</italic> = 0.018; Group S vs Z, <italic>p</italic> = 0.025; Group S vs ZX, <italic>p</italic> = 0.028; Group Z vs X, <italic>p</italic> = 0.006; Group X vs ZX, <italic>p</italic> = 0.009, Mann–Whitney U-test for each\n***Group C vs S, <italic>p</italic> = 0.004; Group C vs Z, <italic>p</italic> = 0.004; Group C vs X, <italic>p</italic> = 0.006; Group C vs ZX, <italic>p</italic> = 0.006; Mann–Whitney U-test for each\n****Group C vs S, <italic>p</italic> = 0.004; Group C vs Z, <italic>p</italic> = 0.004; Group C vs X, <italic>p</italic> = 0.006; Group C vs ZX, <italic>p</italic> = 0.006; Group S vs ZX, <italic>p</italic> = 0.018; Group X vs ZX, <italic>p</italic> = 0.028; Mann–Whitney U-test for each\n<bold>Pre- vs post-operative pachymetry (CCT) comparison within study groups: </bold>Group Z: wilcoxon signed rank test, <italic>p</italic> = 0.028; Group X: wilcoxon signed rank test, <italic>p</italic> = 0.768;<bold/>Group ZX: wilcoxon signed rank test, <italic>p</italic> = 0.043</td></tr></tbody></table></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Collagen fibril diameter and distance between fibrils regarding the groups which were measured by TEM.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Groups</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Collagen diameter in nm, median (range)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Distance between collagens in nm, median (range)</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">N (No intervention [C + S])</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">24,6 (14.1–49.4)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">25.2 (9.1–61.6)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Z</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23.7 (16.7–35.0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">24.3 (11.5–44.6)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">X</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23.1 (15.6–34.9)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">27.7 (16.2–50.4)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">ZX</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23.2 (16.4–32.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">18.3 (9.1–40.9)</td></tr><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">TEM, transmission electron microscope; N, no intervention (C: control and S: sham); Group C, control; Group S, sham; Group Z, ozonated only; Group X, cross-linked only; Group ZX, ozonated and cross-linked</td></tr></tbody></table></table-wrap><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><p>Between groups comparison of collagen fibril diameter and distance between fibrils.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Collagen fibril diameter (</bold>\n<italic><bold>p</bold></italic>\n<bold>-value)<inline-formula><mml:math id=\"M7\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Distance between collagen fibrils (</bold>\n<italic><bold>p</bold></italic>\n<bold>-value)<inline-formula><mml:math id=\"M8\"><mml:msup><mml:mrow/><mml:mi>a</mml:mi></mml:msup></mml:math></inline-formula></bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Group N vs Z</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M9\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.289</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Group N vs X</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M10\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M11\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Group N vs ZX</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M12\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M13\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Group Z vs X</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.029</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M14\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Group Z vs ZX</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.200</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M15\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Group X vs ZX</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.337</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M16\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"left\" colspan=\"3\" rowspan=\"1\">N, no intervention (control and sham); Group Z, ozonated only; Group X, cross-linked only; Group ZX, ozonated and cross-linked; a, Mann–Whitney U-test</td></tr></tbody></table></table-wrap><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Optical Coherence Tomography corneal images of the groups control (a), sham (b), ozone (c), cross-linking (d), ozone-cross-linking (e), respectively. Hyperreflectivity is increased in anterior stroma in groups X and Z, but generalized in ZX.</p></caption><graphic xlink:href=\"jovr-15-289-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Corneal confocal images of groups sham (a), ozone (b), cross-linking (c), ozone-cross-linking (d), respectively. The keratocytes in stromal level shows straight extensions, which are signs of activity in all groups except control, which are exaggerated in ZX group.</p></caption><graphic xlink:href=\"jovr-15-289-g002\"/></fig><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>(a) A section of cornea from control group (HE X 400). (b) Positively stained nuclei in a section from ZX group (Caspase X 400).</p></caption><graphic xlink:href=\"jovr-15-289-g003\"/></fig><fig id=\"F4\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><p>Images taken from transmissional electron microscopy of the corneas from groups (x100K); control (a), ozone (b), cross-linking (c), ozone-cross-linking (d), respectively. Collagen fibers and interlamellar spaces were examined by electron microscopy.</p></caption><graphic xlink:href=\"jovr-15-289-g004\"/></fig><p>CCM demonstrated corneal stromal hyper-reflectivity (group Z, X, and ZX) in all interventional groups; however, the hyper-reflectivity was marked and present in all layers of the stroma in group ZX (Figure 2).</p><p>The cross-linked groups (groups X and ZX) showed higher caspase activity, with significant differences observed in caspase-3 staining between groups C and ZX, between groups S and X, and between groups S and ZX (<italic>P</italic> = 0.025, <italic>P</italic> = 0.021, <italic>P</italic> = 0.009, respectively) (Table 1, Figure 3).</p><p>The TEM findings and images revealed similar collagen fibril organization and undulation in groups C and S. The fibrils were compact and randomly distributed in group Z, oriented in a parallel pattern in group X, more compact and tightly arranged in group ZX (Figure 4).</p><p>The collagen fibril diameters were higher in the non-interventional groups (groups C and S). In the interventional groups, the collagen fibrils were thicker in group Z than in the other groups (Tables 2 and 3). The distance between collagen fibrils was lowest in group ZX and highest in group X (Tables 2 and 3).</p></sec><sec sec-type=\"section\"><title> DISCUSSION</title><p>Oxygen radicals are present in normal tissues and cells. However, their effect is dose-dependent with a possible destructive effect on the cell wall structure and genetic material.<sup>[<xref rid=\"B18\" ref-type=\"bibr\">18</xref>]</sup> Ozone (O<inline-formula><mml:math id=\"M17\"><mml:msub><mml:mrow/><mml:mn>3</mml:mn></mml:msub></mml:math></inline-formula>) is normally found in the stratosphere and is used for conventional drinking water production at a concentration of 1–3 µl/mL.<sup>[<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup> It is a highly reactive, unstable gas that spontaneously degrades to oxygen radicals and acts as an oxygen provider.<sup>[<xref rid=\"B17\" ref-type=\"bibr\">17</xref>]</sup> There are three main types of molecular ozone reactions: (1) Electron transfer reactions, resulting in free oxygen radicals; (2) Oxygen-atom transfer reactions mainly occurring in inorganic materials; (3) Ozone addition reactions, which are the primary reactions occurring in organic compounds, resulting in double bond formation.<sup>[<xref rid=\"B20\" ref-type=\"bibr\">20</xref>]</sup> Our study revealed that oxygen levels in the corneal stroma were higher in ozonated groups, which supports our primary goal, the oxygen-providing effect.</p><p>Several studies have evaluated the effects of ozone molecules on the eye. An experimental study revealed that ozone resulted in a decrease in goblet cell density and an increase in inflammatory cytokines on the ocular surface.<sup>[<xref rid=\"B21\" ref-type=\"bibr\">21</xref>]</sup> Our study showed that corneal thickness was decreased in the ozonated groups (groups Z and ZX). This observation can be explained by the use of liquid ozone which induced this decrease in thickness owing to its osmotic effect. To minimize this effect, we preferred the maximum possible concentration and the lowest exposure time. Wu et al<sup>[<xref rid=\"B22\" ref-type=\"bibr\">22</xref>]</sup> have shown that ozone decomposes rapidly to diatomic oxygen and has a short half-life of 1–10 min in water.<sup>[<xref rid=\"B22\" ref-type=\"bibr\">22</xref>]</sup> We applied ozone through a silica tube with an 8 mm aperture to limit the contact time within this range and prevent corneal edema.</p><p>Kamaew et al have shown that the stromal oxygen concentrations decrease within the first 15 sec of UVA application and then rise to normal levels 3 min after cessation of UVA.<sup>[<xref rid=\"B12\" ref-type=\"bibr\">12</xref>]</sup> Oxygen is necessary for the photochemical reaction to occur during the CXL procedure.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> In our study, the microprobe measurements showed higher corneal stromal oxygen levels in ozonated groups (groups Z and ZX), suggesting penetration of the topically applied ozone to the stroma. This assumption, however, needs further refinement.</p><p>The standard CXL procedures lasts up to 60 min and a pulsed accelerated protocol has been developed to shorten the treatment duration without losing the therapeutic efficacy.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>,<xref rid=\"B23\" ref-type=\"bibr\">23</xref>]</sup> Therefore, we aimed to augment the CXL effect in the pulsed accelerated protocol and, in the case of encouraging findings, implement this modification to clinical settings.</p><p>Early demarcation was previously described in an experimental study by Zhu et al; they postulated that the early apoptosis of keratocytes could induce increased light scattering and demarcation between the affected and unaffected stroma.<sup>[<xref rid=\"B24\" ref-type=\"bibr\">24</xref>]</sup> We detected that demarcation lines in groups Z and ZX were similar to those in group X, indicating that the ozone itself triggers a chemical reaction similar to that observed in CXL. Post-interventional CCM findings demonstrated that increased stromal reflectivity and matrix striation were prominent in the anterior part in groups Z and X, whereas these changes were present throughout the stromal layer in group ZX. These results confirmed that ozone can potentially augment the cross-linking effect of the classical CXL procedure.</p><p>Another study has indicated anterior stromal keratocyte apoptosis following CXL.<sup>[<xref rid=\"B25\" ref-type=\"bibr\">25</xref>]</sup> We detected apoptosis with a higher caspase activity in cross-linking groups (groups X and ZX), which demonstrates the efficacy of CXL.</p><p>CXL allows the reorganization of the corneal lamellar tissue by modifying the stromal collagen diameter and distance between collagen fibrils and proteoglycans.<sup>[<xref rid=\"B26\" ref-type=\"bibr\">26</xref>]</sup> Clinical studies have revealed a mean collagen diameter of 30.8 nm and 32.2 nm in individuals younger and older than 65 years, respectively.<sup>[<xref rid=\"B27\" ref-type=\"bibr\">27</xref>]</sup> It reveals that a natural cross-linking occurs non-enzymatically via free radicals with increasing age.<sup>[<xref rid=\"B28\" ref-type=\"bibr\">28</xref>]</sup>\n</p><p>Furthermore, we examined the effect of these procedures on collagen fibrils by utilizing TEM. The obtained images supported our hypothesis regarding the distribution pattern of collagen fibrils. In the ozonated group (group Z), the fibrils were denser than the controls and demonstrated a random distribution pattern. In the CXL group (group X), the fibrils were markedly condensed and presented an undulated pattern; in the ZX group, the undulation was less prominent and the distribution was more uniform and dense. These findings showed that the ozone application had an augmenting effect on the CXL procedure. Conversely, our measurements of fibril diameter and interfibrillar distance were not in accordance with our hypothesis. The fibril diameters of the study groups were similar but smaller than those of the control and sham groups, and interfibrillar distance was decreased from group X to group Z to group ZX in descending order (Table 2). The small number of eyes in the groups, the random selection of four ultrastructural counting areas, and manual counting could be the principal explanations for this contradicting finding.<sup>[<xref rid=\"B29\" ref-type=\"bibr\">29</xref>]</sup>\n</p><p>Ozone is an inert, cheap, and easily available molecule that induces covalent chemical bonds resulting in cross-linking. Our study is the first to evaluate the effects of this molecule on the results of the CXL procedure. Our findings in cadaveric eyes can be regarded as preliminary to potential clinical applications. However, further animal studies are warranted to determine the most appropriate concentration, dosage, and duration of application of ozone as well as safety issues. These studies should evaluate the osmotic effects of the gaseous ozone form and the biomechanical properties of the treated corneas.</p></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>This work was funded by the Scientific and Technological Research Council of Turkey (TUBITAK) [grant numbers: 115S862].</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Chen S, Mienaltowski MJ, Birk DE. Regulation of corneal stroma extracellular matrix assembly. <italic>Exp Eye Res</italic> 2015;133:69–80.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">Gomes JA, Tan D, Rapuano CJ, Belin MW, Ambrósio R Jr, Guell JL, et al. Group of panelists for the Global Delphi Panel of Keratoconus and Ectatic Diseases. 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] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864067</article-id><article-id pub-id-type=\"pmc\">PMC7431726</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7455</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Low-contrast Pattern-reversal Visual Evoked Potential in Different Spatial Frequencies</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Hassankarimi</surname><given-names>Homa</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Jafarzadehpur</surname><given-names>Ebrahim</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mohammadi</surname><given-names>Alireza</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Noori</surname><given-names>Seyed Mohammad Reza</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Department of Medical Physics, School of Medicine, Iran University of Medical Sciences, Tehran, Iran</aff><aff id=\"I2\">\n<sup>2</sup>Department of Optometry, School of Rehabilitation Science, Iran University of Medical Sciences, Tehran, Iran</aff><aff id=\"I3\">\n<sup>3</sup>Departments of Medical Physics and Biomedical Engineering, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Ebrahim Jafarzadehpur, PhD. Department of Optometry,\nRehabilitation Faculty, Iran University of Medical\nSciences (IUMS), Shahnazary St., Mohseni Sq.,\nMirdamad Blvd, Tehran 15459, Iran.\nE-mail: Jafarzadehpour.e@iums.ac.ir\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>362</fpage><lpage>371</lpage><history><date date-type=\"received\"><day>28</day><month>1</month><year>2019</year></date><date date-type=\"accepted\"><day>09</day><month>3</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Hassankarimi et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><sec><title>Purpose</title><p>To evaluate the pattern-reversal visual evoked potential (PRVEP) in low-contrast, spatial frequencies in time, frequency, and time-frequency domains.</p></sec><sec><title>Methods</title><p>PRVEP was performed in 31 normal eyes, according to the International Society of Electrophysiology of Vision (ISCEV) protocol. Test stimuli had checkerboard of 5% contrast with spatial frequencies of 1, 2, and 4 cycles per degree (cpd). For each VEP waveform, the time domain (TD) analysis, Fast Fourier Transform(FFT), and discrete wavelet transform (DWT) were performed using MATLAB software. The VEP component changes as a function of spatial frequency (SF) were compared among time, frequency, and time–frequency dimensions.</p></sec><sec><title>Results</title><p>As a consequence of increased SF, a significant attenuation of the P100 amplitude and prolongation of P100 latency were seen, while there was no significant difference in frequency components. In the wavelet domain, an increase in SF at a contrast level of 5% enhanced DWT coefficients. However, this increase had no meaningful effect on the 7P descriptor.</p></sec><sec><title>Conclusion</title><p> At a low contrast level of 5%, SF-dependent changes in PRVEP parameters can be better identified with the TD and DWT approaches compared to the Fourier approach. However, specific visual processing may be seen with the wavelet transform.</p></sec></abstract><kwd-group><kwd>Discrete Wavelet Transform</kwd><kwd> Fast Fourier Transform</kwd><kwd> Spatial Frequency</kwd><kwd> Visual Evoked Potential</kwd></kwd-group><counts><fig-count count=\"2\"/><table-count count=\"1\"/><ref-count count=\"55\"/><page-count count=\"10\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> Introduction</title><p>Contrast is the main issue in visual perception.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> As the first mechanism for visual detection, discrimination and perception may be affected by the contrast level of objects.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup> High-contrast objects and symbols are used in the visual examination room as E or similar acuity charts.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> However, objects in the real world do not show high contrast. Therefore, in the real world, visual function is usually in a low to moderate contrast condition.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> Evaluation of the visual system in a low-contrast situation may indicate its performance in the natural visual environment.</p><p>Visual evoked potential (VEP) is a noninvasive and objective electrophysiological test for evaluating human visual function.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup> Pattern-reversal visual evoked potential (PRVEPs) directly mirrors neural activities or the extent of stimulated neural network in each eye using the afferent impulse toward the primary visual cortex (V1).<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> The most prominent and strongest peak in VEP is P100, which has minimal variation and high repeatability. Amplitude and latency of P100 depend on the stimulus conditions, such as the size, luminance, contrast, and spatial frequency (SF).<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> Several studies examining the effects of SF changes on the time domain (TD) parameters of VEP have shown that SF has a significant impact on VEP responses.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>,<xref rid=\"B11\" ref-type=\"bibr\">11</xref>,<xref rid=\"B12\" ref-type=\"bibr\">12</xref>]</sup>\n</p><p>Decomposition of the time function into its particular frequencies, amplitudes, and phases by means of the Fourier transform is an objective and common method for the VEP analysis.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>,<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup> The Fourier technique was successfully applied to determine features of steady-state VEP (SSVEP) and transient VEP (TVEP).<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B15\" ref-type=\"bibr\">15</xref>,<xref rid=\"B16\" ref-type=\"bibr\">16</xref>,<xref rid=\"B17\" ref-type=\"bibr\">17</xref>,<xref rid=\"B18\" ref-type=\"bibr\">18</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>,<xref rid=\"B20\" ref-type=\"bibr\">20</xref>,<xref rid=\"B21\" ref-type=\"bibr\">21</xref>]</sup> The power of each frequency band in the frequency domain relates to signal amplitudes in the TD, and phase spectrum provides precise estimation of latencies in TD.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup> The Fast Fourier Transform (FFT) has been employed to measure the VEP phase and amplitude spectrum of the even harmonic response to determine reliability of amplitude and for estimation latency, determine the neural mechanisms in frequency domain, and develop a fast and reliable TVEP technique.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B21\" ref-type=\"bibr\">21</xref>]</sup> Zemon et al presented a set of frequency domain measurements that fully obtained the response content and demonstrated that their novel indices may be performed as a more powerful tool to evaluate the visual function. They offered that quantitative and objective measurements in the frequency domain provide a more precise and efficient method for the assessment of the visual system in healthy and diseased brains.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup>\n</p><p>The wavelet transform (WT) is a valuable and efficient approach of biosignal processing. This method is widely applied in different studies to analyze, denoise, and extract new parameters of evoked potential signals (EPs), SSVEP, multifocal VEP (mfVEP), TVEP, and PRVEP responses, all of which have totally emphasized on the effectiveness and usefulness of this method.<sup>[<xref rid=\"B22\" ref-type=\"bibr\">22</xref>,<xref rid=\"B23\" ref-type=\"bibr\">23</xref>,<xref rid=\"B24\" ref-type=\"bibr\">24</xref>,<xref rid=\"B25\" ref-type=\"bibr\">25</xref>,<xref rid=\"B26\" ref-type=\"bibr\">26</xref>,<xref rid=\"B27\" ref-type=\"bibr\">27</xref>,<xref rid=\"B28\" ref-type=\"bibr\">28</xref>,<xref rid=\"B29\" ref-type=\"bibr\">29</xref>,<xref rid=\"B30\" ref-type=\"bibr\">30</xref>,<xref rid=\"B31\" ref-type=\"bibr\">31</xref>,<xref rid=\"B32\" ref-type=\"bibr\">32</xref>,<xref rid=\"B33\" ref-type=\"bibr\">33</xref>]</sup> WT provides simultaneous estimation of time and frequency of VEP signals, which yields noteworthy diagnostic information.<sup>[<xref rid=\"B34\" ref-type=\"bibr\">34</xref>]</sup> Experiments on the SSVEP analysis in time, frequency, and time–frequency domains have suggested that time–frequency and frequency analyses of these waveforms are more efficient than the TD analysis.<sup>[<xref rid=\"B35\" ref-type=\"bibr\">35</xref>,<xref rid=\"B36\" ref-type=\"bibr\">36</xref>]</sup>\n</p><p>Although several experiments have already evaluated how SF changes affect VEP amplitudes and peak times in TD and VEP amplitude and phase spectrum in the frequency domain, to the best of our knowledge, this issue has not been investigated for the parameters of frequency or time–frequency domains, which are considered in this study. Moreover, the efficiency of the three mentioned dimensions in representing changes of these parameters as a function of SF has not been compared. In the present study, we focused on the relationship between SF and extracted parameters for the PRVEP analysis in time, frequency, and time–frequency domains, and also compared the efficiency of these dimensions in revealing changes.</p></sec><sec sec-type=\"section\"><title> Methods</title><p>Thirty-one healthy individuals (19 men and 12 women; mean age, 25.6 <inline-formula><mml:math id=\"M1\"><mml:mo>±</mml:mo></mml:math></inline-formula> 6.26 years) participated in this study. All subjects underwent ophthalmic tests and showed a normal visual acuity (minimum and maximum, 0.1 and 0.3 logMAR, respectively). All procedures involving human participants were done in accordance with the ethical standards of the Iran University of Medical Sciences and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Written informed consent was obtained from all participants after informing them about the purpose of the study.</p><p>Considering the International Society for Clinical Electrophysiology of Vision (ISCEV) protocol, PRVEPs were performed by Metrovision MonPack One (Metrovision Company, Pérenchies, France) with gold-plated cupola electrodes, according to the 10–20 system. Active and reference electrodes were placed on occiput (O<inline-formula><mml:math id=\"M2\"><mml:msub><mml:mrow/><mml:mi>z</mml:mi></mml:msub></mml:math></inline-formula> location) and frontal (F<inline-formula><mml:math id=\"M3\"><mml:msub><mml:mrow/><mml:mi>z</mml:mi></mml:msub></mml:math></inline-formula> location) zones, respectively. The ear lobe served as ground. The PRVEP signals were amplified 2,000 times, filtered in the range of 1–100 Hz and sampled at 1,024 Hz using 240 data points.</p><p>A checkerboard pattern alternating at a rate of 2.5 times per second (temporal frequency of 2.5 Hz) was utilized as a stimulus. Test stimuli comprised of spatial frequencies of 1, 2, and 4 cycles per degree (cpd) (corresponding to check sizes of 30, 15, and 7 min of arc, respectively) and contrast level of 5% for each SF. The average sweep numbers per trial was 60.</p><p>All VEP waveforms were analyzed in time, frequency, and time–frequency domains using MATLAB software (MATLAB R2015b, The Mathworks, Inc., Natick, Massachusetts, USA). The P100 amplitudes and latencies were evaluated in TD. Following signal normalization, the FFT and discrete wavelet transform (DWT) of P100 peak of all waveforms were carried out in MATLAB environment. MATLAB (matrix laboratory) is a high-level language for high performance numerical computation and visualization. It is an extremely useful, powerful, and popular simulation tool with immense utility in biosignal processing.<sup>[<xref rid=\"B37\" ref-type=\"bibr\">37</xref>]</sup>\n</p><p>The FFT and power spectral density (PSD) help determine the frequency components and distribution in fine detail. The mean frequency (F<inline-formula><mml:math id=\"M4\"><mml:msub><mml:mrow/><mml:mi> mean </mml:mi></mml:msub></mml:math></inline-formula>) was derived from FFT, and the mode frequency (F<inline-formula><mml:math id=\"M5\"><mml:msub><mml:mrow/><mml:mi> mod </mml:mi></mml:msub></mml:math></inline-formula>) was extracted from Welch PSD of VEP responses. F<inline-formula><mml:math id=\"M6\"><mml:msub><mml:mrow/><mml:mi> mean </mml:mi></mml:msub></mml:math></inline-formula> stands for the average frequency in terms of the sampling frequency. F<inline-formula><mml:math id=\"M7\"><mml:msub><mml:mrow/><mml:mi> mod </mml:mi></mml:msub></mml:math></inline-formula> stands for the most common frequency and refers to the frequency of maximum value in the power spectrum. Hence, F<inline-formula><mml:math id=\"M8\"><mml:msub><mml:mrow/><mml:mi> mod </mml:mi></mml:msub></mml:math></inline-formula> demonstrates the dominant frequency in the PSD.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Results of one-way analysis of variance (ANOVA) for three groups of spatial frequency</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"4\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Component</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Group</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Mean ± SD**</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold><inline-formula><mml:math id=\"M9\"><mml:mi>P</mml:mi></mml:math></inline-formula>-value</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.09 ± 3.49</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Amplitude (µV)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3.45 ± 3.1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.001*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.529 ± 2.24</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">120.9 ± 10.31</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Latency (ms)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">129.81 ± 15.01</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.021*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">131.03 ± 19.35</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.15 ± 3.85</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">F<inline-formula><mml:math id=\"M10\"><mml:msub><mml:mrow/><mml:mrow><mml:mi>m</mml:mi><mml:mi>e</mml:mi><mml:mi>a</mml:mi><mml:mi>n</mml:mi></mml:mrow></mml:msub></mml:math></inline-formula> (Hz)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.50 ± 4.92</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.842</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.07 ± 5.30</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.06 ± 0.36</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">F<inline-formula><mml:math id=\"M11\"><mml:msub><mml:mrow/><mml:mrow><mml:mi>m</mml:mi><mml:mi>o</mml:mi><mml:mi>d</mml:mi></mml:mrow></mml:msub></mml:math></inline-formula> (Hz)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 ± 0.00</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.372</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 ± 0.00</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–4.14 ± 7.27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Approximation coefficient</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.516 ± 8.922</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.005*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2.969 ± 9.24</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–0.914 ± 0.18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Detail coefficient</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–0.101 ± 0.205</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.008*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.0411 ± 0.204</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">30.971 ± 16.41</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Descriptor 7P***</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">30.618 ± 15.81</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.051</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">*Significant difference (<italic>P</italic>\n<inline-formula><mml:math id=\"M12\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05); **Standard deviation; ***Descriptor of P100 amplitude</td></tr></tbody></table></table-wrap><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Pattern-reversal visual evoked potentials for one participant at 1, 2, and 4 cpd in the time domain.</p></caption><graphic xlink:href=\"jovr-15-362-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>F<inline-formula><mml:math id=\"M13\"><mml:msub><mml:mrow/><mml:mi> mod </mml:mi></mml:msub></mml:math></inline-formula> extracted from the Power Spectral Density (PSD) of pattern-reversal visual evoked potentials at 1, 2, and 4 cpd. F<inline-formula><mml:math id=\"M14\"><mml:msub><mml:mrow/><mml:mi> mod </mml:mi></mml:msub></mml:math></inline-formula> is the peak frequency in the PSD (approximately 2 Hz).</p></caption><graphic xlink:href=\"jovr-15-362-g002\"/></fig><p>The WT is a convolution of frequency contents of the signal (scale) with the wavelet function, which describes a more useful signal information. Discrete wavelet transform decomposes a signal into “detail” coefficients (high-pass filter components) and “approximation” coefficients (low-pass filter components). In DWT, the mother wavelet (Ψ (t)) is decimated by a factor of two and is shifted. The discrete wavelet coefficients can be written as: </p><disp-formula><mml:math id=\"M15\"><mml:mrow><mml:msub><mml:mi>γ</mml:mi><mml:mrow><mml:mi>j</mml:mi><mml:mo>,</mml:mo><mml:mi>k</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msubsup><mml:mo>∫</mml:mo><mml:mrow><mml:mo>−</mml:mo><mml:mi>∞</mml:mi></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mi>∞</mml:mi></mml:mrow></mml:msubsup><mml:mi>x</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mi>t</mml:mi><mml:mo>)</mml:mo></mml:mrow><mml:msup><mml:mn>2</mml:mn><mml:mrow><mml:mo>−</mml:mo><mml:mi>j</mml:mi><mml:mo>/</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msup><mml:mi>Ψ</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:msup><mml:mn>2</mml:mn><mml:mrow><mml:mo>−</mml:mo><mml:mi>j</mml:mi></mml:mrow></mml:msup><mml:mi>t</mml:mi><mml:mo>−</mml:mo><mml:mi>k</mml:mi><mml:mo>)</mml:mo></mml:mrow><mml:mi>d</mml:mi><mml:mi>t</mml:mi></mml:mrow></mml:math></disp-formula><p>\nWhere integers <italic>j</italic> and <italic>k</italic> represent the scale and shift parameters, and <italic>x (t)</italic> denotes the original signal with the finite length <italic>N</italic>.<sup>[<xref rid=\"B38\" ref-type=\"bibr\">38</xref>,<xref rid=\"B39\" ref-type=\"bibr\">39</xref>]</sup>\n</p><p>The Daubechies wavelets (Db) are the most important and popular family of wavelets in DWT.<sup>[<xref rid=\"B40\" ref-type=\"bibr\">40</xref>]</sup> With respect to the high resemblance between Daubechies wavelet order of 4 (db4) and PRVEP waveforms, db4 was considered as the proper mother wavelet function for discrete decomposition of PRVEPs in this study.</p><p>In all cases, the detail coefficients of levels less than 7 were discarded, as the frequency content of these bands was higher than the P100 frequencies. The approximation coefficients, detail coefficients, and 7P descriptor of all responses in level 7 were computed. The 7P descriptor is the energy percentage of a single wavelet coefficient to the total energy level at predetermined time intervals at level 7 and is extracted from the DWT scalogram. The approach of calculating and extracting the 7P descriptor from the DWT scalograms was previously explained by Hassankarimi et al.<sup>[<xref rid=\"B32\" ref-type=\"bibr\">32</xref>]</sup>\n</p><p>All data of TD, FFT, PSD, and DWT were analyzed using the Statistical Package for the Social Sciences for Windows, version 22.0 (Inc., Chicago, IL, USA). After testing the normality of all data, the effect of SF changes on all mentioned parameters was evaluated through a one-way analysis of variance (ANOVA) with the post-hoc Fisher's Least Significant Difference (LSD) test. Spatial frequencies of 1, 2, and 4 (cpd) were considered as groups 1, 2, and 3, respectively.</p></sec><sec sec-type=\"section\"><title> Results</title><p>Table 1 shows the results of one-way ANOVA for comparisons of time, frequency, and wavelet domain parameters among three groups of spatial frequencies. For all spatial frequencies, increasing SF resulted in a decrease in the P100 amplitude (Figure 1). The LSD test revealed that differences between 1 and 2 (cpd) groups (<italic>P </italic>= 0.001) and 1 and 4 (cpd) groups (<italic>P </italic>= 0.001) were significant. The latency component also showed a change with increasing spatial frequencies. However, this change was in favor of increasing delay time (Figure 1). Marked latency differences were observed between groups 1 and 2 (<italic>P </italic>= 0.025), and groups 1 and 3 (<italic>P </italic>= 0.011).</p><p>Comparing the results of the DWT coefficients revealed that the mean value of both approximation and detail coefficients considerably tended to increase with the increase of SF (Table 1). According to the results of the LSD test, approximation coefficients differed significantly between groups 1 and 2 (<italic>P </italic>= 0.034), and groups 1 and 3 (<italic>P </italic>= 0.001). The increase in these coefficients was much greater by changing the frequency from 1 cpd to 2 cpd than from 2 cpd to 4 cpd. There was a meaningful influence of SF on detail coefficients between groups 3 and 1 (<italic>P </italic>= 0.010) and groups 3 and 2 (<italic>P </italic>= 0.006). The magnitude of the 7P energy descriptor, extracted from the DWT scalograms, showed no observable differences.</p><p>The frequency domain components did not change significantly. At all spatial frequencies, the peak frequency (F<inline-formula><mml:math id=\"M16\"><mml:msub><mml:mrow/><mml:mi> mod </mml:mi></mml:msub></mml:math></inline-formula>) had almost a constant value of approximately 2 Hz (Figure 2).</p></sec><sec sec-type=\"section\"><title> Discussion</title><p>In the present study, the relationship between SF increase and VEP parameters of time, frequency, and wavelet domains at the contrast level of 5% were investigated. The 5% contrast was considered as the contrast threshold, given the decrease in VEP amplitude due to reduction in contrast and the low signal to noise ratio and negligible VEP responses at the contrast levels <inline-formula><mml:math id=\"M17\"><mml:mo><</mml:mo></mml:math></inline-formula> 5%.<sup>[<xref rid=\"B41\" ref-type=\"bibr\">41</xref>]</sup>\n</p><p>In agreement with previous studies, our results revealed dramatic changes in TD parameters as a function of SF. An increase in SF resulted in P100 amplitude reduction and latency prolongation (Figure 1).<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>,<xref rid=\"B12\" ref-type=\"bibr\">12</xref>,<xref rid=\"B42\" ref-type=\"bibr\">42</xref>,<xref rid=\"B43\" ref-type=\"bibr\">43</xref>,<xref rid=\"B44\" ref-type=\"bibr\">44</xref>]</sup> It has been supposed that differences in the speed of information processing and conduction along the visual pathways, which are preferentially activated by specific spatial frequencies, lead to the sequential visual processing from low to high range of SFs.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> Several studies demonstrated that two or more parallel pathways from the retina to the primary visual cortex (V1) are involved in VEP formation. At low contrasts, the magnocellular (MC) pathway dominantly contributes to the VEP responses, whereas at high contrasts, MC, parvocellular (PC), and koniocellular (KC) pathways involve VEP. The MC neurons preferentially detect the low SF and the high temporal frequency stimuli. They have a high contrast sensitivity, high temporal resolution, and short impulse conduction time, whereas PC neurons with a smaller receptive field are sensitive to low temporal and high spatial frequencies.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B8\" ref-type=\"bibr\">8</xref>,<xref rid=\"B12\" ref-type=\"bibr\">12</xref>,<xref rid=\"B35\" ref-type=\"bibr\">35</xref>,<xref rid=\"B42\" ref-type=\"bibr\">42</xref>,<xref rid=\"B45\" ref-type=\"bibr\">45</xref>,<xref rid=\"B46\" ref-type=\"bibr\">46</xref>,<xref rid=\"B47\" ref-type=\"bibr\">47</xref>,<xref rid=\"B48\" ref-type=\"bibr\">48</xref>,<xref rid=\"B49\" ref-type=\"bibr\">49</xref>]</sup> Therefore, MC signals (high SFs) are conveyed to V1 more rapidly than PC signals (low SFs). At high SFs, the optical properties of the eye noticeably reduce the retinal contrast, resulting in decreased amplitude and delayed latency.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> It has been demonstrated that visual sensitivity progressively weakens with increase in SF or decrease in the size of the object.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B50\" ref-type=\"bibr\">50</xref>]</sup> It can be caused by pre-neural factors, such as the optical quality of the eye or by contribution of higher levels of visual processing (beyond the lateral geniculate nucleus) for VEP formation.<sup>[<xref rid=\"B51\" ref-type=\"bibr\">51</xref>]</sup> It is also suggested that quantal fluctuations in light may give rise to sensitivity loss at high SFs.<sup>[<xref rid=\"B52\" ref-type=\"bibr\">52</xref>]</sup>\n</p><p>To the best of our knowledge, F<inline-formula><mml:math id=\"M18\"><mml:msub><mml:mrow/><mml:mi> mean </mml:mi></mml:msub></mml:math></inline-formula> and F<inline-formula><mml:math id=\"M19\"><mml:msub><mml:mrow/><mml:mi> mod </mml:mi></mml:msub></mml:math></inline-formula> of PRVEPs were not evaluated in previous studies. Our results of the frequency domain analysis showed that changes in SF have no obvious effect on frequency parameters (Table 1). Frequency stability is a significant feature of normal VEP signals. No significant change in F<inline-formula><mml:math id=\"M20\"><mml:msub><mml:mrow/><mml:mi> mean </mml:mi></mml:msub></mml:math></inline-formula> in all SFs can be explained by the fact that all recorded VEPs in this study were normal. The almost constant value of F<inline-formula><mml:math id=\"M21\"><mml:msub><mml:mrow/><mml:mi> mod </mml:mi></mml:msub></mml:math></inline-formula> recordings may indicate that, in all groups, VEP responses were generated by the same subsystems and mechanisms. With respect to the stimulus conditions of this study (5% contrast), we conclude that the MC neuron activity dominantly contribute to eliciting the VEPs.<sup>[<xref rid=\"B41\" ref-type=\"bibr\">41</xref>]</sup>\n</p><p>Unlike frequency parameters, mean value approximation and detail coefficients represent marked increase as a function of SF (Table 1). Based on the capability of the WT in representing the signal frequency contents locally in time<sup>[<xref rid=\"B53\" ref-type=\"bibr\">53</xref>]</sup> and clear P100 latency prolongation with an increase in SF, considerable differences in time–frequency parameters as a function of SF were expected.</p><p>The PRVEPs induced by different SF stimuli originate from segregated neural activities in the visual system. In DWT, approximation coefficients consist of the low-frequency components and the identity of the signal, while the detail coefficients correspond to high-frequency components and fine details of the signal. Statistically significant differences of approximation coefficients between SF groups reflect that the high frequency VEP components at 4 cpd have different origins, generation mechanisms, and visual processing areas compared to other SFs. On the other hand, the low-frequency contents (detail coefficients) at SF of 1 cpd are elicited by different mechanisms compared to the spatial frequencies of 2 and 4 cpd. A possible explanation is that the processing of medium and high SF information occurs in the primary visual cortex (V1), while low spatial frequencies are mainly processed in the secondary visual area (V2).<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B35\" ref-type=\"bibr\">35</xref>]</sup> Furthermore, SFs <inline-formula><mml:math id=\"M22\"><mml:mo>></mml:mo></mml:math></inline-formula> 1.5 cpd generally elicit VEPs that are contrast specific in nature, whereas SFs <inline-formula><mml:math id=\"M23\"><mml:mo><</mml:mo></mml:math></inline-formula> 1.5 cpd elicit VEPs that are mainly originated from local luminance changes.<sup>[<xref rid=\"B51\" ref-type=\"bibr\">51</xref>]</sup> Moreover, since the stimulus conditions have a significant impact on the neural responses,<sup>[<xref rid=\"B54\" ref-type=\"bibr\">54</xref>]</sup> under our stimulus conditions, an increase in the DWT coefficients can result in simultaneous stimulation of similar neuronal circuits in the visual cortex and inner cortex interaction between neurons outside the receptive field. As mentioned earlier, MC and PC contribute to VEP response formation. It has been proven that in the 4c layer in V1, the nerve endings projected by MC and PC axon terminals have significant overlapping. Nonselective stimuli activate both magno and parvo systems and give rise to anatomical and functional overlapping.<sup>[<xref rid=\"B51\" ref-type=\"bibr\">51</xref>,<xref rid=\"B55\" ref-type=\"bibr\">55</xref>]</sup> In the present experiment, although stimuli contrast was low, selective spatial frequencies had not been chosen specifically to activate the MC neurons. Therefore, neuron activities were not exclusively recorded via VEP responses. Considering the results of the wavelet analysis, it seems that specific SF activates specific receptive field, and, in addition, other factors are also involved in the response formation mechanisms.</p><p>In summary, the obtained results indicate that optical information processing is performed through parallel pathways in the visual system. In addition, the visual system can select a dedicated channel for processing of specific information according to different optical properties. This system has distinct spatial and contrast filters, and this filtration is associated with stimulus condition.</p><p>In conclusion, the authors evaluated the SF effect on PRVEP features in time, frequency, and time–frequency domains and concluded that the TD and DWT approaches are more efficient compared to the FFT and PSD approaches to detect the impact of SF on the VEP parameters at a contrast level of 5%. Furthermore, sources, mechanisms, and pathways involved in evoking and processing PRVEP responses are SF dependent. We suggest further research on more subjects with stimuli of different contrasts, using other wavelet functions.</p></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>Nil.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec><sec sec-type=\"section\"><title> Appendix</title><sec sec-type=\"subsection\"><title>MATLAB Codes</title><sec sec-type=\"subsubsection\"><title>Code for normalizing the input</title><p>%Normalization range is [-2 2]</p><p>function [normalized_output] = Normalization(input)</p><p>temp03 =.5 * (max(input) + min(input));</p><p>temp04 =.5 * (max(input) – min(input));</p><p>input = 2 * (input - temp03) / temp04;</p><p>t05 = isnan(input);</p><p>input(t05) = 0;</p><p>t06 = input <inline-formula><mml:math id=\"M24\"><mml:mo>></mml:mo></mml:math></inline-formula> 2;</p><p>input(t06) = 2;</p><p>normalized_output = input;</p></sec><sec sec-type=\"subsubsection\"><title>Time domain analysis</title><p>X = p;</p><p>Y = t;</p><p>Z = x;</p><p>Fs = 1024;       % Sampling frequency</p><p>T = 1/Fs;       % Sampling period</p><p>L = 240;       % Length of signal</p><p>t = (0:L-1)*T*1000;       % Time vector</p><p>subplot(3,1,1);plot(t,X,'b');</p><p>subplot(3,1,2);plot(t,Y,'b');</p><p>subplot(3,1,3);plot(t,Z,'b');xlabel('time(ms)');ylabel('amplitude (µV)');</p></sec><sec sec-type=\"subsubsection\"><title>Frequency domain analysis and power spectral density </title><p>X = d;</p><p>Fs = 1024;       % Sampling frequency</p><p>T = 1/Fs;       % Sampling period</p><p>L = 240;       % Length of signal</p><p>tm = (0:L-1)*T*1000;      % Time vector</p><p>subplot(2,2,1);plot(tm,X,'r');</p><p>Y = fft(X);</p><p>P2 = abs(Y/L);</p><p>P1 = P2(1:L/2+1);</p><p>fr = Fs*(0:(L/2))/L;</p><p>subplot(2,2,2);plot(fr,P1);</p><p>title('Single-Sided Amplitude Spectrum of X(t)')</p><p>xlabel('f (Hz)')</p><p>ylabel('|P1(f)|');</p><p>M = meanfreq(X,Fs);</p><p>pxx = pwelch(X);</p><p>subplot(2,2,3);plot(pxx);xlabel('f (Hz)');</p></sec><sec sec-type=\"subsubsection\"><title>Calculate approximation and detail coefficients of Discrete Wavelet Transform </title><p>data = Normalization(b);</p><p>wname = 'db4'; % Wavelet Mather Functidn Name</p><p>nLevel = 7; % Wavelxt Decomposition Level</p><p>[C, L] = wavedec(data,nLevel,wname);% Wavelet Decomposition</p><p>A7 = appcoef(C,L,wname,nLevel);    % Approximation Coefficients</p><p>D7 = detcoef(C,L,7);       % Detail Coefficients of Level 7</p><p>D6 = detcoef(C,L,6);       % Detail Coefficients of Level 6</p><p>D5 = detcoef(C,L,5);       % Detail Coefficients of Level 5</p><p>D4 = detcoef(C,L,4);       % Detail Coefficients of Level 4</p><p>D3 = detcoef(C,L,3);       % Detail Coefficients of Level 3</p><p>D2 = detcoef(C,L,2);       % Detail Coefficients of Level 2</p><p>D1 = detcoef(C,L,1);       % Detail Coefficients of Level 1</p></sec><sec sec-type=\"subsubsection\"><title>Calculate d7 descriptor</title><p>d7 = D7(1, 6)<inline-formula><mml:math id=\"M25\"><mml:mo>∧</mml:mo></mml:math></inline-formula>2;</p><p>power_D7 = sum(D7.<inline-formula><mml:math id=\"M26\"><mml:mo>∧</mml:mo></mml:math></inline-formula>2);</p><p>pd7 = d7/power_D7 *100;</p></sec></sec></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Hess RF. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864066</article-id><article-id pub-id-type=\"pmc\">PMC7431727</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7454</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Choroidal Thickness in Different Types of Inherited Retinal Dystrophies</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Sabbaghi</surname><given-names>Hamideh</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ahmadieh</surname><given-names>Hamid</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Jalili</surname><given-names>Jalil</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"I4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Behnaz</surname><given-names>Nazanin</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Fakhri</surname><given-names>Maryam</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Suri</surname><given-names>Fatemeh</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kheiri</surname><given-names>Bahareh</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rajabpour</surname><given-names>Mojtaba</given-names></name><degrees>BS</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Entezari</surname><given-names>Morteza</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Daftarian</surname><given-names>Narsis</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I5\">\n<sup>5</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Ophthalmic Epidemiology Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran</aff><aff id=\"I2\">\n<sup>2</sup>Department of Optometry, School of Rehabilitation, Shahid Beheshti University of Medical Sciences, Tehran, Iran</aff><aff id=\"I3\">\n<sup>3</sup>Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran</aff><aff id=\"I4\">\n<sup>4</sup>Medical Physics and Biomedical Engineering Department, School of Medicine, Tehran University of Medical Sciences, Tehran,\nIran</aff><aff id=\"I5\">\n<sup>5</sup>Ocular Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Narsis Daftarian, MD. Ocular Tissue Engineering\nResearch Center, Research Institute for Ophthalmology\nand Vision Science, Shahid Beheshti University of\nMedical Sciences, No 23, Paidarfard St., Boostan 9 St.,\nPasdaran Ave., Tehran 16666, Iran.\nE-mail: nardaftarian@hotmail.com\nNazanin Behnaz, MD. Ophthalmic Research Center,\nResearch Institute for Ophthalmology and Vision\nScience, Shahid Beheshti University of Medical\nSciences, No 23, Paidarfard St., Boostan 9 St., Pasdaran\nAve., Tehran 16666, Iran.\nE-mail: n.behnaz1990@gmail.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>351</fpage><lpage>361</lpage><permissions><copyright-statement>Copyright © 2020 Sabbaghi et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><sec><title>Purpose</title><p>To compare the choroidal thickness among eyes with retinitis pigmentosa (RP), Stargardt disease, Usher syndrome, cone-rod dystrophy, and healthy eyes of sex- and age-matched individuals.</p></sec><sec><title>Methods</title><p>In this comparative study, 503 eyes with RP (<italic>n</italic> = 264), cone-rod dystrophy (<italic>n </italic>= 109), Stargardt disease (<italic>n</italic> = 76), and Usher syndrome (<italic>n</italic> = 54) were included. To validate the data, 109 healthy eyes of 56 sex- and age-matched individuals were studied as controls. Choroidal imaging was performed using enhanced depth imaging-optical coherence tomography. Choroidal thickness was measured manually using MATLAB software at 13 points in nasal and temporal directions from the foveal center with the interval of 500 µm and the choroidal area encompassing the measured points was calculated automatically.</p></sec><sec><title>Results</title><p>The mean age was 36.33 <inline-formula><mml:math id=\"M1\"><mml:mo>±</mml:mo></mml:math></inline-formula> 13.07 years (range, 5 to 72 years). The mean choroidal thickness at 13 points of the control eyes was statistically significantly higher than that in eyes with RP (<italic>P </italic>\n<inline-formula><mml:math id=\"M2\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001) and Usher syndrome (<italic>P</italic>\n<inline-formula><mml:math id=\"M3\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05), but not significantly different from that in eyes with Stargardt disease and cone-rod dystrophy. Among different inherited retinal dystrophies (IRDs), the choroidal thickness was the lowest in eyes with RP (<italic>P </italic>\n<inline-formula><mml:math id=\"M4\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001). Choroidal thickness in the subfoveal area correlated negatively with best-corrected visual acuity (<italic>r</italic> = <inline-formula><mml:math id=\"M5\"><mml:mo>-</mml:mo></mml:math></inline-formula>0.264, <italic>P </italic>\n<inline-formula><mml:math id=\"M6\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001) and the duration of ocular symptoms (<italic>r</italic> = <inline-formula><mml:math id=\"M7\"><mml:mo>-</mml:mo></mml:math></inline-formula>0.341, <italic>P </italic>\n<inline-formula><mml:math id=\"M8\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001) in all studied IRDs. No significant correlation was observed between the subfoveal choroidal thickness and central macular thickness (<italic>r</italic> = <inline-formula><mml:math id=\"M9\"><mml:mo>-</mml:mo></mml:math></inline-formula>0.24, <italic>P </italic>= 0.576).</p></sec><sec><title>Conclusion</title><p>Choroidal thinning in four different types of IRDs does not follow a similar pattern and depends on the type of IRD and the duration of ocular symptoms. A larger cohort is required to verify these findings</p></sec></abstract><kwd-group><kwd>Choroidal Thickness</kwd><kwd> Cone-rod Dystrophy</kwd><kwd> Enhanced Depth Optical Coherence Tomography</kwd><kwd> Inherited Retinal Dystrophy</kwd><kwd> Retinitis Pigmentosa</kwd><kwd> Stargardt Disease</kwd><kwd> Usher Syndrome</kwd></kwd-group><counts><fig-count count=\"4\"/><table-count count=\"2\"/><ref-count count=\"25\"/><page-count count=\"11\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> INTRODUCTION</title><p>Photoreceptors and retinal pigment epithelial (RPE) cells as their protectors are the primary units for light photon translation into neural electric codes.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> Choroid is a complex vascular tissue that supplies oxygen and nutrients to these cells with a high metabolism rate. Therefore, a healthy choroidal vasculature could provide optimum blood flow to RPE and photoreceptor cells.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> Inherited retinal dystrophies (IRDs) are ocular diseases that primarily involve progressive degeneration of RPE and/or photoreceptor cells.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup> Retinitis pigmentosa (RP) is the most prevalent type of IRD that has been estimated to affect approximately 1.5 million individuals worldwide.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup> Stargardt disease is the most prevalent inherited macular dystrophy with an estimated prevalence of 1 per 10,000 individuals.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> A recent study showed that mutations in more than 120 causative genes are responsible for different types of IRDs<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> and these mutations can be transmitted to the next generation by different Mendelian patterns of inheritance.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup> Generally, it appears that the outer retina and RPE cells are primarily involved, resulting in death of these cells.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>,<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> Choriocapillaris may also be involved in the late stages of the disease, manifesting as chorioretinal atrophy in fundus examination.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>,<xref rid=\"B10\" ref-type=\"bibr\">10</xref>]</sup>\n</p><p>Currently, there is no definite treatment for IRDs. However, recent advances have been reported in the field of gene therapy for RP, Leber's congenital amaurosis, and Stargardt disease.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> If gene therapy may be speculated to repair the\ndefect in gene function, healthy choriocapillaries are necessary to provide enough blood flow to compensate for the normal metabolic needs of the outer retina.</p><p>Enhanced depth imaging (EDI) by spectral domain optical coherence tomography (SD-OCT), is a technique used to visualize the detailed structure of the choroidal tissue from the nasal region adjacent to the optic nerve to the subfoveal choroid and the temporal region of the choroid and can be used to measure the choroidal thickness.<sup>[<xref rid=\"B12\" ref-type=\"bibr\">12</xref>]</sup> Measurement of the choroidal thickness can be informative in determining the pathophysiology and natural course of IRDs.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>,<xref rid=\"B14\" ref-type=\"bibr\">14</xref>,<xref rid=\"B15\" ref-type=\"bibr\">15</xref>]</sup>\n</p><p>Some studies have reported a significant reduction in the choroidal thickness in RP,<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B15\" ref-type=\"bibr\">15</xref>,<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup> Stargardt disease,<sup>[<xref rid=\"B17\" ref-type=\"bibr\">17</xref>]</sup> and cone dystrophy<sup>[<xref rid=\"B18\" ref-type=\"bibr\">18</xref>]</sup> when compared with healthy controls. However, no difference was observed in other studies.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup> Due to this discrepancy, the aim of the present study was to measure the choroidal thickness in a large Iranian cohort with RP, Stargardt disease, cone-rod dystrophy, and Usher syndrome and to compare it with age-matched healthy subjects using EDI-optical coherence tomography (EDI-OCT). The study also aimed to evaluate the relationship of choroidal thickness with the best-corrected visual acuity (BCVA), central macular thickness (CMT), and the duration of ocular symptoms.</p></sec><sec sec-type=\"section\"><title> METHODS</title><p>In this comparative study, 503 eyes of 253 patients diagnosed with IRDs including RP (264 eyes of 133 patients), Stargardt disease (76 eyes of 38 patients), cone-rod dystrophy (109 of 55 patients), and Usher syndrome (54 eyes of 27 patients) were included. For comparison, 109 normal eyes of 56 healthy subjects were included as controls. Healthy controls were matched based on patients' age and sex. Data were extracted from the Iranian National Registry of IRDs (IRDRegⓇ). Patients were recalled for additional examinations and imaging according to the standard protocol. This study was conducted at Labbafinejad Medical Center, Tehran, Iran, from January 2016 to August 2018.</p><p>The Ethics Committee of the Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran approved this study and all procedures were in compliance with the tenets of the Declaration of Helsinki. Written informed consent was obtained from all subjects for diagnostic procedures and choroidal imaging.</p><sec sec-type=\"subsection\"><title>Visual and Ocular Examinations</title><p>Initially, all patients were interviewed to identify the age of disease manifestations and the common signs and symptoms including photophobia, color vision deficiency, nyctalopia, nystagmus, restricted visual field, and previous general and ocular conditions. All subjects underwent complete ophthalmic examination including BCVA assessment, color vision testing using Ishihara Pseudoisochromatic plates, slit-lamp biomicroscopy, measurement of the intraocular pressure using the Goldmann applanation tonometer, and dilated fundus examination using a +78D lens. In addition, visual field testing was performed with Humphrey visual field (Carl Zeiss Meditec Inc., Dublin, CA, USA) using 30-2 Swedish Interactive Threshold Algorithm standard method. Additionally, SD-OCT scanning was performed and choroidal thickness was also measured using the EDI-OCT scan (Spectralis, Heidelberg Engineering, Heidelberg, Germany). Fundus photographs were obtained by a digital stereoscopic camera (Visucam Pro NM, Carl Zeiss Meditec AG, Germany). Infrared imaging, fundus autofluorescence, and fluorescein angiography (Heidelberg Engineering GmbH, Heidelberg, Germany) were also performed. In addition, electrophysiological examinations including electroretinography (ERG) and/or electro-oculography (RETIport 21 system, version 7/03, Roland Consult, Osaka, Japan) were conducted to confirm the clinical diagnosis. Based on fundus examination, macular involvement was defined as the presence of any kind of macular abnormality from reduced foveal reflex to bull's eye pattern or beaten bronze appearance.</p></sec><sec sec-type=\"subsection\"><title>Inclusion and Exclusion Criteria</title><p>Patients with syndromic RP, IRD cases having optic atrophy due to other etiologies, visually significant cataract or other media opacities, high refractive errors (myopia <inline-formula><mml:math id=\"M10\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 5.00 D, hyperopia <inline-formula><mml:math id=\"M11\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 3.00 D, and astigmatism <inline-formula><mml:math id=\"M12\"><mml:mo>≥</mml:mo></mml:math></inline-formula> 3.00 D), nystagmus or wandering gaze, poor image quality, and any other associated retinal pathologies were excluded. Patients with cystoid macular edema were also excluded from the analysis. We also excluded patients with systemic diseases affecting the choroidal thickness such as systemic hypertension, diabetes, and renal failure. The control group included sex- and age-matched healthy subjects with no ocular and systemic diseases and without high refractive errors. None of the controls had a positive family history of IRDs.</p></sec><sec sec-type=\"subsection\"><title>Final Diagnosis</title><p>The Final diagnosis of retinal dystrophy was obtained based on clinical examinations, retinal multimodal imaging, and psychophysical tests such as ERG, color vision, and visual fields. Additionally, retinal dystrophy was confirmed by genetic findings in 98 patients (19.5%). Cross-validation of patients' response with clinical records was performed to increase the data validity.</p></sec><sec sec-type=\"subsection\"><title>Choroidal Thickness Measurement </title><p>Each patient underwent an EDI-OCT scan after dilation of the pupil by 1% tropicamide eye drop (Figure 1, A1 to F2). EDI-OCT automatically sets the choroid closer to the zero-delay line and thus, theoretically provides better visualization of the choroidoscleral interface. One horizontal 9mm high-quality line scan through the fovea was obtained for each eye. The line scan was saved for analysis after averaging of 100 frames. Choroidal measurements were performed using MATLAB 2016b program (MathWorks, Natick, MA, USA) at 13 points subfoveally and in nasal and temporal directions with the interval of 500 µm in a length of 6000 µm (Figure 1, B1 and B2).</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Representative enhanced depth imaging (EDI) of four different inherited retinal dystrophies and of a healthy control.\nA1: Right eye of a healthy control, A2: left eye of a healthy control\nB1, B2: Examples of choroidal thickness measurement manually and automatically in the right and the left eyes, respectively, of a healthy subject\nC1, C2: EDI of the right and the left eyes, respectively, of a patient with retinitis pigmentosa D1, D2: EDI of the right and the left eyes, respectively, of a patient with Usher syndrome\nE1, E2: EDI of the right and the left eyes, respectively, of a patient with Stargardt disease\nF1, F2: EDI of the right and the left eyes, respectively, of a patient with cone-rod dystrophy</p></caption><graphic xlink:href=\"jovr-15-351-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Mean choroidal thickness in the subfoveal area and at six different spots along the nasal and the temporal directions in different types of inherited retinal dystrophies.\nRP, retinitis pigmentosa</p></caption><graphic xlink:href=\"jovr-15-351-g002\"/></fig><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>Mean total choroidal thickness (at 13 spots of measurement) in different types of inherited retinal dystrophies.\nRP, retinitis pigmentosa\n*Significant <italic>P</italic>-values between 0.01 and 0.05; ***Significant <italic>P</italic>-values <inline-formula><mml:math id=\"M13\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</p></caption><graphic xlink:href=\"jovr-15-351-g003\"/></fig><fig id=\"F4\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><p>Mean choroidal area in different types of inherited retinal dystrophies\nRP, retinitis pigmentosa\n**Significant <italic>P</italic>-values between 0.01 and 0.001\n***Significant <italic>P</italic>-values <inline-formula><mml:math id=\"M14\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</p></caption><graphic xlink:href=\"jovr-15-351-g004\"/></fig><p>All choroidal measurements were done by an ophthalmologist familiar with the MATLAB program. All measurements were performed from the outer portion of the hyper-reflective line corresponding to the RPE cells-Bruch's membrane complex as the inner border to the hypo-reflective line corresponding to the sclerochoroidal interface as the outer border. Subsequently, the program automatically measured choroidal thickness in the specified spots according to the spaces defined in the preceding step, measured the area of choroid in the specified region, and provided the information in an excel file. The aforementioned application was designed to work as follows.</p><p>1. An EDI-OCT image was displayed in a window after loading the image into the application. The coordinates of all pixels and their intensities were shown in the image.</p><p>2. The program allowed the physicians to manually select the center of macula and the choroidal region on the EDI-OCT images.</p><p>3. Based on the specified center and the area, six points with 500 μm intervals on each side of the center (13 points including the center) were automatically marked and the choroidal thickness was calculated at these points.</p><p>4. The program also provided charts related to changes in the choroidal thickness and other additional information including the area of the choroidal region.</p><p>All EDI-OCT images from the patients and the controls were obtained from 2:00 to 6:00 pm to reduce the possible effect of diurnal variation on choroidal thickness. Finally, two other board-certified retina specialists independently rechecked the measurements to avoid disagreements (<italic>Cronbach's <inline-formula><mml:math id=\"M15\"><mml:mi>α</mml:mi></mml:math></inline-formula></italic> = 0.794). In a few images with disagreement, the RPE-Bruch's membrane complex and the sclerochoroidal interface were rechecked and the measurements were repeated to be confirmed by the two retina specialists.</p></sec><sec sec-type=\"subsection\"><title>Statistical Analysis</title><p>Data were presented as mean and standard deviation, median and range, and frequency and percentage. To compare the subject characteristics between the IRD and the control groups, we used Dunnet's correction for multiple comparisons. To determine the possible correlation of the patients' characteristics or the IRD characteristics with the choroidal thickness and the choroidal area, generalized estimating equation analysis was used. All statistical analyses were performed using IBM SPSS Statistics for Windows, version 25.0. (IBM Corp., Armonk, NY, USA). All tests were two-sided and a <italic>P</italic>-values <inline-formula><mml:math id=\"M16\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05 were considered statistically significant.</p></sec></sec><sec sec-type=\"section\"><title> RESULTS</title><p>In this comparative study, 503 eyes with a confirmed diagnosis of IRD including three types of diffuse photoreceptor dystrophies (RP, Usher syndrome, and cone-rod dystrophy) and one macular dystrophy (Stargardt disease) were included. The age and gender distribution of the patients and controls are presented in Table 1.</p><p>We observed that for most of the cases, the duration since first recognition of the disease was 10 to 20 years (30.5%, <italic>P </italic>= 0.04). The distribution of the IRD duration in 10-year periods was not significantly different among the types of IRDs.</p><p>Table 2 summarizes the clinical characteristics of the study subjects. Comparison of central vision among different types of IRDs showed that the mean BCVA of patients with cone-rod dystrophy was significantly lower than that of patients with Stargardt disease (<italic>P </italic>= 0.009) and Usher syndrome (<italic>P </italic>\n<inline-formula><mml:math id=\"M17\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001).</p><p>The mean CMT values of patients with Stargardt disease and cone-rod dystrophy were significantly lower than those of patients from other groups (<italic>P </italic>\n<inline-formula><mml:math id=\"M18\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001).</p><p>Figure 2 shows the linear comparison of mean choroidal thickness at each of the 13 different spots in different IRDs and the mean choroidal thickness in the control group. The mean subfoveal choroidal thickness in healthy controls (336.79 <inline-formula><mml:math id=\"M19\"><mml:mo>±</mml:mo></mml:math></inline-formula> 82.75 µm) was greater than that in RP patients (<italic>P</italic>\n<inline-formula><mml:math id=\"M20\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001), but did not differ significantly from mean subfoveal choroidal thickness in other IRDs including Usher syndrome, cone-rod dystrophy, and Stargardt disease.</p><p>The mean total choroidal thickness (mean choroidal thickness at 13 points) in eyes with RP (<italic>P</italic>\n<inline-formula><mml:math id=\"M21\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001) and Usher syndrome (<italic>P</italic> = 0.042) was significantly lower than that in healthy eyes. There was no significant difference in mean total choroidal thickness between patients with Stargardt disease and cone-rod dystrophy vs. healthy controls (<italic>P</italic> = NS). The mean choroidal thickness in RP patients was less than that in patients with Stargardt disease (<italic>P</italic>\n<inline-formula><mml:math id=\"M22\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001), cone-rod dystrophy (<italic>P</italic>\n<inline-formula><mml:math id=\"M23\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001), and Usher syndrome (<italic>P</italic> = 0.028) (Figure 3).</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Basic characteristics of the study subjects</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"9\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" colspan=\"5\" rowspan=\"1\"><bold>Groups</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Parameters</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Levels</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Total (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 612)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>RP (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 264)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Stargardt (<italic><bold>n</bold></italic><bold> = 76)</bold></bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Cone- Rod Dystrophy (<italic><bold>n</bold></italic><bold> = 109)</bold></bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Usher Syndrome (<italic><bold>n</bold></italic><bold> = 54)</bold></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Control (<italic><bold>n</bold></italic><bold> = 109)</bold></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold/>\n<italic><bold>P</bold></italic>\n<bold>-value</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Age (years)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Mean <inline-formula><mml:math id=\"M24\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">35.75 <inline-formula><mml:math id=\"M25\"><mml:mo>±</mml:mo></mml:math></inline-formula> 12.81</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">40.21 <inline-formula><mml:math id=\"M26\"><mml:mo>±</mml:mo></mml:math></inline-formula> 13.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29.47 <inline-formula><mml:math id=\"M27\"><mml:mo>±</mml:mo></mml:math></inline-formula> 8.82</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">32.33 <inline-formula><mml:math id=\"M28\"><mml:mo>±</mml:mo></mml:math></inline-formula> 13.52</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">35.04 <inline-formula><mml:math id=\"M29\"><mml:mo>±</mml:mo></mml:math></inline-formula> 11.01</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32.78 <inline-formula><mml:math id=\"M30\"><mml:mo>±</mml:mo></mml:math></inline-formula> 11.07</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M31\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.075*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Median (range)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">34 (5 to 72)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">39 (8 to 72)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30 (9 to 45)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">32 (8 to 64)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">36 (5 to 53)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">31 (11 to 65)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Sex (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Male</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">149 (48.4%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">69 (51.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17 (44.7%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30 (54.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 (37.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23 (41.8%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.41*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Female</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">159 (51.6%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">64 (48.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21 (55.3%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25 (45.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17 (63.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32 (58.2%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Incidence Age (years)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Mean <inline-formula><mml:math id=\"M32\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.87 <inline-formula><mml:math id=\"M33\"><mml:mo>±</mml:mo></mml:math></inline-formula> 13.09</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20.65 <inline-formula><mml:math id=\"M34\"><mml:mo>±</mml:mo></mml:math></inline-formula> 14.16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17.05 <inline-formula><mml:math id=\"M35\"><mml:mo>±</mml:mo></mml:math></inline-formula> 9.46</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17.08 <inline-formula><mml:math id=\"M36\"><mml:mo>±</mml:mo></mml:math></inline-formula> 12.89</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16.38 <inline-formula><mml:math id=\"M37\"><mml:mo>±</mml:mo></mml:math></inline-formula> 11.85</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">____</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.171*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Median (range)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17 (0 to 63)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18 (0 to 63)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15 (6 to 40)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16 (0 to 46)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (1 to 40)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Duration of Disease (years)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Mean <inline-formula><mml:math id=\"M38\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.96 <inline-formula><mml:math id=\"M39\"><mml:mo>±</mml:mo></mml:math></inline-formula> 13.21</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22.22 <inline-formula><mml:math id=\"M40\"><mml:mo>±</mml:mo></mml:math></inline-formula> 14.57</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.42 <inline-formula><mml:math id=\"M41\"><mml:mo>±</mml:mo></mml:math></inline-formula> 8.8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.83 <inline-formula><mml:math id=\"M42\"><mml:mo>±</mml:mo></mml:math></inline-formula> 11.34</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.73 <inline-formula><mml:math id=\"M43\"><mml:mo>±</mml:mo></mml:math></inline-formula> 10.08</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">_____</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M44\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001*</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Median (range)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16.5 (0 to 61)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21 (0 to 61)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11 (1 to 35)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (0 to 45)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16.5 (4 to 40)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Duration of Disease</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 – 10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">69 (28.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29 (22.7%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17 (44.7%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17 (32.7%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (15.4%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">_____</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.04<inline-formula><mml:math id=\"M45\"><mml:mo>†</mml:mo></mml:math></inline-formula>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 – 20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">75 (30.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">34 (26.6%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12 (31.6%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19 (36.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 (38.5%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">20 – 30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">49 (19.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27 (21.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (18.4%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8 (15.4%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (26.9%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">30 – 40</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">31 (12.6%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18 (14.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (5.3%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (13.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (15.4%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">40 – 50</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17 (6.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15 (11.7%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (1.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (3.8%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">50 – 60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5 (2.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5 (3.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" colspan=\"9\" rowspan=\"1\">RP, retinitis pigmentosa; SD, standard deviation; n, number\n*<italic>P</italic>-value is based on ANOVA (in all the above analysis, multiple comparison correction have been done with Bonferroni method)\n<inline-formula><mml:math id=\"M46\"><mml:mo>†</mml:mo></mml:math></inline-formula>Based on Chi-square test</td></tr></tbody></table></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Clinical characteristics of the study subjects</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"9\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" colspan=\"5\" rowspan=\"1\"><bold>Groups</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Parameters</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Level</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Total (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 612)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>RP (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 264)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Stargardt (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 76)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Cone- Rod Dystrophy (<italic><bold>n</bold></italic><bold> = 109)</bold></bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Usher Syndrome (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 54)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Control (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 109)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold/>\n<italic><bold>P</bold></italic>\n<bold>-value</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">BCVA (LogMAR)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Mean <inline-formula><mml:math id=\"M47\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.04 <inline-formula><mml:math id=\"M48\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.91</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.19 <inline-formula><mml:math id=\"M49\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.98</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.02 <inline-formula><mml:math id=\"M50\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.47</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.43 <inline-formula><mml:math id=\"M51\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.87</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.84 <inline-formula><mml:math id=\"M52\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.69</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 <inline-formula><mml:math id=\"M53\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M54\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Median (range)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.8 (0 to 2.79)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.8 (0 to 2.79)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.9 (0.1 to 2.31)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.31 (0 to 2.7)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.74 (0.1 to 2.7)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0 to 0)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">SE (D)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Mean <inline-formula><mml:math id=\"M55\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–1.0 <inline-formula><mml:math id=\"M56\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.78</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–0.88 <inline-formula><mml:math id=\"M57\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.79</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–1.54 <inline-formula><mml:math id=\"M58\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.33</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–0.88 <inline-formula><mml:math id=\"M59\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.07</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–1.67 <inline-formula><mml:math id=\"M60\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.94</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–0.33 <inline-formula><mml:math id=\"M61\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.94</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M62\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Median (range)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–0.75 (–6.63 to 2.75)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–0.5 (–5.75 to 2.75)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–1.25 (–5.25 to 0.75)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–0.31 (–6.63 to 2.63)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–1.5 (–5.75 to 1.5)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–0.38 (–2 to 2)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">CMT (µm)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Mean <inline-formula><mml:math id=\"M63\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">215.99 <inline-formula><mml:math id=\"M64\"><mml:mo>±</mml:mo></mml:math></inline-formula> 72.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">230.66 <inline-formula><mml:math id=\"M65\"><mml:mo>±</mml:mo></mml:math></inline-formula> 68.81</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">165.54 <inline-formula><mml:math id=\"M66\"><mml:mo>±</mml:mo></mml:math></inline-formula> 65.04</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">186.03 <inline-formula><mml:math id=\"M67\"><mml:mo>±</mml:mo></mml:math></inline-formula> 48.59</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">244.88 <inline-formula><mml:math id=\"M68\"><mml:mo>±</mml:mo></mml:math></inline-formula> 99.87</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">258.72 <inline-formula><mml:math id=\"M69\"><mml:mo>±</mml:mo></mml:math></inline-formula> 15.71</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M70\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Median (range)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">213 (80 to 690)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">222 (95 to 674)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">141 (80 to 378)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">186.5 (88 to 317)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">229.5 (90 to 690)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">259.5 (220 to 285)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Color Vision</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Normal</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">164 (28.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">40 (16.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6 (8.3%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (1.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (14.6%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">109 (100.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M71\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">CVD</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">213 (36.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">71 (28.6%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">53 (73.6%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">62 (57.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27 (56.3%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">207 (35.4%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">137 (55.2%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13 (18.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">43 (40.2%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (29.2%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Cataract Type (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">376 (62.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">101 (38.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">69 (95.8%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">80 (73.4%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17 (31.5%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">109 (100.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">CC</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M72\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">NS</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">44 (7.3%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29 (11.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11 (10.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (7.4%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">PSC</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">169 (27.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">120 (45.8%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 (4.2%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16 (14.7%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30 (55.6%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">PCIOL</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (2.3%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12 (4.6%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (1.8%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Posterior Polar</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 (0.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 (5.6%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Optic Atrophy (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">46 (9.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 (3.8%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22 (28.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (12.8%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M73\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">457 (90.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">254 (96.2%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">54 (71.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">95 (87.2%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">54 (100.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Macular Involvement (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">122 (19.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11 (4.2%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (1.8%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">109 (100.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<inline-formula><mml:math id=\"M74\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">490 (80.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">253 (95.8%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">76 (100.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">107 (98.2%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">54 (100.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0 (0.0%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" colspan=\"9\" rowspan=\"1\"> RP, retinitis pigmentosa; BCVA, best corrected visual acuity; LogMAR, logarithm minimum angle of resolution; SE, spherical equivalent; D, diopter; CMT, central macular thickness; µm, micrometer; CVD, color vision defect; N/A, not applicable; CC, cortical cataract; NS, nuclear sclerotic; PSC, posterior subcapsular; SD, standard deviation; n, number\n*These parameters are presented based on monocular findings</td></tr></tbody></table></table-wrap><p>The mean total choroidal area in different types of IRD and in healthy controls is illustrated in Figure 4. The mean total choroidal area in healthy subjects (1.9 <inline-formula><mml:math id=\"M75\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.4 µm<inline-formula><mml:math id=\"M76\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula>) was significantly more than that in RP (1.4 <inline-formula><mml:math id=\"M77\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.5 µm<inline-formula><mml:math id=\"M78\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula>, <italic>P</italic>\n<inline-formula><mml:math id=\"M79\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001) and Usher syndrome (1.6 <inline-formula><mml:math id=\"M80\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.5 µm<inline-formula><mml:math id=\"M81\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula>, <italic>P</italic> = 0.006). Among different IRDs, the mean total choroidal area was less in RP patients than in patients with Stargardt disease (<italic>P</italic>\n<inline-formula><mml:math id=\"M82\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001) and cone-rod dystrophy (<italic>P </italic>\n<inline-formula><mml:math id=\"M83\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001).</p><p>Additionally, the subfoveal choroidal thickness was inversely correlated with BCVA (<italic>r</italic> = <inline-formula><mml:math id=\"M84\"><mml:mo>-</mml:mo></mml:math></inline-formula>0.264, <italic>P</italic>\n<inline-formula><mml:math id=\"M85\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001) and the duration of ocular symptoms (<italic>r</italic> = <inline-formula><mml:math id=\"M86\"><mml:mo>-</mml:mo></mml:math></inline-formula>0.341, P <inline-formula><mml:math id=\"M87\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.001) in all IRDs. However, no statistically significant correlation was observed between the subfoveal choroidal thickness and CMT (<italic>r</italic> = <inline-formula><mml:math id=\"M88\"><mml:mo>-</mml:mo></mml:math></inline-formula>0.24, <italic>P</italic> = 0.576) in all IRDs.</p></sec><sec sec-type=\"section\"><title> DISCUSSION</title><p>In the present comparative study, choroidal thickness was measured in a large group of patients with different types of IRDs including RP, Stargardt disease, Usher syndrome, and cone-rod dystrophy. We included age- and sex-matched controls for comparison.</p><p>The mean choroidal thickness measured at 13 different spots in the nasal and in the temporal direction was significantly lower in patients with RP and Usher syndrome when compared with healthy controls and patients with Statgardt disease and cone-rod dystrophy. Other studies have also reported similar findings.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B13\" ref-type=\"bibr\">13</xref>,<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup> However, no significant reduction in choroidal thickness was reported in RP patients in the study by Chhablani et al.<sup>[<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup> This discrepancy could be attributed to the different characteristics of the study population including better BCVA (0.99 <inline-formula><mml:math id=\"M89\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.94 LogMAR) and younger age (31.09 <inline-formula><mml:math id=\"M90\"><mml:mo>±</mml:mo></mml:math></inline-formula> 13.40 years) than the BCVA (1.19 <inline-formula><mml:math id=\"M91\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.98 LogMAR) and age (40.21 <inline-formula><mml:math id=\"M92\"><mml:mo>±</mml:mo></mml:math></inline-formula> 13.03 years) of patients in our study. High number of patients with RP (<italic>n</italic> = 264) was a strength of the present study. The number of RP patients was higher than those included in previous studies.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B13\" ref-type=\"bibr\">13</xref>,<xref rid=\"B16\" ref-type=\"bibr\">16</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup> Sodi et al reported findings consistent with our results with no difference in mean choroidal thickness between healthy controls and patients with Stargardt disease.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> They described a statistically significant correlation between lower subfoveal choroidal thickness and longer duration of ocular symptoms; this finding was also observed in the current study.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup>\n</p><p>To the best of our knowledge, this is the first study that compared the choroidal thickness among different types of IRDs. Our investigation showed that the lowest choroidal thickness was observed in patients with RP. This finding could be explained by the possible trophic role of RPE cells, which support proper choroidal function through secretion of growth factors. Conversely, the reduction in oxygen demand of the degenerating photoreceptors will subsequently result in reduced blood flow to the retina and the choroid, which is called primary vascular dysregulation in RP patients.<sup>[<xref rid=\"B20\" ref-type=\"bibr\">20</xref>,<xref rid=\"B21\" ref-type=\"bibr\">21</xref>]</sup> Additionally, RP has been associated with an imbalance in oxidant/antioxidant status and subclinical inflammatory processes that may stimulate the excessive production of endothelin-1 (ET-1).<sup>[<xref rid=\"B16\" ref-type=\"bibr\">16</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup> Therefore, retinal degeneration and choroidal thinning in patients with RP seem to interact with each other.</p><p>The difference in the amount of choroidal thinning between RP and other IRDs can be explained by the pathogenesis of RP.<sup>[<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup> Most of the mutations in RP are attributed to gene coding of proteins involved in the vision cycle at the level of photoreceptors and RPE cells. These mutations cause apoptosis and degeneration of photoreceptors and subsequent outer retinal thinning.<sup>[<xref rid=\"B16\" ref-type=\"bibr\">16</xref>,<xref rid=\"B22\" ref-type=\"bibr\">22</xref>]</sup> Photoreceptor atrophy results in reduced oxygen demand and blood flow. Furthermore, trophic role of RPE cells through secretion of growth factors such as vascular endothelial growth factor<sup>[<xref rid=\"B23\" ref-type=\"bibr\">23</xref>]</sup> that support proper choroidal function is diminished. Thus, choroidal thinning can be explained by reduced blood flow and RPE cell degeneration.</p><p>Yoshida et al observed cell, flare, and inflammatory cytokines in aqueous humor and vitreous fluid of patients with RP.<sup>[<xref rid=\"B24\" ref-type=\"bibr\">24</xref>]</sup> Confirmation of reduced level of antioxidants in this group of patients by Martinez et al is suggestive of the oxidative stress in RP.<sup>[<xref rid=\"B25\" ref-type=\"bibr\">25</xref>]</sup> Subclinical ocular inflammatory process and hypoxic-oxidative stress elicit increased ocular and plasma levels of ET-1.<sup>[<xref rid=\"B16\" ref-type=\"bibr\">16</xref>,<xref rid=\"B22\" ref-type=\"bibr\">22</xref>]</sup>\n</p><p>ET-1 is the most powerful endogenous vasoconstrictor of small and large vessels. In the eye, its synthesis and secretion is performed by different tissues such as cornea, uveal tissue, retinal microvascular pericytes, RPE cells, and optic nerve. Increased levels of ET-1 and vasoconstriction effect result in vascular dysgenesis and impaired ocular blood flow. This vicious cycle contributes to the amplification of the inflammatory response, altered intraocular perfusion, relative ischemia, and consequent degeneration of outer retinal layer and choroidal thinning.<sup>[<xref rid=\"B22\" ref-type=\"bibr\">22</xref>]</sup>\n</p><p>In the present study, a negative correlation was observed between the subfoveal choroidal thickness and BCVA and between subfoveal choroidal thickness and disease duration of the IRDs. However, no correlation was observed between CMT and choroidal thickness. Some studies have reported a correlation between choroidal thickness and BCVA and between choroidal thickness and the duration of ocular symptoms in IRDs including RP and Stargardt disease.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>][<xref rid=\"B5\" ref-type=\"bibr\">5</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup> However, other studies did not find such correlations.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>,<xref rid=\"B13\" ref-type=\"bibr\">13</xref>,<xref rid=\"B16\" ref-type=\"bibr\">16</xref>,<xref rid=\"B18\" ref-type=\"bibr\">18</xref>]</sup> This difference may be due to a higher number of subjects and a longer duration of ocular symptoms in the present study. Ayton et al<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup> reported that choroid becomes thinner with increasing duration of RP symptoms, which is consistent with the findings of the present study. However, Sodi et al<sup>[<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup> claimed that age might have more effect on choroidal thickness than the duration of RP itself. They suggested that this lack of association might be due to a very strict criterion for the definition of age at the onset of the disease. In the present study, additional age-adjusted analysis confirmed that age could not be a confounding variable and the findings may be directly related to the IRD entity.</p><p>Previous studies have found a significant correlation between CMT and choroidal thickness in Stargardt disease<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> and no relationship between them in RP patients.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>][<xref rid=\"B16\" ref-type=\"bibr\">16</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup> In the present study with a larger sample size, no correlation was observed between CMT and subfoveal choroidal thickness in RP and in Stargardt disease. As previously stated, patients with cystoid macular edema were not included in the statistical analysis.</p><p>Total choroidal area in the eyes of healthy subjects was significantly higher than that in eyes of patients with RP and Usher syndrome. The choroid was generally thinner in RP patients when compared with patients having Stargardt disease and cone-rod dystrophy.</p><p>One of the strengths of the present study is the comparison of choroidal thickness among four relatively common types of IRDs including RP, Stargardt disease, Usher syndrome, and cone-rod dystrophy. Additionally, a larger sample size compared to the sample size in previous studies is another merit of this study. The design of the novel software using MATLAB computer programming for automatic measurement of the choroidal parameters minimizes the chance of inter-operator and intra-operator errors. The measurement of the total choroidal area as well as the choroidal thickness at 13 different spots (including the center of the fovea) 6000 µm along the nasal and the temporal directions from the center of the fovea is another merit of the present study.</p><p>The present study has some limitations including the lack of accessibility to the genetic data for all study subjects. Another limitation of this study was relying on patients' self-report for identification of the age of disease onset. Of course, cross-validation of patients' response with clinical records was performed to increase the data validity.</p><p>In conclusion, with the same duration of ocular symptoms, generalized choroidal thinning was observed in RP and Usher syndrome, but not in Stargardt disease and cone-rod dystrophy. Therefore, different pathophysiologic and blood flow mechanisms may be implicated in each IRD, which demands further cohort studies with special consideration of choroidal blood flow as a potential therapeutic target.</p></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>The study data were extracted from the Iranian National Registry of IRDs (IRDRegⓇ), which is financially supported by the Deputy of Research and Technology of the Iranian Ministry of Health and Medical Education, as well as Shahid Beheshti University of Medical Sciences, Tehran, Iran.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Srinivasan VJ, Dubra A. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864069</article-id><article-id pub-id-type=\"pmc\">PMC7431728</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7457</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Perspective</subject></subj-group></article-categories><title-group><article-title>Ocular Gene Therapy with Adeno-associated Virus Vectors: Current Outlook for Patients and Researchers</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Casey</surname><given-names>Geoffrey A.</given-names></name><degrees>BS (EE), BS (MolBiol)</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Papp</surname><given-names>Kimberly M.</given-names></name><degrees>BS</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>MacDonald</surname><given-names>Ian M.</given-names></name><degrees>MDCM</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I3\">\n<sup>3</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Canada</aff><aff id=\"I2\">\n<sup>2</sup>Faculty of Science, University of Alberta, Canada</aff><aff id=\"I3\">\n<sup>3</sup>Department of Ophthalmology, Faculty of Medicine and Dentistry, University of Alberta, Canada</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Ian M. MacDonald, MDCM, Department of\nOphthalmology and Visual Sciences, University of\nAlberta, 7-030 Katz Bldg. Edmonton, Alberta T6G2E1,\nCanada.\nEmail: macdonal@ualberta.ca\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>396</fpage><lpage>399</lpage><permissions><copyright-statement>Copyright © 2020 Casey et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><p>In this “Perspective”, we discuss ocular gene therapy – the patient's perspective, the various strategies of gene replacement and gene editing, the place of adeno-associated virus vectors, routes of delivery to the eye and the remaining question - “why does immunity continue to limit efficacy?” Through the coordinated efforts of patients, researchers, granting agencies and industry, and after many years of pre-clinical studies, biochemical, cellular, and animal models, we are seeing clinical trials emerge for many previously untreatable heritable ocular disorders. The pathway to therapies has been led by the successful treatment of the RPE65 form of Leber congenital amaurosis with LUXTURNA<inline-formula><mml:math id=\"M1\"><mml:msup><mml:mrow/><mml:mi> TM </mml:mi></mml:msup></mml:math></inline-formula>. In some cases, immune reactions to the vectors continue to occur, limiting efficacy. The underlying mechanisms of inflammation require further study, and new vectors need to be designed that limit the triggers of immunity. Researchers studying ocular gene therapies and clinicians enrolling patients in clinical trials must recognize the current limitations of these therapies to properly manage expectations and avoid disappointment, but we believe that gene therapies are well on their way to successful, widespread utilization to treat heritable ocular disorders.</p></abstract><counts><ref-count count=\"12\"/><page-count count=\"4\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> INTRODUCTION</title><p>Patients with hereditary retinal disorders are keenly interested in gene therapy. Many recent media presentations feature patients who have experienced apparent benefit from gene therapy treatments, increasing public awareness of gene therapy. It is true that gene therapy may conceptually be an ideal method to treat heritable eye diseases before irreparable damage has occurred to the eye, but it is crucially important to manage patient perspectives around their participation in clinical trials. The leading successful example of ocular gene therapy is LUXTURNA<inline-formula><mml:math id=\"M2\"><mml:msup><mml:mrow/><mml:mi> TM </mml:mi></mml:msup></mml:math></inline-formula> (voretigene neparvovec-rzyl), an adeno-associated virus (AAV) vector treatment for Leber congenital amaurosis. LUXTURNA<inline-formula><mml:math id=\"M3\"><mml:msup><mml:mrow/><mml:mi> TM </mml:mi></mml:msup></mml:math></inline-formula> is now approved as a treatment in the USA and the European Union. Unfortunately, many patients believe that enrollment in a clinical trial means “early access” to a treatment or cure, when the trials have not yet reported fully their outcomes. As an example, some patients do not fully understand that LUXTURNA<inline-formula><mml:math id=\"M4\"><mml:msup><mml:mrow/><mml:mi> TM </mml:mi></mml:msup></mml:math></inline-formula> is designed to treat a single genetic disorder caused by biallelic pathogenic variants in the <italic>RPE65</italic> gene. Patients with a progressive heritable eye disease are a distinctly vulnerable population. They may have incomplete knowledge of their condition and partially formed opinions on risks and benefits of experiments such as gene therapy. They carry with them the hope that a trial may lead to treatment but may also conflate the trial with finding a cure. After the patient passes the screening and consent process, they may face significant personal challenges of a rigorous and frequent follow-up schedule. They (or their families) may not be completely prepared for the possibility of harm or the systemic consequences of high-dose immunosuppression. According to the partner of one patient in the choroideremia clinical trial in Alberta, “[The prednisone] was the hardest part for me. I was angry and upset and after my husband had to start a second round of it. I was kind of thinking, `was this the right thing to do?'<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>]</sup> Careful consideration of the patient's experience throughout the clinical trial should be considered in the planning stages to avoid miscommunication and psychological harm.</p><sec sec-type=\"subsection\"><title>Gene augmentation vs gene editing</title><p>Gene therapies typically employ a gene augmentation or gene editing strategy. In the case of gene editing, the entities introduced into patient cells (for example, a CRISPR/Cas9 system) are designed to correct the mutation in the endogenous copy (or copies) of the gene. In augmentation gene therapy, the mutated/non-functional copies of the gene are ignored, and patient cells are supplemented with a functional copy of the gene.<sup>[<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> This strategy depends upon sufficiently healthy residual cells being present that can be “rescued” by the therapy. If photoreceptors have already been lost, then alternate approaches must be utilized. In a gene therapy technique called “optogenetics”, non-photoreceptor cells are made photosensitive by expressing channel proteins. A clinical trial of the optogenetic approach is currently recruiting patients with retinitis pigmentosa regardless of the genetic etiology of their condition (ClinicalTrials.gov NCT03326336).</p><p>Clinical trials of ocular gene therapies to treat several monogenic disorders (choroideremia, X-linked retinoschisis, X-linked retinitis pigmentosa, achromatopsia, and Leber hereditary optic neuropathy) are underway across North America. AAV is a common choice as a vector for gene delivery. Despite initial hope that AAV vectors would be minimally immunogenic, preliminary results are available and adverse events have been reported in response to the vectors in some cases.</p></sec><sec sec-type=\"subsection\"><title>Routes of vector administration</title><p>The route of administration for an ocular gene therapy vector depends on the target cells and the tropism of the vector itself, but will generally fall into one of two categories: intravitreal injection or subretinal injection.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup> Wild-type AAV vector capsids do not penetrate deeply into the retina when administered intravitreally, but the ability of novel AAV capsids (produced via directed evolution) to transduce the posterior cells of the retina is under investigation.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> An additional barrier to successful transduction of any retinal cells via intravitreal injection is the humoral immune system; intravitreal injection of non-human primates with both the AAV2 and AAV8 serotypes was found to stimulate production of neutralizing antibodies in subjects with and without pre-existing immunity to the serotype.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> Conversely, subretinal injection directly exposes the posterior cells of the retina to the vector and evades humoral immune surveillance; however, the retinal detachment required to create space for vector administration triggers microglial activation and photoreceptor death.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup>\n</p></sec><sec sec-type=\"subsection\"><title>The remaining question of immune response</title><p>The natural triggers of immunity from the viral vectors remain problematic in experimental therapies.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> In our center, in an investigator-sponsored (Phase 1) trial of a subretinally injected AAV gene therapy for choroideremia, one subject experienced a serious loss of vision.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> Unfortunately, the disease progressed without apparent benefit in all patients. We now question why this serious adverse event occurred? Could it have been prevented by an alternative vector design? Why was steroid (given perioperatively) not sufficient to manage inflammation? In our trial, all subjects were treated with high-dose oral steroid (prednisone 1 mg/kg/day 3 days prior to surgery, continuing for 21 days). The steroid was given as a prophylactic to suppress the potential risk to the eye of the subretinal injection. Steroid has both local and systemic anti-inflammatory and immunosuppressive effects; briefly, prednisone inhibits neutrophil migration.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>]</sup> It decreases mononuclear phagocyte chemotaxis and the production of interleukins and TNF-α. It causes the redistribution of CD4+ and CD8+ T-lymphocytes, and inhibits T-lymphocyte activation, proliferation, and lymphokine production. At high doses, it inhibits immunoglobulin production by B-lymphocytes.</p><p>While prophylactic steroids may aid in regulating players of adaptive immunity such as T-lymphocytes and B-lymphocytes, these cells would not occupy retinal tissue unless a severe breach had occurred in the blood–retinal barrier. For the purposes of retinal gene therapy, local innate immune factors are the primary influencers of an inflammatory response and are of therapeutic importance. To illustrate, our choroideremia gene therapy trial utilized a subretinal injection procedure to introduce an AAV vector into the retina. Retinal detachment induces local TNF-α secretion which in turn may promote autophagy of photoreceptors by resident microglia.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>]</sup> Even without considering the immunological effects of the viral vector or the diseased microenvironment of the patients' retinas, the gene therapy procedure itself calls for the incorporation of a local immune regulator to prevent such adverse events.</p><p>Both a diseased microenvironment and retinal detachment from the AAV injection “prime” the local immune state to recognize and respond to viral vectors more effectively. AAV viruses are DNA-based and are thus recognized by toll-like receptor 9 (TLR9). TLR9 stimulation in retinal pigment epithelial cells (RPE) by CpG-DNA induces secretion of the pro-inflammatory cytokine IL-8, which represents the initiation of an inflammatory cascade.<sup>[<xref rid=\"B12\" ref-type=\"bibr\">12</xref>]</sup> Preventing this TLR9-initiated inflammatory cascade is essential when designing an immunity regulator in AAV-based retinal gene therapy. Our team is investigating a modified AAV vector that blocks the dimerization and immune signaling of TLR9. Should this vector be effective in preventing RPE from releasing pro-inflammatory cytokines in response to AAV treatment, we will move from cell culture models to animal models to confirm safety of the vector before attempting a second choroideremia gene therapy clinical trial.</p><p>Experimental vector gene therapies for hereditary ocular disorders will continue to improve, gain popular exposure, and march toward regulatory approval. A better understanding of the impacts of innate immunity in the retina will improve the safety profile of these therapies and improve the probability of positive outcomes for patients.</p></sec></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>Alberta Innovates Health Solutions, Canadian Institutes of Health Research, Fighting Blindness Canada.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Brooks S, Benjaminy S, Bubela T. Participant perspectives on phase I/II ocular gene therapy trial (NCT02077361). <italic>Ophthalmic Genet</italic> 2019;40:276–281.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">Anguela X, High K. Entering the Modern Era of Gene Therapy. <italic>Ann Rev Med</italic> 2019;70:273–288.</mixed-citation></ref><ref id=\"B3\"><label>3</label><mixed-citation publication-type=\"other\">Li Q, Miller R, Han P, Pang J, Dinculescu A, Chiodo V, et al. Intraocular route of AAV2 vector administration defines humoral immune response and therapeutic potential. <italic>Mol Vis</italic> 2008;14:1760–1769.</mixed-citation></ref><ref id=\"B4\"><label>4</label><mixed-citation publication-type=\"other\">Woodard K, Liang K, Bennett W, Samulski R. Heparan sulfate binding promotes accumulation of intravitreally delivered adeno-associated viral vectors at the retina for enhanced transduction but weakly influences tropism. <italic>J Virol</italic> 2016;90:9878–9888.</mixed-citation></ref><ref id=\"B5\"><label>5</label><mixed-citation publication-type=\"other\">Kay C, Ryals R, Aslanidi G, Min S, Ruan Q, Sun J, et al. Targeting photoreceptors via intravitreal delivery using novel, capsid-mutated AAV vectors. <italic>PLOS ONE</italic> 2013;8:e62097.</mixed-citation></ref><ref id=\"B6\"><label>6</label><mixed-citation publication-type=\"other\">Reichel F, Peters T, Wilhelm B, Biel M, Ueffing M, Wissinger B, et al. Humoral immune response after intravitreal but not after subretinal AAV8 in primates and patients. <italic>Invest Ophthalmol Vis Sci </italic>2018;59:1910–1915.</mixed-citation></ref><ref id=\"B7\"><label>7</label><mixed-citation publication-type=\"other\">Nakazawa T, Hisatomi T, Nakazawa C, Kosuke N, Marayuma K, She H, et al. Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis. <italic>PNAS</italic> 2007;104:2425–2430.</mixed-citation></ref><ref id=\"B8\"><label>8</label><mixed-citation publication-type=\"other\">Xiong W, Wu DM, Xue Y, Wang SK, Chung MJ, X Ji, et al. AAV cis-regulatory sequences are correlated with ocular toxicity. <italic>PNAS </italic>2019;116:5785–5794.</mixed-citation></ref><ref id=\"B9\"><label>9</label><mixed-citation publication-type=\"other\">Dimopoulos IS, Hoang SC, Radziwon A, Binczyk NM, Seabra MC, MacLaren RE, et al. Two-year results after AAV2-mediated gene therapy for choroideremia: the alberta experience. <italic>Am J Ophthalmol</italic> 2018;193:130–142.</mixed-citation></ref><ref id=\"B10\"><label>10</label><mixed-citation publication-type=\"other\">Nussenblatt R, Whitcup S. Philosophy, goals, and approaches to medical therapy. In: Uveitis. 4th Edition. Maryland Heights, USA: Mosby; 2010.</mixed-citation></ref><ref id=\"B11\"><label>11</label><mixed-citation publication-type=\"other\">Xie J, Zhu R, Peng Y, Gao W, Du J, Zhao L, et al. Tumour necrosis factor-alpha regulates photoreceptor cell autophagy after retinal detachment. <italic>Sci Rep </italic>2017;7:17108.</mixed-citation></ref><ref id=\"B12\"><label>12</label><mixed-citation publication-type=\"other\">Ebihara N, Chen L, Tokura T, Ushio H, Iwatsu M, Murakami A. Distinct functions between Toll-like Receptors 3 and 9 in retinal pigment epithelial cells. <italic>Ophthalmic Res </italic>2006;39:155–163.</mixed-citation></ref></ref-list></back></article>\n"
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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864061</article-id><article-id pub-id-type=\"pmc\">PMC7431729</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7449</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Influence of Intraocular Lens Asphericity and Blue Light Filtering on Visual Outcome, Contrast Sensitivity, and Aberrometry after Uneventful Cataract Extraction</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Tzamalis</surname><given-names>Argyrios</given-names></name><degrees>MD, PhD, MA, FEBO</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kynigopoulos</surname><given-names>Myron</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Pallas</surname><given-names>Grigoris</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"I2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Tsinopoulos</surname><given-names>Ioannis</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ziakas</surname><given-names>Nikolaos</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"I1\">\n<sup>1</sup>\n</xref></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>2nd Department of Ophthalmology, Aristotle University of Thessaloniki, Papageorgiou General Hospital, Thessaloniki, Greece</aff><aff id=\"I2\">\n<sup>2</sup>Department of Ophthalmology, Clinic Pallas, Olten, Switzerland</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Argyrios Tzamalis, MD, PhD, MA, FEBO. 2nd Department\nof Ophthalmology, Aristotle University of Thessaloniki,\nPapageorgiou General Hospital, Thessaloniki, Greece.\nE-mail: argyriostzamalis@yahoo.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>308</fpage><lpage>317</lpage><history><date date-type=\"received\"><day>19</day><month>1</month><year>2019</year></date><date date-type=\"accepted\"><day>31</day><month>12</month><year>2019</year></date></history><permissions><copyright-statement>Copyright © 2020 Tzamalis et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><sec><title>Purpose</title><p> To evaluate the effect of asphericity and blue light filter (BLF) of three different intraocular lenses (IOLs) on the visual performance, second- and third-order aberrations (defocus, coma, trefoil), and contrast sensitivity after uneventful cataract surgery.</p></sec><sec><title>Methods</title><p>One hundred and twenty eyes of 60 patients with clinically significant cataract were randomly assigned to receive one of the three IOL types: Bioline Yellow Accurate (aspheric, with BLF, i-medical, Germany), BioAcryl 60125 (spherical, without BLF, Biotech, France), and H65C/N (aspheric, without BLF, PhysIOL, Belgium). Each IOL was implanted in 40 eyes. Complete ophthalmologic examination, functional acuity contrast testing and wavefront analysis were performed 60 days postoperatively.</p></sec><sec><title>Results</title><p> The mean postoperative best-corrected visual acuity (BCVA) was 0.95 <inline-formula><mml:math id=\"M1\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.08, not differing statistically among the IOL groups (<italic>P</italic> = 0.83). Mean defocus and coma values did not yield any statistically significant difference through the IOL groups varying from –0.784 to –0.614 μm and 0.129 to 0.198 μm (<italic>P</italic> = 0.79 and 0.34, respectively). Bioline Yellow Accurate IOL presented less trefoil aberrations, 0.108 <inline-formula><mml:math id=\"M2\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.05 μm, compared to the other two IOL types (BioAcryl [0.206 <inline-formula><mml:math id=\"M3\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.19 μm] and Physiol [0.193 <inline-formula><mml:math id=\"M4\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.17 μm], <italic>P</italic>\n<inline-formula><mml:math id=\"M5\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05). Contrast sensitivity values did not differ among the groups under all lighting conditions. Bioline Yellow IOL showed a statistically higher loss of contrast sensitivity (between mesopic and mesopic with glare conditions) compared to the BioAcryl and PhysIOL in 12 and 3 cpd spatial frequencies, respectively (<italic>P</italic>\n<inline-formula><mml:math id=\"M6\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05).</p></sec><sec><title>Conclusion</title><p>Bioline Yellow IOL indicated lower contrast sensitivity under mesopic conditions when glare was applied but resulted in less trefoil aberrations after uneventful cataract surgery. No further differences were noted in postoperative visual performance among three IOL groups.</p></sec></abstract><kwd-group><kwd>Aberrometry</kwd><kwd> Asphericity</kwd><kwd> Blue-light Filtering</kwd><kwd> Contrast Sensitivity</kwd><kwd> Intraocular Lens</kwd></kwd-group><counts><fig-count count=\"3\"/><table-count count=\"4\"/><ref-count count=\"32\"/><page-count count=\"10\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> INTRODUCTION</title><p>Modern cataract surgery has recently evolved into a refractive procedure aimed at improving visual quality in addition to increasing the visual acuity. Therefore, it is routinely combined with the implantation of intraocular lenses (IOLs) of various materials and designs.</p><p>Since the initial introduction of IOLs, there has been great debate over the importance of light filtration.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> Ultraviolet (UV) light-filtering lenses have been the dominant IOLs used in the modern era since growing evidence indicated that UV light could result in photic retinopathy and other retinal pathologies.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup> There has recently though been support for increasing the range of light absorption by IOLs. The rationale was that UV light-filtering IOLs cannot offer protection to the retina from phototoxic damage induced by the high-energy, short-wavelength blue light (400–480 nm), which is considered to contribute to the pathogenesis of age-related macular degeneration (AMD).<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> In response to this potential damage, blue light-filtering (BLF) IOLs were introduced in 1996 and have been thereafter widely used, especially in cataract surgery candidates with signs of AMD as a possible measure of preventing associated retinal pathology.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B9\" ref-type=\"bibr\">9</xref>,<xref rid=\"B10\" ref-type=\"bibr\">10</xref>,<xref rid=\"B11\" ref-type=\"bibr\">11</xref>,<xref rid=\"B12\" ref-type=\"bibr\">12</xref>]</sup> The yellow tint of BLF IOLs replicates the spectral transmission properties of the aged human crystalline lens in a much closer manner than the UV light-filtering IOLs do.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>]</sup>\n</p><p>While the possible visual benefits of BLF IOLs are still under debate, controversy has been raised whether a yellow-tinted IOL could modify the visual performance of patients, specifically regarding the postoperative best-corrected visual acuity (BCVA), contrast sensitivity, color vision, and glare.</p><p>In addition to BLF, another recently commercialized property of IOLs that has become increasingly popular is asphericity. Spherical aberration has a strong influence on image quality.<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup> It is well-established that conventional-spherical IOLs degrade image quality by increasing the spherical higher-order aberrations (HOAs), and several authors have published studies indicating that aspheric IOLs may improve retinal image quality and mesopic contrast sensitivity at low spatial frequencies.<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>,<xref rid=\"B16\" ref-type=\"bibr\">16</xref>,<xref rid=\"B17\" ref-type=\"bibr\">17</xref>,<xref rid=\"B18\" ref-type=\"bibr\">18</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>,<xref rid=\"B20\" ref-type=\"bibr\">20</xref>,<xref rid=\"B21\" ref-type=\"bibr\">21</xref>]</sup>\n</p><p>However, the combination of BLF and asphericity in IOLs has not been clearly investigated regarding their effect on contrast sensitivity, aberrometry, and quality of vision. The purpose of this prospective randomized study was to evaluate the effect of blue light-filtering and aphericity of IOL on visual quality by comparing the three different IOL types: one aspherical IOL with BLF, one aspherical IOL without BLF, and one spherical IOL without BLF.</p></sec><sec sec-type=\"section\"><title> METHODS</title><p>This prospective, randomized clinical study comprised patients who underwent bilateral cataract surgery for visually significant cataract. Sixty patients were randomly assigned to receive one of the three IOL types. Group A received Bioline Yellow Accurate IOL (aspheric with BLF, i-medical, Germany), Group B had H65C/N IOL (aspheric without BLF, PhysIOL, Belgium), and Group C had BioAcryl60125 IOL (spherical, without BLF). Each IOL was implanted in 40 eyes of 20 randomly selected patients. All patients were recruited from the outpatient anterior segment unit of the Clinic Pallas Ophthalmology Department in Olten, Switzerland. The study was performed in adherence with the Declaration of Helsinki for research involving human subjects after approval was obtained from the Institutional Review Boards of Pallas Clinic.</p><p>Patients with bilateral cataract with visual disturbance and no history of color vision deficiency were eligible for inclusion in the study. The exclusion criteria were ocular diseases such as corneal opacity or irregularity, astigmatism greater than 2.5 D, dry eye syndrome, inadequate visualization of the fundus, amblyopia, anisometropia, calculated IOL power less than 10.0 diopters (D) or more than 30.0 D, surgical complications, IOL tilt, previous or current use of medications known to cause color vision deficiencies, and incomplete follow-up. Also, patients with a history of uveitis and current intraocular inflammation, uncontrollable glaucoma, proliferative diabetic retinopathy, or retinal detachment were excluded from the study.</p><p>One experienced surgeon (GP) performed all surgeries with standard small incision phacoemulsification and IOL implantation in the capsular bag. The time between first eye surgery and second eye surgery was six–eight days in all cases. All eyes were targeted for emmetropia. Table 1 shows the characteristics of the IOLs implanted during the study period. All patients were given combined antibiotic–steroid eye drops for four weeks postoperatively. Patients were examined before surgery and one, seven, and one to three months postoperatively. At all visits, the best corrected-distance visual acuity (BCDVA) and uncorrected distance visual acuity (UDVA) were measured. Contrast sensitivity assessment and aberrometry by means of wavefront analysis were evaluated at the baseline (preoperatively) and last follow-up visit which was performed one to three months postoperatively.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Main characteristics and special features of the intraocular lenses used in the study</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"4\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Bioline Yellow</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>H65C/N</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Bioacryl</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Optic material</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Hydrophilic acrylic copolymer 26 % water</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hydrophilic acrylic copolymer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hydrophilic acrylic</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Optic design</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Biconvex aspherical</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Biconvex aspherical</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Spherical</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Optic diameter (mm)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">6.0</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Length (mm)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.5</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Design</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">360º square edge</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">360º reinforced edge design \"pco-barrier\"</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">360º square edge</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Haptic angulation (º)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">5</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Ultraviolet filter</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">With BLF</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Without BLF</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Without BLF</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>A-constant</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">118.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">118.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">118.0</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Refractive index</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.465</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.46</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1.462</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Estimated anterior chamber depth (mm)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.98</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.99</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4.96</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">BLF, blue light filtering</td></tr></tbody></table></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Main demographics and clinical characteristics of the study participants</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"6\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Bioline Yellow</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>H65C/N</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Bioacryl 60125</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold/>\n<italic><bold>P</bold></italic>\n<bold>-value<inline-formula><mml:math id=\"M7\"><mml:mo>†</mml:mo></mml:math></inline-formula></bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Total</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Gender</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21F/19M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22/18M</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19F/21M</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.79</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">62F/58M</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Age (years)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">70.3 <inline-formula><mml:math id=\"M8\"><mml:mo>±</mml:mo></mml:math></inline-formula> 8.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">74.6 <inline-formula><mml:math id=\"M9\"><mml:mo>±</mml:mo></mml:math></inline-formula> 8.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">72.5 <inline-formula><mml:math id=\"M10\"><mml:mo>±</mml:mo></mml:math></inline-formula> 6.4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.53</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">72.4 <inline-formula><mml:math id=\"M11\"><mml:mo>±</mml:mo></mml:math></inline-formula> 9.5</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Axial length (mm)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24.1 <inline-formula><mml:math id=\"M12\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23.6 <inline-formula><mml:math id=\"M13\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23.7 <inline-formula><mml:math id=\"M14\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.59</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">23.8 <inline-formula><mml:math id=\"M15\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.1</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Preoperative SE</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.19 <inline-formula><mml:math id=\"M16\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.45 <inline-formula><mml:math id=\"M17\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.39 <inline-formula><mml:math id=\"M18\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.68</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.34 <inline-formula><mml:math id=\"M19\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.5</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Postoperative SE</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–0.32 <inline-formula><mml:math id=\"M20\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–0.34 <inline-formula><mml:math id=\"M21\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–0.31 <inline-formula><mml:math id=\"M22\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.41</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–0.32 <inline-formula><mml:math id=\"M23\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.4</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>IOL power</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21.1 <inline-formula><mml:math id=\"M24\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22.5 <inline-formula><mml:math id=\"M25\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22.2 <inline-formula><mml:math id=\"M26\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">21.8 <inline-formula><mml:math id=\"M27\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.5</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>BCVA preop, LogMAR (Decimal)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.38 (0.42 <inline-formula><mml:math id=\"M28\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.16)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.29 (0.53 <inline-formula><mml:math id=\"M29\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.12)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.32 (0.48 <inline-formula><mml:math id=\"M30\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.18)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.59</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.33 (0.47 <inline-formula><mml:math id=\"M31\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.17)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>BCVA postop, LogMAR (Decimal)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.018 (0.96 <inline-formula><mml:math id=\"M32\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.07)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.031 (0.93 <inline-formula><mml:math id=\"M33\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.09)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.022 (0.95 <inline-formula><mml:math id=\"M34\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.08)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.59</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.023 (0.948 <inline-formula><mml:math id=\"M35\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.08)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>CR preop</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.65 <inline-formula><mml:math id=\"M36\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.64 <inline-formula><mml:math id=\"M37\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.66 <inline-formula><mml:math id=\"M38\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.87</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.65 <inline-formula><mml:math id=\"M39\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.3</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>CR postop</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.66 <inline-formula><mml:math id=\"M40\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.66 <inline-formula><mml:math id=\"M41\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.69 <inline-formula><mml:math id=\"M42\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.93</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.67 <inline-formula><mml:math id=\"M43\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.3</td></tr><tr><td align=\"left\" colspan=\"6\" rowspan=\"1\">\n<inline-formula><mml:math id=\"M44\"><mml:mo>†</mml:mo></mml:math></inline-formula>\n<italic>P</italic>-value (significance level) was calculated by means of chi-square test, ANOVA-test and Kruskal–Wallis test.\nSE, spherical equivalent; BCVA, best-corrected visual acuity; CR, corneal radius LogMAR, logarithm of the minimum angle of resolution</td></tr></tbody></table></table-wrap><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><p>Comparisons of second- and third-order aberrations among the three IOL groups</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"4\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>IOL (Group)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Defocus (Z200)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Coma (Z311, Z310)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Trefoil (Z331, Z330)</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Bioline Yellow (Group1)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–0.7842 <inline-formula><mml:math id=\"M45\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.9915</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1288 <inline-formula><mml:math id=\"M46\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.1075</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1082 <inline-formula><mml:math id=\"M47\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.049</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>PhysIOL H65C/N (Group2)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–0.6557 <inline-formula><mml:math id=\"M48\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.7235</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1573 <inline-formula><mml:math id=\"M49\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.1186</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1929 <inline-formula><mml:math id=\"M50\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.1736</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>BioAcryl 60125 (Group3)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–0.6136 <inline-formula><mml:math id=\"M51\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.6239</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.1984 <inline-formula><mml:math id=\"M52\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.1599</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.2058 <inline-formula><mml:math id=\"M53\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.1852</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Mean</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–0.69 <inline-formula><mml:math id=\"M54\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.72</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.16 <inline-formula><mml:math id=\"M55\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.16 <inline-formula><mml:math id=\"M56\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.15</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Comparison between groups (</bold>\n<italic><bold>P</bold></italic>\n<bold>-value)</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Group1–Group2</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.52</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.11</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.03</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Group1–Group3</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.59</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.43</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>0.042</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Group2–Group3</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.36</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.82</td></tr></tbody></table></table-wrap><table-wrap id=\"T4\" orientation=\"portrait\" position=\"float\"><label>Table 4</label><caption><p>Mean loss of contrast sensitivity, expressed in logarithmic units, from photopic to mesopic lighting conditions (LC) and under glare conditions in mesopic LC.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"11\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" colspan=\"5\" rowspan=\"1\"><bold>Loss Photopic to Mesopic LC</bold>\n</td><td align=\"center\" colspan=\"5\" rowspan=\"1\"><bold>Loss Mesopic to Mesopic with glare LC</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Spatial Frequency (cpd)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>1.5</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>3</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>6</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>12</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>18</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>1.5</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>3</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>6</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>12</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>18</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>IOL1</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.051</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.105</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.227</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.406</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.569</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.096</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.23</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.233</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.452</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.256</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>IOL2</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.021</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.129</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.185</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.400</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.358</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.165</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.121</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.154</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.217</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.331</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>IOL3</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.030</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.140</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.221</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.521</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.433</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.167</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.139</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.229</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.099</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.206</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold/>\n<italic><bold>P</bold></italic>\n<bold>(ANOVA)<inline-formula><mml:math id=\"M57\"><mml:mo>†</mml:mo></mml:math></inline-formula></bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.69</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.62</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.69</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.69</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.27</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.75</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.02</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.64</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold/>\n<italic><bold>P</bold></italic>\n<bold>(</bold>\n<italic><bold>t</bold></italic>\n<bold>-test)1–2*</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.43</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.59</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.77</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.25</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.13</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.01</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.71</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.27</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.84</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold/>\n<italic><bold>P</bold></italic>\n<bold>(</bold>\n<italic><bold>t</bold></italic>\n<bold>-test)1–3*</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.53</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.31</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.34</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.41</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.84</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.04</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.81</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold/>\n<italic><bold>P</bold></italic>\n<bold>(</bold>\n<italic><bold>t</bold></italic>\n<bold>-test)2–3*</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.92</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.62</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.75</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.98</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.77</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.85</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.30</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.67</td></tr><tr><td align=\"left\" colspan=\"11\" rowspan=\"1\">IOL1, Bioline Yellow Accurate; IOL2, Physiol H65C/N; IOL3, BioAcryl60125; cpd, cycles per degree\n<inline-formula><mml:math id=\"M58\"><mml:mo>†</mml:mo></mml:math></inline-formula>\n<italic>P</italic>-value comparing all three IOL groups with one-way analysis of variance; *<italic>P</italic>-value comparing IOL groups in couples with Student's <italic>t</italic>-test</td></tr></tbody></table></table-wrap><p>Visual acuity was measured using Snellen chart under scotopic conditions (target luminance 1.5 candelas [cd]/m2). Contrast sensitivity was assessed using Functional Acuity Contrast Testing (FACT-Optec6500, Stereo Optical Inc., USA) with spectacle correction under photopic conditions (target luminance value 85 candelas [cd]/m2) and mesopic conditions (target luminance value 3 cd/m<inline-formula><mml:math id=\"M59\"><mml:msup><mml:mrow/><mml:mn>2</mml:mn></mml:msup></mml:math></inline-formula>) with and without glare. Lighting conditions were controlled with a luxometer (Gossen-Starlite). The log base 10 contrast sensitivity values were used to construct a graph for each spatial frequency tested and then presented using the original test scale.</p><p>A Zywave Hartmann-Shack aberrometer (Bausch & Lomb, Germany) was used for all aberrometry measurements. Zywave was used to assess and compensate for the refractive errors, and, eye fogging system was acquired before each wavefront measurement to avoid patient accommodation. Before use, the aberrometer was calibrated by an experienced Bausch & Lomb technician to ensure the accuracy. Five measurements were performed by a single experienced technician to avoid interobserver variability in the results; of these, two measurements with higher deviations from the mean were excluded and the three best measurements were averaged and used for statistical analyses. Patients were instructed to blink between measurements, and acquisition was obtained after a blink to ensure higher quality results by limiting tear film disruption. All results were exported as raw data so that individual Zernike terms could be analyzed independently. The details of the Zernike coefficients up to the third order were recorded and used for the statistical analysis. Zywave measurements were obtained without any pharmacologic mydriasis and dark adaptation. Nonetheless, all measurements were made in certain mesopic lighting conditions and it was confirmed that pupillary diameter was at least 6 mm in every case. Total, corneal, and internal components for each of the high-order aberrations were obtained and used for the analysis.</p><p>Statistical analysis was performed using the SPSS (version 17.0 for Windows, SPSS, Inc. Chicago, IL) and MedCalc statistical software (version 9.3.0.0, MariaKerke, Belgium). Normality was checked using the Kolmogorov–Smirnov test. Since data were not normally distributed in all cases, both parametric and nonparametric methods were used. For normally distributed data, Pearson correlation was used to evaluate the association between two continuous variables and the one-way analysis of variance (ANOVA) was applied to evaluate the influence of a qualitative factor on another continuous variable. The association of not normally distributed data was assessed using Rank correlation calculating Spearman's coefficient rho. When parametric analysis was possible, the Student's <italic>t</italic>-test was used to compare the outcomes between two IOL groups. Categorical variables were compared using the Fisher's exact test. Nonparametric Kruskal–Wallis and Mann–Whitney tests were also used to examine the associations between categorical variables and continuous or ordered outcomes. A <italic>P</italic>-value of <inline-formula><mml:math id=\"M60\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05 was defined for all statistical tests as statistically significant.</p></sec><sec sec-type=\"section\"><title> RESULTS </title><p>A total number of 120 eyes of 60 patients (mean age, 72.4 <inline-formula><mml:math id=\"M61\"><mml:mo>±</mml:mo></mml:math></inline-formula> 9.5 years) who underwent uneventful bilateral cataract surgery were found eligible and were finally enrolled in the statistical analysis. All eyes were divided into one of the three groups, based on the type of IOL they received. The main demographic and clinical characteristics of each group are demonstrated in Table 2.</p><p>There were no statistically significant differences among the study groups in preoperative clinical and refractive values. Preoperative total and internal components of aberrometry showed great deviations between cases, as patients with various degrees and types of cataracts were included. However, the preoperative corneal component of coma, defocus, and trefoil did not have any statistically significant difference among the three groups (<italic>P</italic>\n<inline-formula><mml:math id=\"M62\"><mml:mo>></mml:mo></mml:math></inline-formula> 0.05). The mean Snellen postoperative BCVA was 0.95 <inline-formula><mml:math id=\"M63\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.08 (0.023 LogMAR) with a mean postoperative spherical equivalent of –0.32 <inline-formula><mml:math id=\"M64\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.13D; not differing statistically between the IOL groups (Table 2; <italic>P </italic>\n<inline-formula><mml:math id=\"M65\"><mml:mo>></mml:mo></mml:math></inline-formula> 0.05). The mean\nLogMAR uncorrected VA (UCVA) increased from 0.58 <inline-formula><mml:math id=\"M66\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.25 (Bioline Yellow), 0.54 <inline-formula><mml:math id=\"M67\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.23 (H65C/N), and 0.55 <inline-formula><mml:math id=\"M68\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.26 at screening to 0.23 <inline-formula><mml:math id=\"M69\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.12, 0.22 <inline-formula><mml:math id=\"M70\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.11, and 0.24 <inline-formula><mml:math id=\"M71\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.11, respectively, at postoperative follow-up. There was no statistically significant difference in the postoperative UCVA among the IOL groups. Postoperative values were recorded at a mean time of 63.2 <inline-formula><mml:math id=\"M72\"><mml:mo>±</mml:mo></mml:math></inline-formula>11.7 days after uneventful cataract surgery varying between 38 and 87 days, with no significant difference among the groups in the duration of follow-up (ANOVA, <italic>P</italic> = 0.21). Table 2 demonstrates the preoperative and postoperative refraction data in more details.</p><p>Mean defocus and coma values did not yield any statistically significant difference among IOL groups varying from –0.784 to –0.614 and 0.129 to 0.198, respectively (Table 3). Bioline Yellow Accurate presented less trefoil aberrations, 0.108 <inline-formula><mml:math id=\"M73\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.05 μm compared to the other two IOL types (<italic>P</italic>\n<inline-formula><mml:math id=\"M74\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05). Table 3 illustrates the second- and third-order aberrations as well as their intergroup comparisons.</p><p>There was no statistically significant difference among the three IOL groups in contrast sensitivity at any spatial frequency under all three lighting conditions. Figure 1 (photopic 85 cd/m2), Figure 2 (mesopic 3 cd/m2), and Figure 3 (mesopic with glare) depict postoperative contrast sensitivity for all IOL groups. In a separate analysis, Bioline Yellow was found to have a statistically lower contrast sensitivity under glare conditions compared to the BioAcryl and PhysIOL in 12 and 3 cpd spatial frequencies, respectively (<italic>P</italic>\n<inline-formula><mml:math id=\"M75\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05). Table 4 compares the postoperative contrast sensitivity among the IOL groups.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Postoperative contrast sensitivity (log) under photopic conditions (85 cd/m2) in various spatial frequencies for the IOLs included in the study [IOL1 = Bioline Yellow Accurate (i-medical, Germany), IOL2 = BioAcryl 60125 (Biotech, France), IOL3 = H65C/N (PhysIOL, Belgium)].</p></caption><graphic xlink:href=\"jovr-15-308-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Postoperative contrast sensitivity (log) under mesopic conditions (3 cd/m2) in various spatial frequencies for the IOLs included in the study [IOL1 = Bioline Yellow Accurate (i-medical, Germany), IOL2 = BioAcryl 60125 (Biotech, France), IOL3 = H65C/N (PhysIOL, Belgium)].</p></caption><graphic xlink:href=\"jovr-15-308-g002\"/></fig><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>Postoperative contrast sensitivity (log) under mesopic with glare conditions (3 cd/m2 with glare) in various spatial frequencies for the IOLs included in the study [IOL1 = Bioline Yellow Accurate (i-medical, Germany), IOL2 = BioAcryl 60125 (Biotech, France), IOL3 = H65C/N (PhysIOL, Belgium)].</p></caption><graphic xlink:href=\"jovr-15-308-g003\"/></fig></sec><sec sec-type=\"section\"><title> DISCUSSION</title><p>Modern cataract surgery with the implementation of specially designed IOLs has developed tremendously over the past decades, attempting to meet patients' expectations for optimal visual outcomes.<sup>[<xref rid=\"B11\" ref-type=\"bibr\">11</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>,<xref rid=\"B21\" ref-type=\"bibr\">21</xref>]</sup> Contemporary diagnostic tools have extended our knowledge on the impact of HOAs and contrast sensitivity on the quality of vision. Therefore, in order to achieve the best outcome after phacoemulsification, the IOL implantation should result in minimal aberrations and high-contrast sensitivity.</p><p>IOLs with aspheric optics, designed to optimize postoperative spherical aberration and implants with BLF as a possible measure of preventing associated retinal pathology have gained great popularity. However, there is still great controversy on their potential benefit and the effect of these features on the postoperative visual performance, specifically regarding the ultimate BCVA, contrast sensitivity, color vision, and postoperative aberrations.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>,<xref rid=\"B4\" ref-type=\"bibr\">4</xref>,<xref rid=\"B5\" ref-type=\"bibr\">5</xref>][<xref rid=\"B9\" ref-type=\"bibr\">9</xref>][<xref rid=\"B10\" ref-type=\"bibr\">10</xref>][<xref rid=\"B11\" ref-type=\"bibr\">11</xref>][<xref rid=\"B12\" ref-type=\"bibr\">12</xref>][<xref rid=\"B15\" ref-type=\"bibr\">15</xref>,<xref rid=\"B16\" ref-type=\"bibr\">16</xref>,<xref rid=\"B17\" ref-type=\"bibr\">17</xref>,<xref rid=\"B18\" ref-type=\"bibr\">18</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>,<xref rid=\"B20\" ref-type=\"bibr\">20</xref>,<xref rid=\"B21\" ref-type=\"bibr\">21</xref>]</sup>\n</p><p>The present prospective randomized study attempted to investigate the effect of BLF and aspherical IOL design on the final visual outcome. Therefore, we compared the visual performance after the implantation of three different IOLs; one aspheric, with BLF; one aspheric, without BLF; and one spherical, without BLF. Our results showed that BCVA did not differ statistically significantly among the IOL groups. These results regarding the effect of BLF in postoperative BCVA are in concordance with a recent Cochrane Database Systematic Review which demonstrated, with moderate certainty, that the presence of BLF in IOLs had no clinically meaningful effect on short-term BCVA.<sup>[<xref rid=\"B22\" ref-type=\"bibr\">22</xref>]</sup>\n</p><p>Although no significant difference was found in our study among the different IOL groups in the postoperative BCVA, the group of patients implanted with an aspheric IOL with BLF indicated fewer trefoil aberrations when compared to the other IOL groups included in the study. Notably, the preoperative corneal component of HOAs did not differ significantly among the three IOL groups. Therefore, the lower trefoil measurements shown in this group could be attributed to the internal components, mainly the IOL itself. A postoperative IOL tilt could also be a predisposing factor for increased aberrations.</p><p>Blue-light filtering is an add-on feature of IOLs, considered to offer an extra retinal protection against AMD, although this has not been fully proven so far.<sup>[<xref rid=\"B23\" ref-type=\"bibr\">23</xref>]</sup> IOLs with BLF are supposed to reduce longitudinal chromatic aberrations. Theoretically, such a reduction should not affect spherical aberrations. However, in our study, the yellow-tinted IOL achieved better results in postoperative trefoil when compared not only to the spherical IOL but also to the aspheric one without BLF. It should be noticed that the two aspheric IOLs used in this study had minimal differences in terms of optical design and material being produced by different manufacturers. This fact could also have some impact on the results reported. To the best of our knowledge, there are no published studies evaluating the effect of BLF on spherical HOAs by comparing the same IOL types.</p><p>As far as postoperative contrast sensitivity is concerned, no significant difference was found among IOLs in any spatial frequency under photopic, mesopic, and mesopic with glare-lighting conditions. However, the yellow-tinted aspheric IOL was found to have a statistically higher loss of contrast sensitivity under glare conditions compared to the non-tinted IOLs at some spatial frequencies.</p><p>In recent years, aspheric IOLs have gained increasing popularity among surgeons due to their theoretical advantage of being able to compensate for the spherical aberration of the human cornea, with the aim of restoring the optical performance of the eye.<sup>[<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup> Most studies performed on this task have confirmed this theory reporting that aspheric IOLs implanted have significantly reduced the overall spherical aberrations, hence improving optical performance in certain cases.<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>,<xref rid=\"B17\" ref-type=\"bibr\">17</xref>,<xref rid=\"B18\" ref-type=\"bibr\">18</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>,<xref rid=\"B20\" ref-type=\"bibr\">20</xref>,<xref rid=\"B21\" ref-type=\"bibr\">21</xref>,<xref rid=\"B22\" ref-type=\"bibr\">22</xref>,<xref rid=\"B23\" ref-type=\"bibr\">23</xref>,<xref rid=\"B24\" ref-type=\"bibr\">24</xref>]</sup>\n</p><p>Comparing the aspheric Tecnis ZA9003 IOL with the spherical AcrySof SA60AT IOL (Alcon, Inc.), Kim et al<sup>[<xref rid=\"B25\" ref-type=\"bibr\">25</xref>]</sup> reported a significant improvement in contrast sensitivity under mesopic and photopic conditions with the aspheric IOL; the authors reported that the mean spherical aberration was significantly higher in eyes implanted with the spherical IOL, although total higher order aberrations did not differ significantly between the results of two further prospective randomized studies performed by Rocha et al and Caporrosi et al, who concluded that eyes implanted with aspheric IOLs had less aberrations and performed better under mesopic condition compared to spherical IOLs.<sup>[<xref rid=\"B18\" ref-type=\"bibr\">18</xref>,<xref rid=\"B26\" ref-type=\"bibr\">26</xref>]</sup>\n</p><p>On the contrary, several researchers have reported no statistically significant differences in visual acuity and contrast sensitivity between spherical IOLs and aspheric IOLs.<sup>[<xref rid=\"B27\" ref-type=\"bibr\">27</xref>,<xref rid=\"B28\" ref-type=\"bibr\">28</xref>,<xref rid=\"B29\" ref-type=\"bibr\">29</xref>]</sup> We compared in our study the outcome of the three different IOLs, two aspheric and one spherical and found no statistically significant difference in the second- and third-order aberrations other than trefoil aberrations that was lower in the eyes implanted with aspheric IOL with a BLF. Surprisingly, no difference was noted between the two non-tinted IOL groups, despite one of them having an aspherical design. One may hypothesize that BLF added on yellow-tinted IOLs could reduce some spherical aberrations along with the longitudinal chromatic ones; however, this theory needs to be examined by further prospective randomized studies with larger population sizes to compare aberrations between IOLs of identical design and material.</p><p>In the past decade, several manufacturers and distributors have promoted commercially available IOLs with BLF properties. Theoretically, BLF IOLs may induce a reduction in mesopic and scotopic visual performance attributed to the Purkinje shift, where differing peaks of spectral sensitivity for scotopic and photopic vision are identified.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup> Violet and blue lights are much more important for vision in dim-light environments than in bright-light environments, providing 45% of rod-mediated aphakic scotopic sensitivity but only 7% of photopic sensitivity for an iso-illuminance light source.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> However, the results reported in the literature are controversial regarding postoperative contrast sensitivity after the BLF-IOL implantation and do not indicate a significant decrease in the mesopic and scotopic visual function.</p><p>Kara-Junior et al<sup>[<xref rid=\"B30\" ref-type=\"bibr\">30</xref>]</sup> investigated the long-term possible side effects after implantation of an IOL with a BLF. The authors found no significant differences in color perception, scotopic contrast sensitivity, or photopic contrast sensitivity between the BLF IOL and the IOL with a UV-light filter only. In another study, Greenstein et al<sup>[<xref rid=\"B31\" ref-type=\"bibr\">31</xref>]</sup> investigated contrast sensitivity in nine patients implanted with a BLF IOL (AcrySof SN60AT) in one eye and a UV-only filtering IOL (AcrySof SA60AT) in the fellow eye. In addition, they compared the results with those obtained in nine young phakic patients and found no significant difference in hue discrimination or dark-adapted sensitivity between the two IOLs.<sup>[<xref rid=\"B31\" ref-type=\"bibr\">31</xref>]</sup> These results were comparable to the outcome of a study by Muftuoglu et al<sup>[<xref rid=\"B32\" ref-type=\"bibr\">32</xref>]</sup> who compared photopic and scotopic CS in eyes with an AcrySof SN60AT IOL (with BLF) and eyes with a conventional AcrySof SA60AT IOL (UV-only filtering) and reported no statistically significant differences between the two IOL types. Furthermore, Hayashi et al<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> measured contrast visual acuity in 74 patients implanted bilaterally with either tinted IOL (HOYA YA60BB) or non-tinted IOLs (VA60BB) and reported no significant difference between the IOL groups.</p><p>In concordance with these aforementioned studies, our results showed no statistically significant difference in contrast sensitivity between IOLs with and without BLF. In an additional analysis, we evaluated the loss of contrast sensitivity after glare was applied in mesopic conditions and found that the tinted IOL had a statistically greater loss of contrast sensitivity under glare compared to the non-tinted IOLs, but only in some spatial frequencies. Although this may be an accidental finding, it is noteworthy as most previous studies did not include contrast sensitivity measurement in mesopic conditions under glare, a situation that is rather common in real life, such as night driving, and can substantially affect the patient's quality of life after cataract surgery.</p><p>A weakness worth mentioning of all studies reporting mesopic CS results after the implantation of IOLs with BLF is the fact that all have utilized measures that are a function of only central vision, where macular pigment is also acting as a BLF. Moreover, one should consider that mesopic vision is mediated, at least in part, by cones, and therefore it is less likely to be adversely influenced by the transmittance properties of such blue-blocking IOLs. Other limitations of our study include the relatively small sample size and the lack of a group with implantation of a spherical IOL with BLF; this type of IOL was not commercially available at the time the study was conducted.</p><p>In summary, the present study compared three IOLs varying in terms of asphericity and BLF and showed only minimal differences in postoperative contrast sensitivity and aberrometry. All IOLs achieved comparable results in postoperative visual performance; an aspheric IOL with BLF, however, resulted in less trefoil aberrations and a greater loss of contrast sensitivity in mesopic conditions when glare was applied. Further randomized patient-centered studies are needed to evaluate the long-term results of aspheric IOL design and BLF and to investigate whether these features are desirable for the patients' quality of life.</p></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>Nil.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Mainster MA. Violet and blue light blocking intraocular lenses: photoprotection versus photoreception. <italic>Br J Ophthalmol</italic> 2006;90:784–792.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">Schwiegerling J. 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] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Ophthalmic Vis Res</journal-id><journal-id journal-id-type=\"publisher-id\">\nJOVR\n</journal-id><journal-title-group><journal-title>Journal of Ophthalmic & Vision Research</journal-title></journal-title-group><issn pub-type=\"ppub\">2008-2010</issn><issn pub-type=\"epub\">2008-322X</issn><publisher><publisher-name>PUBLISHED BY KNOWLEDGE E</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864065</article-id><article-id pub-id-type=\"pmc\">PMC7431730</article-id><article-id pub-id-type=\"doi\">10.18502/jovr.v15i3.7453</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Safety of Intravitreal Injection of Stivant, a Biosimilar to Bevacizumab, in Rabbit Eyes</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Lashay</surname><given-names>Alireza</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Faghihi</surname><given-names>Hooshang</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Mirshahi</surname><given-names>Ahmad</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Khojasteh</surname><given-names>Hassan</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Khodabande</surname><given-names>Alireza</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Riazi-Esfahani</surname><given-names>Hamid</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Amoli</surname><given-names>Fahimeh Asadi</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Pour</surname><given-names>Elias Khalili</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Delrish</surname><given-names>Elham</given-names></name><degrees>PhD</degrees></contrib></contrib-group><aff id=\"I1\">\n<sup>1</sup>Translational Ophthalmology Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran</aff><author-notes><corresp id=\"cor1\"><sup>*</sup>Hooshang Faghihi, MD. Eye Research Center, Farabi Eye\nHospital, Tehran University of Medical Sciences, Qazvin\nSquare, South Kargar St., Tehran 13366, Iran.\nEmail: Faghihih@hotmail.com\nHamid Riazi-Esfahani, MD. Eye Research Center, Farabi\nEye Hospital, Tehran University of Medical Sciences,\nQazvin Square, South Kargar St., Tehran 13366, Iran.\nEmail: Hamidriazi@gmail.com\n</corresp></author-notes><pub-date pub-type=\"collection\"><season>Jul-Sep</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>29</day><month>7</month><year>2020</year></pub-date><volume>15</volume><issue>3</issue><fpage>341</fpage><lpage>350</lpage><history><date date-type=\"received\"><day>04</day><month>6</month><year>2019</year></date><date date-type=\"accepted\"><day>05</day><month>2</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Lashay et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\nwhich permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><abstract><title>Abstract</title><sec><title>Purpose</title><p> To evaluate the safety of intravitreal injection of Stivant, a biosimilar to bevacizumab, in rabbits using electrophysiological and histological analysis.</p></sec><sec><title>Methods</title><p>Both eyes of 41 New Zealand albino rabbits were injected with 0.1 mL (2.5 mg) of Stivant. The rabbits were scheduled to be sacrificed 1, 2, 7, 14, and 28 days after injection for histopathological evaluations. Clinical examinations and electroretinography (ERG) were performed at baseline and just before sacrificing the rabbits. Fourteen separate rabbits received a reference drug (Avastin) and were considered as the control group. Furthermore, three other rabbits received the same volume of saline (saline control group). Rabbits of both control groups were sacrificed four weeks after injection. ERG was performed 1, 2, 7, 14, and 28 days after injections.</p></sec><sec><title>Results</title><p>No significant difference was observed in a- and b-wave amplitudes and latency after intravitreal Stivant injection between baseline and different time points. Moreover, there was no statistically significant difference in wave amplitudes and latency between the Stivant and control groups. The histology of rabbit eyes of the Stivant and control groups after intravitreal injections was not distinguishable.</p></sec><sec><title>Conclusion</title><p>The biosimilar Stivant, up to a dose of 2.5 mg, did not appear to be toxic to the retina in albino rabbits. These results suggest that this drug could be a safe and inexpensive alternative to intravitreal bevacizumab. The efficacy of these injections was not investigated in this study and needs to be evaluated in future studies.</p></sec></abstract><kwd-group><kwd>Biosimilar</kwd><kwd> Intravitreal Injection</kwd><kwd> Safety</kwd><kwd> Stivant</kwd></kwd-group><counts><fig-count count=\"1\"/><table-count count=\"5\"/><ref-count count=\"20\"/><page-count count=\"10\"/></counts></article-meta></front><body><sec sec-type=\"section\"><title> INTRODUCTION</title><p>Vascular endothelial growth factor (VEGF) plays a substantial role in angiogenesis and vasculogenesis. Therefore, it is the main target for the treatment of cancers and ophthalmic vascular disorders.<sup>[<xref rid=\"B1\" ref-type=\"bibr\">1</xref>,<xref rid=\"B2\" ref-type=\"bibr\">2</xref>]</sup> The inhibition of VEGF causes regression of aberrant new vessels in experimental models of proliferative vascular retinopathies and neovascularization of the choroid.<sup>[<xref rid=\"B3\" ref-type=\"bibr\">3</xref>]</sup>\n</p><p>In 2004, bevacizumab, a humanized full-length immunoglobulin G against VEGF, was approved by the US Federal Drug Administration for the management of colon cancer.<sup>[<xref rid=\"B4\" ref-type=\"bibr\">4</xref>]</sup> Bevacizumab is now being used as an “off-label” intravitreal agent for wet age-related macular degeneration (AMD), diabetic retinopathy, and retinal vein occlusions globally.<sup>[<xref rid=\"B5\" ref-type=\"bibr\">5</xref>]</sup> Intravitreal injections of ranibizumab and aflibercept are both approved for wet-type AMD and macular edema due to retinal vascular disorders. However, despite similar efficacy to bevacizumab, there are large differences between the costs of these drugs. On an average, bevacizumab is 20 times less expensive than ranibizumab and aflibercept.<sup>[<xref rid=\"B6\" ref-type=\"bibr\">6</xref>]</sup>\n</p><p>A biosimilar is “essentially the same” as a reference biologic in terms of structure, efficacy, safety, and quality, although it has some natural variability owing to its complex nature and production methods.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup> Biosimilars, which are economically more viable, have the potential to reduce healthcare costs relative to reference biologics, thereby increasing treatment access for patients who need them.<sup>[<xref rid=\"B8\" ref-type=\"bibr\">8</xref>,<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> Stivant (CinnaGen Co., Tehran, Iran) has been developed as a biosimilar to a reference product, bevacizumab (Avastin: Genentech, Inc., South San Francisco, CA). Stivant is formulated with the same excipients as the reference product and is provided in the same pharmaceutical form and dosage strength. AvastinⓇ and Stivant have similar safety and efficacy as intravenous injections for metastatic colorectal cancer and have been approved for this use.</p><p>To determine the safety of intravitreal injection of this biosimilar drug, the albino rabbits were injected intravitreally with Stivant and evaluated for any functional and histological changes in the retina.</p></sec><sec sec-type=\"section\"><title> METHODS</title><p>Forty-one albino New Zealand rabbits, weighing between 1.5 and 2.5 kg, were used to evaluate the safety of intravitreal Stivant injection. The rabbits were treated in agreement with the statement for the use of animals in ophthalmic and vision research, proposed by The Association for Research in Vision and Ophthalmology. The study design was approved by the Institutional Review Board of Tehran University of Medical Sciences.</p><p>The rabbits were kept in an air-conditioned room with a 12-hour light–dark cycle and fed with standard processed laboratory food. Seven and eight of the rabbits were scheduled to be sacrificed 24 and 48 hours after injection, respectively, and the remaining were divided into three separate groups to be sacrificed at one (seven rabbits), two (ten rabbits), and four weeks (nine rabbits) after injection. Clinical examinations and electroretinography (ERG) were performed at baseline and just before killing the rabbits (Table 1). Fourteen separate rabbits received a reference drug (Avastin: Genentech, Inc., South San Francisco, CA) and were considered as the control group. Furthermore, three other rabbits received the same volume of normal saline and were considered as the saline control group. Both control groups were sacrificed four weeks after injection; however the ERG for these groups was performed 1, 2, 7, 14, and 28 days after injections (Table 1).</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Number of rabbits in each group</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"3\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Number of rabbits</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Time for enucleation and scarification</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Group 1 (Stivant)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1 day after injection</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Group 2 (Stivant)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 days after injection</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Group 3 (Stivant)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1 week after injection</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Group 4 (Stivant)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2 weeks after injection</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Group 5 (Stivant)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 weeks after injection</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Control (Saline)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 weeks after injection</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Control (Avastin)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4 weeks after injection</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>The amplitudes and latency times of ERG waves from the Stivant-treated rabbits</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Groups</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>a-wave Amplitude</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>b-wave Amplitude</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>a-wave Latency Time</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>b-wave Latency Time</bold>\n</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<bold>24 hours (14 eyes)</bold> *</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–38.5 <inline-formula><mml:math id=\"M1\"><mml:mo>±</mml:mo></mml:math></inline-formula> 5.5 µV **</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">97.1 <inline-formula><mml:math id=\"M2\"><mml:mo>±</mml:mo></mml:math></inline-formula> 12 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13.9 <inline-formula><mml:math id=\"M3\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.6 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32.6 <inline-formula><mml:math id=\"M4\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.3 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–36.1 <inline-formula><mml:math id=\"M5\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.4 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">99.1 <inline-formula><mml:math id=\"M6\"><mml:mo>±</mml:mo></mml:math></inline-formula> 22.1 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13.9 <inline-formula><mml:math id=\"M7\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.7 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32.3 <inline-formula><mml:math id=\"M8\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.1 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Difference in</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 6.2%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Increase 2.1%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 0.1%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Decrease 1%</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<bold>48 hours (16 eyes) %</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–34.8 <inline-formula><mml:math id=\"M9\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.6 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">91.7 <inline-formula><mml:math id=\"M10\"><mml:mo>±</mml:mo></mml:math></inline-formula> 9.5 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13.5 <inline-formula><mml:math id=\"M11\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.5 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.6 <inline-formula><mml:math id=\"M12\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.7 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–32.6 <inline-formula><mml:math id=\"M13\"><mml:mo>±</mml:mo></mml:math></inline-formula> 5.9 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">98.8 <inline-formula><mml:math id=\"M14\"><mml:mo>±</mml:mo></mml:math></inline-formula> 11.9 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.1 <inline-formula><mml:math id=\"M15\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.5 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.1 <inline-formula><mml:math id=\"M16\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.4 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Difference in</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 6.4%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Increase 7.2%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 4.3%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 4.3%</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<bold>1 week (14 eyes) %</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–47.2 <inline-formula><mml:math id=\"M17\"><mml:mo>±</mml:mo></mml:math></inline-formula> 6 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">128.5 <inline-formula><mml:math id=\"M18\"><mml:mo>±</mml:mo></mml:math></inline-formula> 21.1 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13.9 <inline-formula><mml:math id=\"M19\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.6 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.3 <inline-formula><mml:math id=\"M20\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.6 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–37.9 <inline-formula><mml:math id=\"M21\"><mml:mo>±</mml:mo></mml:math></inline-formula> 9.2 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">127.2 <inline-formula><mml:math id=\"M22\"><mml:mo>±</mml:mo></mml:math></inline-formula> 38.3 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.8 <inline-formula><mml:math id=\"M23\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.7 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.3 <inline-formula><mml:math id=\"M24\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.9 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Difference in</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 19.8%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 1.1%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 6.1%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 5.4%</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<bold>2 weeks (20 eyes) %</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–48.2 <inline-formula><mml:math id=\"M25\"><mml:mo>±</mml:mo></mml:math></inline-formula> 10.3 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">140.3 <inline-formula><mml:math id=\"M26\"><mml:mo>±</mml:mo></mml:math></inline-formula> 21.7 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.1 <inline-formula><mml:math id=\"M27\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.7 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">36.6 <inline-formula><mml:math id=\"M28\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.9 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–47.7 <inline-formula><mml:math id=\"M29\"><mml:mo>±</mml:mo></mml:math></inline-formula> 9.5 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">147.5 <inline-formula><mml:math id=\"M30\"><mml:mo>±</mml:mo></mml:math></inline-formula> 21.5 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.3 <inline-formula><mml:math id=\"M31\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.6 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.7 <inline-formula><mml:math id=\"M32\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.1 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Difference in</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 1.1%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Increase 4.9%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 1.4%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 3%</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<bold>4 weeks (18 eyes) %</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–46.5 <inline-formula><mml:math id=\"M33\"><mml:mo>±</mml:mo></mml:math></inline-formula> 7.1 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">127.9 <inline-formula><mml:math id=\"M34\"><mml:mo>±</mml:mo></mml:math></inline-formula>22.7 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.7 <inline-formula><mml:math id=\"M35\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.7 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">36.1 <inline-formula><mml:math id=\"M36\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.3 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–48.6 <inline-formula><mml:math id=\"M37\"><mml:mo>±</mml:mo></mml:math></inline-formula> 14.1 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">115.3 <inline-formula><mml:math id=\"M38\"><mml:mo>±</mml:mo></mml:math></inline-formula> 30.1 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15.1 <inline-formula><mml:math id=\"M39\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.5 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37 <inline-formula><mml:math id=\"M40\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.1 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Difference in %</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Increase 4.4%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 9.9%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 2.7%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 2.5%</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\">After injections; **Data are expressed as mean <inline-formula><mml:math id=\"M41\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD\n<italic>Microvolts</italic> (µV); Milliseconds (ms)</td></tr></tbody></table></table-wrap><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><p>The amplitudes and latency times of ERG waves from the Avastin-treated rabbits (28 eyes)</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Groups</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>a-wave Amplitude</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>b-wave Amplitude</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>a-wave Latency Time</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>b-wave Latency Time</bold>\n</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<bold>24 hours (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 28) *</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–37.8 <inline-formula><mml:math id=\"M42\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3 µV **</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">120.1 <inline-formula><mml:math id=\"M43\"><mml:mo>±</mml:mo></mml:math></inline-formula> 14.7 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.5 <inline-formula><mml:math id=\"M44\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.5 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34.4 <inline-formula><mml:math id=\"M45\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.5 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–35.8 <inline-formula><mml:math id=\"M46\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.8 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">114.8 <inline-formula><mml:math id=\"M47\"><mml:mo>±</mml:mo></mml:math></inline-formula> 13.8 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13.9 <inline-formula><mml:math id=\"M48\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0. 7 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.1 <inline-formula><mml:math id=\"M49\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.3 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Difference in</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 5.3%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 4.5%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Decrease 4.2%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 2%</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<bold>48 hours (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 28) %</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–37.8 <inline-formula><mml:math id=\"M50\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">120.1 <inline-formula><mml:math id=\"M51\"><mml:mo>±</mml:mo></mml:math></inline-formula> 14.7 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.5 <inline-formula><mml:math id=\"M52\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.5 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34.4 <inline-formula><mml:math id=\"M53\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.5 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–34.7 <inline-formula><mml:math id=\"M54\"><mml:mo>±</mml:mo></mml:math></inline-formula> 5.9 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100.8 <inline-formula><mml:math id=\"M55\"><mml:mo>±</mml:mo></mml:math></inline-formula> 13.6 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.7 <inline-formula><mml:math id=\"M56\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.3 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.3 <inline-formula><mml:math id=\"M57\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Difference in</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 8.3%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 16.6%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 1.3%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 8.4%</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<bold>1 week (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 28) %</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–37.8 <inline-formula><mml:math id=\"M58\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">120.1 <inline-formula><mml:math id=\"M59\"><mml:mo>±</mml:mo></mml:math></inline-formula> 14.7 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.5 <inline-formula><mml:math id=\"M60\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.5 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34.4 <inline-formula><mml:math id=\"M61\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.5 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–32.9 <inline-formula><mml:math id=\"M62\"><mml:mo>±</mml:mo></mml:math></inline-formula> 5.8 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">106.5 <inline-formula><mml:math id=\"M63\"><mml:mo>±</mml:mo></mml:math></inline-formula> 21.7 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.3 <inline-formula><mml:math id=\"M64\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.9 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32.5 <inline-formula><mml:math id=\"M65\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.3 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Difference in</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 13 %</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 11.4%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Decrease 1.4%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Decrease 5.6%</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<bold>2 weeks (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 28) %</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–37.8 <inline-formula><mml:math id=\"M66\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">120.1 <inline-formula><mml:math id=\"M67\"><mml:mo>±</mml:mo></mml:math></inline-formula> 14.7 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.5 <inline-formula><mml:math id=\"M68\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.5 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34.4 <inline-formula><mml:math id=\"M69\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.5 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–44.5 <inline-formula><mml:math id=\"M70\"><mml:mo>±</mml:mo></mml:math></inline-formula> 9.9 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">134.4 <inline-formula><mml:math id=\"M71\"><mml:mo>±</mml:mo></mml:math></inline-formula> 22.1 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13.6 <inline-formula><mml:math id=\"M72\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.5 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.1 <inline-formula><mml:math id=\"M73\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.1 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Difference in</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Increase 17%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Increase 11%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Decrease 6.3%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 2%</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<bold>4 weeks (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 28) %</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–37.8 <inline-formula><mml:math id=\"M74\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">120.1 <inline-formula><mml:math id=\"M75\"><mml:mo>±</mml:mo></mml:math></inline-formula> 14.7 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.5 <inline-formula><mml:math id=\"M76\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.5 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34.4 <inline-formula><mml:math id=\"M77\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.5 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–42.6 <inline-formula><mml:math id=\"M78\"><mml:mo>±</mml:mo></mml:math></inline-formula> 10.5 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">110.4 <inline-formula><mml:math id=\"M79\"><mml:mo>±</mml:mo></mml:math></inline-formula> 14.7 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.2 <inline-formula><mml:math id=\"M80\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.2 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.2 <inline-formula><mml:math id=\"M81\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.6 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Difference in %</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Increase 12.6%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 8.1%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Decrease 2.1%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Decrease 3.5%</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">*After injections; **Data are exprssed as mean <inline-formula><mml:math id=\"M82\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD\nMicrovolts (µV); Milliseconds (ms)</td></tr></tbody></table></table-wrap><table-wrap id=\"T4\" orientation=\"portrait\" position=\"float\"><label>Table 4</label><caption><p>The amplitudes and latency times of ERG waves from the Saline-treated rabbits (6 eyes)</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"5\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Groups</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>a-wave Amplitude</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>b-wave Amplitude</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>a-wave Latency Time</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>b-wave Latency Time</bold>\n</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<bold>24 hours (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 6) *</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–34.6 <inline-formula><mml:math id=\"M83\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.1 µV **</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">114.8 <inline-formula><mml:math id=\"M84\"><mml:mo>±</mml:mo></mml:math></inline-formula> 17.7 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.4 <inline-formula><mml:math id=\"M85\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.1 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.2 <inline-formula><mml:math id=\"M86\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.1 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–32.9 <inline-formula><mml:math id=\"M87\"><mml:mo>±</mml:mo></mml:math></inline-formula> 7.8 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">106.4 <inline-formula><mml:math id=\"M88\"><mml:mo>±</mml:mo></mml:math></inline-formula> 12.7 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">13.2 <inline-formula><mml:math id=\"M89\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.1 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34.2 <inline-formula><mml:math id=\"M90\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.3 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Difference in </bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 5%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 6.6%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 6.4%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 3%</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<bold>48 hours (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 6) %</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–34.6 <inline-formula><mml:math id=\"M91\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.1 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">114.8 <inline-formula><mml:math id=\"M92\"><mml:mo>±</mml:mo></mml:math></inline-formula> 17.7 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.4 <inline-formula><mml:math id=\"M93\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.1 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.2 <inline-formula><mml:math id=\"M94\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.1 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–33.5 <inline-formula><mml:math id=\"M95\"><mml:mo>±</mml:mo></mml:math></inline-formula> 8.8 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">101.4 <inline-formula><mml:math id=\"M96\"><mml:mo>±</mml:mo></mml:math></inline-formula> 19.2 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.7 <inline-formula><mml:math id=\"M97\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.7 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.6 <inline-formula><mml:math id=\"M98\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.2 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Difference in </bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 3.2%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 11.7%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 18.5%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 7.2%</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<bold>1 week (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 6) %</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–34.6 <inline-formula><mml:math id=\"M99\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.1 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">114.8 <inline-formula><mml:math id=\"M100\"><mml:mo>±</mml:mo></mml:math></inline-formula> 17.7 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.4 <inline-formula><mml:math id=\"M101\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.1 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.2 <inline-formula><mml:math id=\"M102\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.1 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–40 <inline-formula><mml:math id=\"M103\"><mml:mo>±</mml:mo></mml:math></inline-formula> 8.1 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">129.9 <inline-formula><mml:math id=\"M104\"><mml:mo>±</mml:mo></mml:math></inline-formula> 27.2 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.1 <inline-formula><mml:math id=\"M105\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.6 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.1 <inline-formula><mml:math id=\"M106\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.6 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Difference in </bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Increase 15.6 %</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Increase 13.1%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 13.7%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 5.7%</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<bold>2 weeks (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 6) %</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–34.6 <inline-formula><mml:math id=\"M107\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.1 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">114.8 <inline-formula><mml:math id=\"M108\"><mml:mo>±</mml:mo></mml:math></inline-formula> 17.7 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.4 <inline-formula><mml:math id=\"M109\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.1 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.2 <inline-formula><mml:math id=\"M110\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.1 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–38.6 <inline-formula><mml:math id=\"M111\"><mml:mo>±</mml:mo></mml:math></inline-formula> 8.4 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">135.3 <inline-formula><mml:math id=\"M112\"><mml:mo>±</mml:mo></mml:math></inline-formula> 28.3 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.3 <inline-formula><mml:math id=\"M113\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.7 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.9 <inline-formula><mml:math id=\"M114\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.5 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Difference in </bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Increase 11.5%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Increase 17.8%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 15.3%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 8.1%</td></tr><tr><td align=\"center\" colspan=\"5\" rowspan=\"1\">\n<bold>4 weeks (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 6) %</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Pre-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–34.6 <inline-formula><mml:math id=\"M115\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.1 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">114.8 <inline-formula><mml:math id=\"M116\"><mml:mo>±</mml:mo></mml:math></inline-formula> 17.7 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">12.4 <inline-formula><mml:math id=\"M117\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.1 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.2 <inline-formula><mml:math id=\"M118\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.1 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Post-treatment</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–41.5 <inline-formula><mml:math id=\"M119\"><mml:mo>±</mml:mo></mml:math></inline-formula> 13.3 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">110.3 <inline-formula><mml:math id=\"M120\"><mml:mo>±</mml:mo></mml:math></inline-formula> 20.5 µV</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">14.1 <inline-formula><mml:math id=\"M121\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.6 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.2 <inline-formula><mml:math id=\"M122\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.1 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Difference in %</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Increase 19.8%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Decrease 4%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 13.7%</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Increase 6%</td></tr><tr><td align=\"left\" colspan=\"5\" rowspan=\"1\"> *After injections; **Data are expressed as mean <inline-formula><mml:math id=\"M123\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD\nMicrovolts (µV); Milliseconds (ms)</td></tr></tbody></table></table-wrap><table-wrap id=\"T5\" orientation=\"portrait\" position=\"float\"><label>Table 5</label><caption><p>Comparison of the amplitudes and latency times of rod responses from the Stivant-treated rabbits, saline-treated rabbits, and Avastin-treated rabbits</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td colspan=\"6\" rowspan=\"1\">\n<hr/>\n</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\"><bold>Groups</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>a-wave Amplitude</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>b-wave Amplitude</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>a-wave Latency Time</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>b-wave Latency Time</bold>\n</td></tr><tr><td align=\"center\" colspan=\"6\" rowspan=\"1\">\n<bold>24 hours</bold>\n</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Saline (</bold>\n<italic><bold>n</bold></italic>\n<bold>* = 6)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–32.9 <inline-formula><mml:math id=\"M124\"><mml:mo>±</mml:mo></mml:math></inline-formula> 7.8 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">106.4 <inline-formula><mml:math id=\"M125\"><mml:mo>±</mml:mo></mml:math></inline-formula> 12.7 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13.2 <inline-formula><mml:math id=\"M126\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.1 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">34.2 <inline-formula><mml:math id=\"M127\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.3 ms</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Avastin (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 28)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–35.8 <inline-formula><mml:math id=\"M128\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.8 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">114.8 <inline-formula><mml:math id=\"M129\"><mml:mo>±</mml:mo></mml:math></inline-formula> 13.8 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13.9 <inline-formula><mml:math id=\"M130\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0. 7 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.1 <inline-formula><mml:math id=\"M131\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.3 ms</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Stivant (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 14)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–36.1 <inline-formula><mml:math id=\"M132\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.4 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">99.1 <inline-formula><mml:math id=\"M133\"><mml:mo>±</mml:mo></mml:math></inline-formula> 22.1 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13.9 <inline-formula><mml:math id=\"M134\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.7 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32.3 <inline-formula><mml:math id=\"M135\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.1 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold/>\n<italic><bold>P</bold></italic>\n<bold>-value**</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Stivant/saline</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.43</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.54</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.86</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.71</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Stivant/Avastin</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.73</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.21</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.79</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.69</td></tr><tr><td align=\"center\" colspan=\"6\" rowspan=\"1\">\n<bold>48 hours</bold>\n</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Saline (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 6)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–33.5 <inline-formula><mml:math id=\"M136\"><mml:mo>±</mml:mo></mml:math></inline-formula> 8.8 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">101.4 <inline-formula><mml:math id=\"M137\"><mml:mo>±</mml:mo></mml:math></inline-formula> 19.2 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.7 <inline-formula><mml:math id=\"M138\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.7 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.6 <inline-formula><mml:math id=\"M139\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.2 ms</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Avastin (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 28)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–34.7 <inline-formula><mml:math id=\"M140\"><mml:mo>±</mml:mo></mml:math></inline-formula> 5.9 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100.8 <inline-formula><mml:math id=\"M141\"><mml:mo>±</mml:mo></mml:math></inline-formula> 13.6 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.7 <inline-formula><mml:math id=\"M142\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.3 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.3 <inline-formula><mml:math id=\"M143\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1 ms</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Stivant (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 16)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–32.6 <inline-formula><mml:math id=\"M144\"><mml:mo>±</mml:mo></mml:math></inline-formula> 5.9 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">98.8 <inline-formula><mml:math id=\"M145\"><mml:mo>±</mml:mo></mml:math></inline-formula> 11.9 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.1 <inline-formula><mml:math id=\"M146\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.5 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.1 <inline-formula><mml:math id=\"M147\"><mml:mo>±</mml:mo></mml:math></inline-formula> 3.4 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold/>\n<italic><bold>P</bold></italic>\n<bold>-value**</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Stivant/saline</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.38</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.44</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.88</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.90</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Stivant/Avastin</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.33</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.29</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.81</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.76</td></tr><tr><td align=\"center\" colspan=\"6\" rowspan=\"1\">\n<bold>1 week</bold>\n</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Saline (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 6)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–40 <inline-formula><mml:math id=\"M148\"><mml:mo>±</mml:mo></mml:math></inline-formula> 8.1 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">129.9 <inline-formula><mml:math id=\"M149\"><mml:mo>±</mml:mo></mml:math></inline-formula> 27.2 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.1 <inline-formula><mml:math id=\"M150\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.6 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.1 <inline-formula><mml:math id=\"M151\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.6 ms</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Avastin (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 28)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–32.9 <inline-formula><mml:math id=\"M152\"><mml:mo>±</mml:mo></mml:math></inline-formula> 5.8 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">106.5 <inline-formula><mml:math id=\"M153\"><mml:mo>±</mml:mo></mml:math></inline-formula> 21.7 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.3 <inline-formula><mml:math id=\"M154\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.9 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">32.5 <inline-formula><mml:math id=\"M155\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.3 ms</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Stivant (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 14)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–37.9 <inline-formula><mml:math id=\"M156\"><mml:mo>±</mml:mo></mml:math></inline-formula> 9.2 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">127.2 <inline-formula><mml:math id=\"M157\"><mml:mo>±</mml:mo></mml:math></inline-formula> 38.3 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.8 <inline-formula><mml:math id=\"M158\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.7 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.3 <inline-formula><mml:math id=\"M159\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.9 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold/>\n<italic><bold>P</bold></italic>\n<bold>-value**</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Stivant/saline</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.19</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.33</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.87</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.53</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Stivant/Avastin</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.11</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.75</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.28</td></tr><tr><td align=\"center\" colspan=\"6\" rowspan=\"1\">\n<bold>2 weeks</bold>\n</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Saline (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 6)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–38.6 <inline-formula><mml:math id=\"M160\"><mml:mo>±</mml:mo></mml:math></inline-formula> 8.4 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">135.3 <inline-formula><mml:math id=\"M161\"><mml:mo>±</mml:mo></mml:math></inline-formula> 28.3 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.3 <inline-formula><mml:math id=\"M162\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.7 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.9 <inline-formula><mml:math id=\"M163\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.5 ms</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Avastin (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 28)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–44.5 <inline-formula><mml:math id=\"M164\"><mml:mo>±</mml:mo></mml:math></inline-formula> 9.9 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">134.4 <inline-formula><mml:math id=\"M165\"><mml:mo>±</mml:mo></mml:math></inline-formula> 22.1 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13.6 <inline-formula><mml:math id=\"M166\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.5 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.1 <inline-formula><mml:math id=\"M167\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.1 ms</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Stivant (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 20)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–47.7 <inline-formula><mml:math id=\"M168\"><mml:mo>±</mml:mo></mml:math></inline-formula> 9.5 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">147.5 <inline-formula><mml:math id=\"M169\"><mml:mo>±</mml:mo></mml:math></inline-formula> 21.5 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.3 <inline-formula><mml:math id=\"M170\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.6 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37.7 <inline-formula><mml:math id=\"M171\"><mml:mo>±</mml:mo></mml:math></inline-formula> 1.1 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold/>\n<italic><bold>P</bold></italic>\n<bold>-value** </bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Stivant/saline</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.94</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.72</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Stivant/Avastin</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.81</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.09</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.61</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.66</td></tr><tr><td align=\"center\" colspan=\"6\" rowspan=\"1\">\n<bold>4 weeks</bold>\n</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Saline (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 6)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–41.5 <inline-formula><mml:math id=\"M172\"><mml:mo>±</mml:mo></mml:math></inline-formula> 13.3 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">110.3 <inline-formula><mml:math id=\"M173\"><mml:mo>±</mml:mo></mml:math></inline-formula> 20.5 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.1 <inline-formula><mml:math id=\"M174\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.6 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">35.2 <inline-formula><mml:math id=\"M175\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.1 ms</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Avastin (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 28)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–42.6 <inline-formula><mml:math id=\"M176\"><mml:mo>±</mml:mo></mml:math></inline-formula> 10.5 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">110.4 <inline-formula><mml:math id=\"M177\"><mml:mo>±</mml:mo></mml:math></inline-formula> 14.7 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.2 <inline-formula><mml:math id=\"M178\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.2 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">33.2 <inline-formula><mml:math id=\"M179\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.6 ms</td></tr><tr><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Stivant (</bold>\n<italic><bold>n</bold></italic>\n<bold> = 18)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">–48.6 <inline-formula><mml:math id=\"M180\"><mml:mo>±</mml:mo></mml:math></inline-formula> 14.1 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">115.3 <inline-formula><mml:math id=\"M181\"><mml:mo>±</mml:mo></mml:math></inline-formula> 30.1 µV</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.1 <inline-formula><mml:math id=\"M182\"><mml:mo>±</mml:mo></mml:math></inline-formula> 0.5 ms</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">37 <inline-formula><mml:math id=\"M183\"><mml:mo>±</mml:mo></mml:math></inline-formula> 2.1 ms</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold/>\n<italic><bold>P</bold></italic>\n<bold>-value** </bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Stivant/saline</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.68</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.88</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.67</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Stivant/Avastin</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.58</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.24</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.54</td></tr><tr><td align=\"left\" colspan=\"6\" rowspan=\"1\">Data are expressed as mean <inline-formula><mml:math id=\"M184\"><mml:mo>±</mml:mo></mml:math></inline-formula> SD\n*Number of the injected eyes in each group; **Based on generalized estimating equation (GEE) regression model\n<italic>Microvolts</italic> (µV); Milliseconds (ms)</td></tr></tbody></table></table-wrap><p>Rabbits with documented anterior or posterior segment abnormalities in the eye were excluded. The animals were anesthetized before all procedures using a mixture of xylazine hydrochloride (5 mg/kg) and ketamine hydrochloride (50 mg/kg). Before each examination, the pupil of the eyes was dilated by topical application of tropicamide 0.5% eye drop.</p><sec sec-type=\"subsection\"><title>Preparation and injection of the drugs</title><p>Under standard sterile conditions, standard tuberculin syringes with 29-gauge needles were filled with the drugs or saline. Intravitreal injections of the drugs in each group were performed after baseline examinations and baseline ERG. Rabbits were anesthetized with an intramuscular injection of the mixture of xylazine and ketamine. The eye was prepped with installation of the 5% diluted povidone iodine solution into the fornixes.</p><p>Both eyes of each rabbit in the Stivant and Avastin groups were injected with 0.1 mL (2.5 mg) of the drugs. Intravitreal injections into the mid-vitreous cavity were performed 1.5 mm posterior to the limbus. Anterior chamber paracentesis was done before intravitreal injections with a 29-gauge needle, withdrawing at least 0.05 mL of the aqueous fluid to prevent intraocular pressure rise after injections and also to prevent reflux from the injection site. The same volume (0.1 mL) of normal saline was injected into both eyes of each of the three control rabbits in the same manner. Ciprofloxacin and timolol eye drops were applied to the eyes for the first three days after injections.</p></sec><sec sec-type=\"subsection\"><title>Clinical observations </title><p>The eyes were examined clinically at baseline on the first and second days after injections and at the end of the first, second, and fourth weeks of injections just before enucleation. The following parameters were recorded: injection of the conjunctiva, status of the cornea, appearance of the crystalline lens, any pathologic findings in the retina, and any cell or flare in the anterior and posterior segments of the eye.</p><p>A hand-held slit lamp was used to evaluate the status of the anterior segment. The anterior chamber and vitreous cavity were carefully examined with the highest magnification to detect any cell or flare. At each follow-up, all eyes underwent indirect ophthalmoscopy.</p></sec><sec sec-type=\"subsection\"><title>Electrophysiology</title><p>Electroretinography using the electrophysiological test system (Metrovision, France) was performed on both eyes of each rabbit at baseline and the time points previously mentioned. All rabbits underwent dark adaptation overnight before ERG tests and were prepared for the procedure under red light. The animals were anesthetized, and corneal surface anesthesia was achieved using tetracaine hydrochloride 0.5%. Flashlight intensity of 10 cdsm<inline-formula><mml:math id=\"M185\"><mml:msup><mml:mrow/><mml:mrow><mml:mo>-</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:math></inline-formula> was used for each recording; the average of the responses from four separate light stimuli was documented. ERG was recorded using a corneal contact lens electrode (Metrovision, France). The ground electrode was inserted into the ear, and the negative electrode was attached near the orbital rim.</p><p>The amplitude and implicit time measurements of the a- and b-waves were used to evaluate ERG responses. The a-wave amplitude was measured from the baseline to the first trough; the b-wave amplitude was measured from the a-wave trough to the peak of the b-wave. Latencies of the a- and b-waves were measured from the time of presenting the stimuli.</p></sec><sec sec-type=\"subsection\"><title>Histopathologic examinations</title><p>After the last ophthalmoscopy and electrophysiology in each group, the animals were sacrificed by intravenous injection of 100 mg/kg of sodium pentobarbital while under deep anesthesia. With careful attention to prevent any damage to globe integrity, the eyes were enucleated. Then, each enucleated eye was promptly placed in a separate bottle containing the neutral formalin solution. After seven days, samples were fixed in paraffin. Microtome sections of 5-m thickness were prepared and stained with hematoxylin-eosin. Slides were examined under a light microscope.</p></sec><sec sec-type=\"subsection\"><title>Statistical Analysis</title><p>Statistical analysis was performed using the SPSS software version 24.0. (IBM SPSS Statistics for Windows, Armonk, NY: IBM Corp). One-way analysis of variance was used to determine whether there were any statistically significant differences between the groups, based on the ERG parameters. The generalized estimating equation (GEE) regression model was used to analyze the effect of the injections on the ERG a- and b-wave amplitudes and implicit time between the Stivant and control groups, separately. A <italic>P</italic>-value <inline-formula><mml:math id=\"M186\"><mml:mo><</mml:mo></mml:math></inline-formula> 0.05 was considered statistically significant.</p></sec></sec><sec sec-type=\"section\"><title> RESULTS</title><p>Of 41 rabbits in the Stivant group, 3 were excluded due to death; 1 died just after injection, and the other 2 died five days and three weeks after injection, respectively. Stivant was tolerated well in the remaining rabbits. Two rabbits also died two weeks after injection in the Avastin control group. No obvious changes in food or water intake were observed.</p><sec sec-type=\"subsection\"><title>Clinical Evaluation </title><p>At each anterior segment examination, there was no new significant abnormality in conjunctiva or different layers of the cornea. Anterior chamber or vitreous cells were not detected in any eye at different time points on biomicroscopic examination. There was no iris abnormality in any eye of each group. The crystalline lenses were clear. The vitreous, retina, choroid, and optic nerve seemed normal, based on indirect ophthalmoscopy.</p></sec><sec sec-type=\"subsection\"><title>Electrophysiology</title><p>There was no significant difference between ERG wave amplitudes and implicit times between Stivant, Avastin, and saline groups at baseline. ERG changes were considered significant at each time point if the difference in amplitudes (a- and b-waves) was more than 20% of the baseline values.</p><p>The ERG results of the Stivant, Avastin, and saline groups are presented in Tables 2, 3, and 4, respectively. Dark-adapted bright flash ERG was performed for all rabbits before the intravitreal injection as a baseline standard and then at each time point. Despite a 19.8% decrease in the amplitude of the a-wave on day 7 after Stivant injection, no significant change was found in the a-wave amplitude and implicit time after injections in each group. We also observed a 13% reduction in the a-wave amplitude, one week after Avastin injection. These reductions were reversed just one week later in both groups.</p><p>This study did not show a significant change in the amplitude and latency of b-waves in Stivant and Avastin groups, although, when compared to the baseline, the b-wave amplitude decreased 9.9% and 8.1% in eyes that were evaluated four weeks after Stivant and Avastin injections, respectively.</p><p>There were no statistically significant differences between the Stivant, Avastin, and saline groups, based on ERG parameters. Also, based on the GEE regression model, there was no significant difference between the Stivant-injected eyes and the Avastin and saline control groups at each time point, separately. (Table 5)</p></sec><sec sec-type=\"subsection\"><title>Histological Findings</title><p>Based on histopathological findings, there were no distinguishable changes in both the Stivant and control groups after intravitreal injections. There were no signs of ocular toxicity, based on histological evaluations in the groups.</p><p>In the light microscopic slides, there was no evidence of corneal deposits, thinning, or endothelial cell damage in the cornea. Uveal tissue did not show any inflammation or neovascularization. There was no sign of inflammation in the anterior and posterior segments of the eyes.</p><p>There was no evidence of intraocular hemorrhage in specimens, except the right eye of a rabbit that was sacrificed two weeks after Stivant injection. The right eye showed retinal and vitreous hemorrhage, although there was no significant change in ERG parameters from baseline in this eye.</p><p>All specimens had normal retinal thickness, ganglion cells, photoreceptor morphology, pigmented epithelial cells, and nuclear layers. No evidence of optic nerve edema, neuritis, or atrophy was identified.</p><p>The only positive histopathological finding was congestion of the choroid without hemorrhage in half of the eyes that were injected with Stivant, which were enucleated 24 hours after injection. This finding was not observed in Stivant-injected groups at other time points (Figure 1).</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Representative histopathological sections from injected eyes four weeks after injection: there is preservation of the retinal cytoarchitecture without loss of the inner and outer nuclear layers or the inner and outer plexiform layers in both Avastin-injected (a) and Stivant-injected eyes (b) (Staining: hematoxylin and eosin [H&E], magnification: <inline-formula><mml:math id=\"M187\"><mml:mo>×</mml:mo></mml:math></inline-formula>40).</p></caption><graphic xlink:href=\"jovr-15-331-g001\"/></fig></sec></sec><sec sec-type=\"section\"><title> DISCUSSION</title><p>Our results showed that a single intravitreal injection of the biosimilar to bevacizumab (Stivant) at doses up to 2.5 mg in albino rabbit eyes did not result in apparent vitreoretinal toxicity at 1, 2, 7, 14, and 28 days after injection, based on electrophysiological and histopathological findings. The ERG responses of the experimental and two control group eyes were similar in a- or b-wave amplitudes and implicit times at different time points after injections.</p><p>Anti-VEGF agents play a key role in the management of different retinal conditions, such as wet AMD, diabetic macular edema, and retinal vein occlusion.<sup>[<xref rid=\"B10\" ref-type=\"bibr\">10</xref>,<xref rid=\"B11\" ref-type=\"bibr\">11</xref>,<xref rid=\"B12\" ref-type=\"bibr\">12</xref>]</sup> As most of these patients need multiple anti-VEGF injections, these drugs incur high individual, medical, and societal costs.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>]</sup> By decreasing the cost of therapy, the economic burden on the individual patients, their families, and society will be reduced. In developing countries, the high cost of treatment is an important limiting factor for patient compliance to anti-VEGF agents.<sup>[<xref rid=\"B13\" ref-type=\"bibr\">13</xref>,<xref rid=\"B14\" ref-type=\"bibr\">14</xref>]</sup>\n</p><p>Biosimilar drugs, in comparison with reference products, have the same structure, efficacy, safety, and quality, although there may be slight differences due to the complexities of the production process.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>,<xref rid=\"B8\" ref-type=\"bibr\">8</xref>]</sup> As both the reference and biosimilar drugs have a degree of natural variability, studies are necessary to ensure that these differences do not affect the safety of intravitreal injection of these drugs. These biosimilar drugs can increase the community access to biological drugs, such as anti-VEGF agents, and can also reduce the burden on the healthcare budget.<sup>[<xref rid=\"B15\" ref-type=\"bibr\">15</xref>]</sup>\n</p><p>The biosimilar ranibizumab (RazumabⓇ; Intas Pharmaceuticals) is the first ophthalmic biosimilar that has been developed in India.<sup>[<xref rid=\"B7\" ref-type=\"bibr\">7</xref>]</sup> It has undergone animal safety studies and larger humanized head-to-head studies to ensure close resemblance in pharmacokinetic and pharmacodynamics characteristics, safety, and efficacy to the reference drug. Intravitreal injection of Zybev (a biosimilar of bevacizumab) has also shown to be safe, based on different studies.<sup>[<xref rid=\"B16\" ref-type=\"bibr\">16</xref>]</sup>\n</p><p>The bevacizumab biosimilar (Stivant; CinnaGen Co) is the first biosimilar to an anti-VEGF agent that has been developed in Iran. Large studies in patients with systemic cancers have been conducted to ensure close resemblance in safety and efficacy with the reference product (AvastinⓇ). To assess the safety of intravitreal injection of this biosimilar drug, we performed intravitreal injections of Stivant at the double dosage in albino New Zealand rabbits and investigated the retinal function and histological findings. The intravitreal injection of bevacizumab may be potentially toxic to the eye through the following three mechanisms: first, the vehicle could be toxic itself; second, toxicity could be due to induction of an immune response by injecting an immunoglobulin; and third, toxicity could be due to interference with endogenous VEGF signaling.<inline-formula><mml:math id=\"M189\"><mml:msup><mml:mrow/><mml:mrow><mml:mo>[</mml:mo><mml:mn>27</mml:mn><mml:mo>]</mml:mo></mml:mrow></mml:msup></mml:math></inline-formula>\n</p><p>Manzano et al<sup>[<xref rid=\"B17\" ref-type=\"bibr\">17</xref>]</sup> evaluated the safety of the intravitreal injection of the reference drug (AvastinⓇ). They defined at least a 30% reduction in ERG wave amplitudes to be significant, whereas we considered a lower threshold (20%) for ERG amplitude and latency changes to be significant. The ERG wave amplitude and latency changes were less than 10% at different time points in our study, except for a 19.8% reduction in a wave amplitude at week 1 after Stivant injection. This reduction was not observed two and four weeks after injections. One week after Avastin injection, we also observed a 13% reduction in the a-wave amplitude, which was reversed in the following weeks. These reductions may be due to a transient effect of the drugs on photoreceptors in the early post-injection period. We observed a similar but earlier reduction in the wave amplitude after saline injection; 48 hours post injection.</p><p>In this study, we report that intravitreal injection of a high dose of the biosimilar bevacizumab (Stivant) in rabbit eyes is well tolerated, at least in the short-term. The vitreous volume of a rabbit eye is approximately 1.5 mL and that of a human eye is approximately 5 mL. As the dose of 1.25 mg of bevacizumab is frequently used in humans, the doses of 1.25 and 2.5 mg of bevacizumab in rabbits result in approximately 3.3 and 6.6 times concentration of the medication in human eyes, respectively. Similar to some previous studies that evaluated the safety profile of a new anti-VEGF on the retinal tissue, we used 2.5 mg of this biosimilar drug to evaluate the safety of a higher concentration.<sup>[<xref rid=\"B9\" ref-type=\"bibr\">9</xref>]</sup> However, the maximum safe dose of bevacizumab was not determined in this study.</p><p>This study has some limitations. The endpoint ERG of the study group did not have a significant change in parameters from baseline. However, this study evaluated the short-term changes after a single intravitreal injection and did not rule out the possibility that long-term follow-up, especially with more injections, might demonstrate inappropriate side effects. As ERG is primarily a functional test of the status of the photoreceptors and bipolar cells, normal ERG results do not exclude possible damage at the level of the retinal ganglion cells or their axons, although this was not seen in histological evaluations.<sup>[<xref rid=\"B18\" ref-type=\"bibr\">18</xref>,<xref rid=\"B19\" ref-type=\"bibr\">19</xref>]</sup> Conversely, safety based on histological findings by light microscopy does not rule out possible changes at the submicroscopic level.<sup>[<xref rid=\"B20\" ref-type=\"bibr\">20</xref>]</sup> Therefore, it is better to design a study to perform immunocytochemical analysis on the histopathologic sections to evaluate the possible damage to retinal microstructures. Another limitation of the study was the disparity between the study group and the control groups in terms of the number of anesthesia sessions before enucleation and lack of tissue for histopathological evaluation in the control groups at time points earlier than four weeks after injections.</p><p>In summary, a single intravitreal injection of the biosimilar to bevacizumab (Stivant) up to a dose of 2.5 mg (high concentration) in the eyes of albino rabbits did not appear to be toxic for the retina. These results suggest that this drug could be a safe and cost-effective alternative to the reference drug. However, further investigations are needed to evaluate the long-term safety and efficacy of this biosimilar drug.</p></sec><sec sec-type=\"section\"><title> Financial Support and Sponsorship</title><p>The study received support from the Eye Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences, TUMS#38687.</p></sec><sec sec-type=\"COI-statement\"><title> Conflicts of Interest</title><p>There are no conflicts of interest.</p></sec></body><back><ref-list><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"other\">Shinoda K, Ishida S, Kawashima S, Wakabayashi T, Uchita M, Matsuzaki T, et al. Clinical factors related to the aqueous levels of vascular endothelial growth factor and hepatocyte growth factor in proliferative diabetic retinopathy. <italic>Curr Eye Res</italic> 2000;21:655–661.</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"other\">Rezzola S, Nawaz MI, Cancarini A, Semeraro F, Presta M. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Antimicrob Resist Infect Control</journal-id><journal-id journal-id-type=\"iso-abbrev\">Antimicrob Resist Infect Control</journal-id><journal-title-group><journal-title>Antimicrobial Resistance and Infection Control</journal-title></journal-title-group><issn pub-type=\"epub\">2047-2994</issn><publisher><publisher-name>BioMed Central</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32811557</article-id><article-id pub-id-type=\"pmc\">PMC7431751</article-id><article-id pub-id-type=\"publisher-id\">796</article-id><article-id pub-id-type=\"doi\">10.1186/s13756-020-00796-5</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Research</subject></subj-group></article-categories><title-group><article-title>Estimating length of stay and inpatient charges attributable to hospital-acquired bloodstream infections</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Yuzheng</given-names></name><address><email>zhang.yuzheng@hku.hk</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Du</surname><given-names>Mingmei</given-names></name><address><email>dumingm@163.com</email></address><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Johnston</surname><given-names>Janice Mary</given-names></name><address><email>jjohnsto@hku.hk</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Andres</surname><given-names>Ellie Bostwick</given-names></name><address><email>eandres@hku.hk</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Suo</surname><given-names>Jijiang</given-names></name><address><email>sjj301@qq.com</email></address><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yao</surname><given-names>Hongwu</given-names></name><address><email>arenewu@163.com</email></address><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Huo</surname><given-names>Rui</given-names></name><address><email>huoray@xinglin-tech.com</email></address><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Liu</surname><given-names>Yunxi</given-names></name><address><email>liuyunxi301@qq.com</email></address><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-4523-608X</contrib-id><name><surname>Fu</surname><given-names>Qiang</given-names></name><address><email>mpazy@sina.com</email></address><xref ref-type=\"aff\" rid=\"Aff4\">4</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.194645.b</institution-id><institution-id institution-id-type=\"ISNI\">0000000121742757</institution-id><institution>School of Public Health, </institution><institution>The University of Hong Kong, </institution></institution-wrap>Patrick Manson Building, 7 Sassoon Road, Pokfulam, Hong Kong, China </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.414252.4</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1761 8894</institution-id><institution>Department of Infection Management and Disease Control, </institution><institution>Chinese PLA General Hospital, </institution></institution-wrap>No. 28 Fuxing Road, Haidian District, Beijing, China </aff><aff id=\"Aff3\"><label>3</label>XingLin Information Technology Company, No. 57 Jianger Road, Binjiang District, Zhejiang, Hangzhou China </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.433167.4</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 6068 0087</institution-id><institution>China National Health Development Research Center, </institution></institution-wrap>No.9 Chegongzhuang Street, Xicheng District, Beijing, China </aff><aff id=\"Aff5\"><label>5</label>National Center for Healthcare Associated Infection Prevention and Control, Beijing, China </aff></contrib-group><pub-date pub-type=\"epub\"><day>18</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>18</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>9</volume><elocation-id>137</elocation-id><history><date date-type=\"received\"><day>19</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>5</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>. The Creative Commons Public Domain Dedication waiver (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/publicdomain/zero/1.0/\">http://creativecommons.org/publicdomain/zero/1.0/</ext-link>) applies to the data made available in this article, unless otherwise stated in a credit line to the data.</license-p></license></permissions><abstract id=\"Abs1\"><sec><title>Background</title><p id=\"Par1\">Hospital-acquired bloodstream infection (BSI) is associated with high morbidity and mortality and increases patients’ length of stay (LOS) and hospital charges. Our goals were to calculate LOS and charges attributable to BSI and compare results among different models.</p></sec><sec><title>Methods</title><p id=\"Par2\">A retrospective observational cohort study was conducted in 2017 in a large general hospital, in Beijing. Using patient-level data, we compared the attributable LOS and charges of BSI with three models: 1) conventional non-matching, 2) propensity score matching controlling for the impact of potential confounding variables, and 3) risk set matching controlling for time-varying covariates and matching based on propensity score and infection time.</p></sec><sec><title>Results</title><p id=\"Par3\">The study included 118,600 patient admissions, 557 (0.47%) with BSI. Six hundred fourteen microorganisms were cultured from patients with BSI. <italic>Escherichia coli</italic> was the most common bacteria (106, 17.26%). Among multi-drug resistant bacteria, carbapenem-resistant <italic>Acinetobacter baumannii</italic> (CRAB) was the most common (42, 38.53%). In the conventional non-matching model, the excess LOS and charges associated with BSI were 25.06 days (<italic>P</italic> < 0.05) and US$22041.73 (<italic>P</italic> < 0.05), respectively. After matching, the mean LOS and charges attributable to BSI both decreased. When infection time was incorporated into the risk set matching model, the excess LOS and charges were 16.86 days (<italic>P</italic> < 0.05) and US$15909.21 (<italic>P</italic> < 0.05), respectively.</p></sec><sec><title>Conclusion</title><p id=\"Par4\">This is the first study to consider time-dependent bias in estimating excess LOS and charges attributable to BSI in a Chinese hospital setting. We found matching on infection time can reduce bias.</p></sec></abstract><kwd-group xml:lang=\"en\"><title>Keywords</title><kwd>Hospital-acquired bloodstream infection</kwd><kwd>Length of stay</kwd><kwd>Hospital charge</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Background</title><p id=\"Par5\">Bloodstream infection (BSI) is a serious adverse event associated with high morbidity and mortality. Studies in Canada demonstrated that 28% of BSIs are nosocomial [<xref ref-type=\"bibr\" rid=\"CR1\">1</xref>], and data from North America and Europe showed BSI ranks among the top seven causes of death [<xref ref-type=\"bibr\" rid=\"CR2\">2</xref>]. Our previous study in a Chinese tertiary hospital indicated an increasing incidence of BSI from 0.53 to 0.65 per 1000 patient-days over 5 years (2013–2017) [<xref ref-type=\"bibr\" rid=\"CR3\">3</xref>]. BSIs are also associated with increased length of stay (LOS) and medical costs [<xref ref-type=\"bibr\" rid=\"CR4\">4</xref>–<xref ref-type=\"bibr\" rid=\"CR6\">6</xref>]. However, infection control programs are often regarded as cost centers and potential areas for budget cuts rather than revenue generators [<xref ref-type=\"bibr\" rid=\"CR7\">7</xref>]. In fact, reduced healthcare associated infections (HAI) and LOS could increase the bed turnover rate and hospital revenue [<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>]. Appropriate analysis methods can help decision-makers understand the clinical and economic burden of BSIs and choose the most cost-effective infection control strategies.</p><p id=\"Par6\">Estimations of BSI hospital related costs vary widely. Most studies do not account for the time-varying nature of HAIs, thereby overestimating the cost of BSI episodes [<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>, <xref ref-type=\"bibr\" rid=\"CR10\">10</xref>]. In conventional methods, the time spent in the hospital prior to the occurrence of the HAI is incorrectly attributed to the HAI, thus inflating attributable LOS and cost [<xref ref-type=\"bibr\" rid=\"CR11\">11</xref>]. Two methods have been used to overcome the overestimation problem: multistate model and matching on infection time [<xref ref-type=\"bibr\" rid=\"CR11\">11</xref>]. Multistate models treat the infection as one of several mutually exclusive states (e.g. discharge/death) after a patient’s admission. The method of matching on infection time (risk-set matching) matches each BSI case with a comparable control patient at risk of infection at the cases’ infection time [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>].</p><p id=\"Par7\">Variance in health insurance and reimbursement systems also impact attributable LOS and cost of BSI. Currently, there are few economic studies about HAI in Mainland China. Studies from Hubei and Sichuan demonstrated that the attributable cost of HAI were US$6173.02 and $2439.77 per case, respectively [<xref ref-type=\"bibr\" rid=\"CR13\">13</xref>, <xref ref-type=\"bibr\" rid=\"CR14\">14</xref>]. Another Chinese study of catheter-related bloodstream infections indicated the total cost attributable was US $3528.6 per case [<xref ref-type=\"bibr\" rid=\"CR15\">15</xref>]. However, these HAI cost studies in Mainland China did not account for disease severity or time-dependent bias. Hence, our primary objective is to estimate the LOS and hospital charges attributable to BSI with time-varying exposures, and to compare our results to those obtained using conventional methods to understand the magnitude of the time-dependent biases.</p></sec><sec id=\"Sec2\"><title>Methods</title><sec id=\"Sec3\"><title>Data sources</title><p id=\"Par8\">A retrospective observational cohort study was conducted at a tertiary hospital with 3800 beds in Beijing. The hospital conducted hospital-wide HAI surveillance with a real-time nosocomial infection surveillance system (RT-NISS). The study included patients admitted and discharged between January 1st and December 31st 2017. We excluded patients in outpatient settings, physical examination centers, and day surgery centers. There was no age limit. Data was divided between two groups: (1) cases, including patients with nosocomial bloodstream infections and (2) controls, comprised of patients without BSIs (or other HAIs). To protect patient privacy, the study excluded sensitive patient identifiers (e.g. name and identification numbers). Ethical approval (number: S2019–142-02) was obtained from the Medical Ethical Committee of the Chinese PLA General Hospital.</p></sec><sec id=\"Sec4\"><title>Case definition</title><p id=\"Par9\">BSIs were identified should meet the following criteria: (1) isolation of bacteria from at least one blood culture, (2) exclusion of contaminated blood samples during the collection and culture, (3) one of the following clinical symptoms: fever (> 38 °C), chills, or hypotension. Only one blood culture positive of the common skin commensals organisms (e.g. coagulase-negative staphylococcus [CoNS], non-<italic>diphtheriae Corynebacterium</italic> spp., <italic>Bacillus</italic> spp., <italic>Propionibacterium</italic> spp., viridans group streptococci, <italic>Aerococcus</italic> spp., and <italic>Micrococcus</italic> spp.) were excluded [<xref ref-type=\"bibr\" rid=\"CR16\">16</xref>]. Based on the BSI criteria, hospital-acquired BSIs were defined as the first positive blood culture obtained≥48 h after hospital admission and with no evidence of infection at admission. Time of infection was defined as the day of physician confirmation, which generally corresponded with the collection time of first positive blood sample.</p></sec><sec id=\"Sec5\"><title>Microbiological test</title><p id=\"Par10\">Blood was cultured with BacT/ALERT 3D system (Becton-Dickinson, Sparks, MD, USA). Microorganism species were identified using the VITEK 2 system (BioMérieux, Marcy 1 Étoile, France). Antibiotic susceptibility testing was determined by the VITEK 2 system or the Kirby-Bauer Disk Diffusion method (Oxford, UK) in accordance with the guideline proposed by the Clinical and Laboratory Standards Institute (CLSI). According to the World Health Organization (WHO) priority list of antibiotic-resistant bacteria, the study included corresponding antibiotic-resistant bacteria responsible for BSI, such as: carbapenem-resistant <italic>Acinetobacter baumannii</italic>, carbapenem-resistant <italic>Pseudomonas aeruginosa</italic> [<xref ref-type=\"bibr\" rid=\"CR17\">17</xref>].</p></sec><sec id=\"Sec6\"><title>Variables of interest</title><p id=\"Par11\">Demographic information (age, sex, region, insurance type), diagnosis code (based on ICD-10), intensive care unit (ICU), and procedure-related information (receiving surgery, central line, urinary catheter, ventilator) were collected. The Charlson comorbidity index was calculated for each patient to capture the severity of comorbidities [<xref ref-type=\"bibr\" rid=\"CR18\">18</xref>].</p><p id=\"Par12\">The outcomes of interest were LOS and inpatient charges. Hospital charges are the hospital fees for services, medicine, and materials, etc. The patients’ individual hospital charges were retrieved from the hospital information system. Mean charge was used for two patients with missing hospital charge data. Medical charges were collected in the Chinese currency Renminbi and converted into US dollars ($) according to the exchange rate (1USD = 6.53 RMB) issued by the Bank of China on 31 December 2017.</p></sec><sec id=\"Sec7\"><title>Statistical analysis</title><p id=\"Par13\">We conducted the analyses with three different models. Model 1, conventional non-matching, compares the BSI patients’ charges and LOS with those of patients without BSI. This method ignores confounders and the time-dependent nature of costs and LOS.</p><p id=\"Par14\">Model 2, conventional propensity score matching (PSM), estimates the propensity scores with logistic regression based on the variables listed in Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>. The matching variables (i.e. status of central line, urinary catheter, and ventilator before infection) include only information available at time of admission (or within the first 2 days). Instead of exact matching the BSI and non-BSI group on the dependent variables, the PSM matches by the propensity score at a 1:1 ratio. We used nearest-neighbor matching with a caliper width of 0.25.</p><p id=\"Par15\">Model 3, risk-set matching, matches patients who experienced a HAI on a specific day to similar patients who have not yet experienced HAIs at that point in their hospital admission. We estimated the risk-set propensity score using Cox proportional hazards regression. The survival outcome was either BSI (i.e., an event) occurring and its corresponding time or BSI not occurring (i.e., a censored event) and the length of stay in the hospital (i.e., censoring time). The same dependent variables included in Model 2 were used in Model 3, except for some time-varying covariates. These variables (receiving central line, urinary catheter, ventilator) were recoded weekly. The risk-set propensity scores were estimated by linear prediction with the time-varying Cox regression model, which was described in previous studies [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>, <xref ref-type=\"bibr\" rid=\"CR19\">19</xref>]. A nearest-neighbor matching was applied with the risk-set propensity score. Each BSI case infected at time T<sub>1</sub> is matched to a patient not yet infected at time T<sub>1</sub> rather than a never-infected patient. The matched patient in the control group may acquire an infection later than T<sub>1</sub>, in which case they would be classified in both the infected and uninfected group.</p><p id=\"Par16\">The degree of balance between the matched pairs were evaluated using standardized mean differences (SMD). The cutoff for interpreting the magnitude of SMD was defined as followings: small, 0.2; medium, 0.5; and large, 0.8 [<xref ref-type=\"bibr\" rid=\"CR20\">20</xref>]. Continuous and categorical variables were compared between BSI and non-BSI groups using t-test and Chi-square test, respectively. Moreover, a sensitivity analysis was conducted with defining the infection time 2 days before the original infection date to account for a 48-h incubation period. Statistical analyses were performed using R version 3.4.3.</p></sec></sec><sec id=\"Sec8\"><title>Results</title><sec id=\"Sec9\"><title>Patients and matching</title><p id=\"Par17\">In total, 118,600 patient admissions were included in the study and 557 (0.47%) BSI were identified. Patient characteristics are presented in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>. Mean age of all patients was 51.12 years and 53.88% were male. The majority of patients (60.79%) were from northern China (Beijing, Tianjin, Shanxi, Hebei, Inner Mongolia). 33,580 (28.31%) patients had public insurance, 77.19% of which were from Beijing. The remaining 22.81% patients with public insurance cannot claim medical fees from the government since they do not reside in Beijing (public insurance is tied to resident location).\n<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Patient Characteristics before and after matching between the case and control groups, 2017</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\"/><th rowspan=\"2\">BSI (<bold><italic>N</italic></bold> = 557)</th><th colspan=\"2\">Model 1</th><th colspan=\"2\">Model 2</th><th colspan=\"2\">Model 3</th></tr><tr><th>No BSI (<bold><italic>N</italic></bold> = 118,043)</th><th>SMD<sup><bold>*</bold></sup></th><th>No BSI (<bold><italic>N</italic></bold> = 557)</th><th>SMD<sup><bold>*</bold></sup></th><th>Control (<bold><italic>N</italic></bold> = 557)</th><th>SMD<sup><bold>*</bold></sup></th></tr></thead><tbody><tr><td>Age (mean, SD)</td><td>57.02 (21.3)</td><td>51.09 (18.4)</td><td char=\".\" align=\"char\">0.297</td><td>56.75 (18.1)</td><td char=\".\" align=\"char\">0.013</td><td>56.29 (17.82)</td><td char=\".\" align=\"char\">0.036</td></tr><tr><td>Sex, female (n, %)</td><td>195 (35.0)</td><td>54,505 (46.2)</td><td char=\".\" align=\"char\">0.229</td><td>206 (37.0)</td><td char=\".\" align=\"char\">0.041</td><td>224 (40.2)</td><td char=\".\" align=\"char\">0.108</td></tr><tr><td>Insurance, yes (n, %)</td><td>164 (29.4)</td><td>33,386 (28.3)</td><td char=\".\" align=\"char\">0.026</td><td>157 (28.2)</td><td char=\".\" align=\"char\">0.028</td><td>126 (29.3)</td><td char=\".\" align=\"char\">0.156</td></tr><tr><td>Region (n, %)</td><td/><td/><td char=\".\" align=\"char\">0.161</td><td/><td char=\".\" align=\"char\">0.111</td><td/><td char=\".\" align=\"char\">0.078</td></tr><tr><td> Northeast</td><td>52 (9.3)</td><td>13,192 (11.2)</td><td/><td>67 (12.0)</td><td/><td>47 (8.4)</td><td/></tr><tr><td> Eastern</td><td>80 (14.4)</td><td>18,091 (15.3)</td><td/><td>86 (15.4)</td><td/><td>77 (13.8)</td><td/></tr><tr><td> Northern</td><td>348 (62.5)</td><td>71,757 (60.8)</td><td/><td>337 (60.5)</td><td/><td>356 (63.9)</td><td/></tr><tr><td> Central</td><td>30 (5.4)</td><td>8387 (7.1)</td><td/><td>23 (4.1)</td><td/><td>32 (5.7)</td><td/></tr><tr><td> Southern</td><td>9 (1.6)</td><td>598 (0.5)</td><td/><td>9 (1.6)</td><td/><td>5 (0.9)</td><td/></tr><tr><td> Southwest</td><td>10 (1.8)</td><td>1698 (1.4)</td><td/><td>8 (1.4)</td><td/><td>10 (1.8)</td><td/></tr><tr><td> Northwest</td><td>28 (5.0)</td><td>4320 (3.7)</td><td/><td>27 (4.8)</td><td/><td>30 (5.4)</td><td/></tr><tr><td>Charlson score (mean, SD)</td><td>2.2 (2.6)</td><td>1.6 (2.7)</td><td char=\".\" align=\"char\">0.216</td><td>2.4 (2.9)</td><td char=\".\" align=\"char\">0.066</td><td>2.4 (2.9)</td><td char=\".\" align=\"char\">0.076</td></tr><tr><td>ICU (n, %)</td><td>91 (16.2)</td><td>4577 (3.9)</td><td char=\".\" align=\"char\">0.422</td><td>84 (15.3)</td><td char=\".\" align=\"char\">0.030</td><td>79 (16.9)</td><td char=\".\" align=\"char\">0.060</td></tr><tr><td colspan=\"8\">Receive any procedures</td></tr><tr><td> Surgery (n, %)</td><td>153 (27.5)</td><td>41,014 (34.7)</td><td char=\".\" align=\"char\">0.158</td><td>155 (27.8)</td><td char=\".\" align=\"char\">0.008</td><td>126 (22.6)</td><td char=\".\" align=\"char\">0.112</td></tr><tr><td> Central line catheter (n, %)</td><td>246 (44.2)</td><td>10,207 (8.6)</td><td char=\".\" align=\"char\">0.880</td><td>253 (45.4)</td><td char=\".\" align=\"char\">0.025</td><td>213 (38.2)</td><td char=\".\" align=\"char\">0.121</td></tr><tr><td> Urinary catheter (n, %)</td><td>235 (42.2)</td><td>29,255 (24.8)</td><td char=\".\" align=\"char\">0.375</td><td>220 (39.5)</td><td char=\".\" align=\"char\">0.055</td><td>213 (38.2)</td><td char=\".\" align=\"char\">0.081</td></tr><tr><td> Ventilator (n, %)</td><td>132 (23.7)</td><td>4328 (3.7)</td><td char=\".\" align=\"char\">0.609</td><td>136 (24.4)</td><td char=\".\" align=\"char\">0.017</td><td>92 (16.5)</td><td char=\".\" align=\"char\">0.180</td></tr><tr><td>Primary discharge diagnosis (ICD-10) (n, %)</td><td/><td/><td char=\".\" align=\"char\">0.665</td><td/><td char=\".\" align=\"char\">0.175</td><td/><td char=\".\" align=\"char\">0.259</td></tr><tr><td> A00-B99</td><td>16 (2.9)</td><td>796 (0.7)</td><td/><td>15 (2.7)</td><td/><td>14 (2.5)</td><td/></tr><tr><td> C00-D48</td><td>126 (22.6)</td><td>24,090 (20.4)</td><td/><td>145 (26.0)</td><td/><td>130 (23.3)</td><td/></tr><tr><td> D50-D89</td><td>5 (0.9)</td><td>384 (0.3)</td><td/><td>5 (0.9)</td><td/><td>3 (0.5)</td><td/></tr><tr><td> E00-E90</td><td>1 (0.2)</td><td>2910 (2.5)</td><td/><td>0</td><td/><td>2 (0.4)</td><td/></tr><tr><td> F00-F99</td><td>0</td><td>194 (0.2)</td><td/><td>0</td><td/><td>0</td><td/></tr><tr><td> G00-G99</td><td>13 (2.3)</td><td>2331 (2.0)</td><td/><td>12 (2.2)</td><td/><td>11 (2.0)</td><td/></tr><tr><td> H00-H59</td><td>0</td><td>3146 (2.7)</td><td/><td>0</td><td/><td>0</td><td/></tr><tr><td> H60-H95</td><td>0</td><td>2290 (1.9)</td><td/><td>0</td><td/><td>0</td><td/></tr><tr><td> I00-I99</td><td>54 (9.7)</td><td>15,562 (13.2)</td><td/><td>49 (8.8)</td><td/><td>37 (6.6)</td><td/></tr><tr><td> J00-J99</td><td>42 (7.5)</td><td>3118 (2.6)</td><td/><td>37 (6.6)</td><td/><td>25 (4.5)</td><td/></tr><tr><td> K00-K93</td><td>80 (14.4)</td><td>7673 (6.5)</td><td/><td>81 (14.5)</td><td/><td>84 (15.1)</td><td/></tr><tr><td> L00-L99</td><td>1 (0.2)</td><td>860 (0.7)</td><td/><td>0</td><td/><td>1 (0.2)</td><td/></tr><tr><td> M00-M99</td><td>15 (2.7)</td><td>8178 (6.9)</td><td/><td>10 (1.8)</td><td/><td>8 (1.4)</td><td/></tr><tr><td> N00-N99</td><td>25 (4.5)</td><td>7336 (6.2)</td><td/><td>18 (3.2)</td><td/><td>25 (4.5)</td><td/></tr><tr><td> O00-O99</td><td>3 (0.5)</td><td>2841 (2.4)</td><td/><td>1 (0.2)</td><td/><td>5 (0.9)</td><td/></tr><tr><td> P00-P96</td><td>6 (1.1)</td><td>656 (0.6)</td><td/><td>8 (1.4)</td><td/><td>8 (1.4)</td><td/></tr><tr><td> Q00-Q99</td><td>4 (0.7)</td><td>1760 (1.5)</td><td/><td>6 (1.1)</td><td/><td>3 (0.5)</td><td/></tr><tr><td> R00-R99</td><td>5 (0.9)</td><td>729 (0.6)</td><td/><td>5 (0.9)</td><td/><td>6 (1.1)</td><td/></tr><tr><td> S00-T98</td><td>34 (6.1)</td><td>3265 (2.8)</td><td/><td>29 (5.2)</td><td/><td>27 (4.8)</td><td/></tr><tr><td> Z00-Z99</td><td>127 (22.8)</td><td>29,913 (25.3)</td><td/><td>136 (24.4)</td><td/><td>168 (30.2)</td><td/></tr></tbody></table><table-wrap-foot><p>Model 1, no matching; Model 2, conventional propensity score matching; Model 3, risk set matching. <italic>BSI</italic> Bloodstream infection, <italic>SMD</italic> Standardized mean difference. <italic>A00-B99</italic> Certain infectious and parasitic diseases, <italic>C00-D48</italic> Neoplasms, <italic>D50-D89</italic> Diseases of the blood and blood-forming organs and certain disorders involving the immune mechanism, <italic>E00-E90</italic> Endocrine, nutritional and metabolic diseases, <italic>F00-F99</italic> Mental and behavioral disorders, <italic>G00-G99</italic> Diseases of the nervous system, <italic>H00-H59</italic> Diseases of the eye and adnexa, <italic>H60-H95</italic> Diseases of the ear and mastoid process, <italic>I00-I99</italic> Diseases of the circulatory system, <italic>J00-J99</italic> Diseases of the respiratory system, <italic>K00-K93</italic> Diseases of the digestive system, <italic>L00-L99</italic> Diseases of the skin and subcutaneous tissue, <italic>M00-M99</italic> Diseases of the musculoskeletal system and connective tissue, <italic>N00-N99</italic> Diseases of the genitourinary system, <italic>O00-O99</italic> Pregnancy, childbirth and the puerperium, <italic>P00-P96</italic> Certain conditions originating in the perinatal period, <italic>Q00-Q99</italic> Congenital malformations, deformations and chromosomal abnormalities, <italic>R00-R99</italic> Symptoms, signs and abnormal clinical and laboratory findings, not elsewhere classified, <italic>S00-T98</italic> Injury, poisoning and certain other consequences of external causes, <italic>Z00-Z99</italic> Factors influencing health status and contact with health services</p></table-wrap-foot></table-wrap></p><p id=\"Par18\">Before matching, statistically significant differences (SMD > 0.2) existed between case and control groups for almost all variables except insurance, region and receiving surgery. Matching mitigated these imbalances through both conventional PSM and risk set matching. However, compared to the risk set matching, conventional PSM achieved greater balance between case and control groups.</p></sec><sec id=\"Sec10\"><title>LOS and charges</title><p id=\"Par19\">The results for LOS and charges in the different models are presented in Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>. In model 1, the attributable mean LOS and charges for those with and without BSI were 25.06 days and US$22041.73, respectively. The results in model 2 and 3 were both smaller than the baseline (model 1). Attributable LOS was less in the risk set matching model (16.86 days) than in the conventional propensity score matching model (21.27 days). Likewise, charges attributable to BSI were less in model 3 ($15,909.21) than model 2 ($18,549.47). Regrading to the component of additional hospital charges in the three models revealed that western medicine accounted for the largest proportion of charges, followed by laboratory and treatment (without surgery) fee.\n<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Comparison of outcomes with different models, 2017</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\">Outcome</th><th rowspan=\"2\">BSI</th><th colspan=\"2\">Model 1</th><th colspan=\"2\">Model 2</th><th colspan=\"2\">Model 3</th></tr><tr><th>Mean</th><th>Difference</th><th>Mean</th><th>Difference</th><th>Mean</th><th>Difference</th></tr></thead><tbody><tr><td>LOS, days</td><td>34.45</td><td>9.39</td><td>25.06<sup>*</sup></td><td>13.18</td><td>21.27<sup>*</sup></td><td>17.59</td><td>16.86<sup>*</sup></td></tr><tr><td>Total charges, USD</td><td>27,102.16</td><td>5060.43</td><td>22,041.73<sup>*</sup></td><td>8552.69</td><td>18,549.47<sup>*</sup></td><td>11,192.94</td><td>15,909.21<sup>*</sup></td></tr><tr><td colspan=\"8\">Treatment, USD</td></tr><tr><td> Treatment (no-surgery)</td><td>3119.80</td><td>357.47</td><td>2762.32<sup>*</sup></td><td>744.72</td><td>2375.08<sup>*</sup></td><td>1140.00</td><td>1979.80<sup>*</sup></td></tr><tr><td> Surgery</td><td>551.73</td><td>426.62</td><td>125.11<sup>*</sup></td><td>564.72</td><td>−12.99</td><td>620.70</td><td>−68.79</td></tr><tr><td> Nursing</td><td>223.48</td><td>37.14</td><td>186.34<sup>*</sup></td><td>63.54</td><td>159.94<sup>*</sup></td><td>96.03</td><td>127.45<sup>*</sup></td></tr><tr><td> Anesthesia</td><td>82.26</td><td>54.21</td><td>28.05<sup>*</sup></td><td>70.68</td><td>11.58</td><td>69.87</td><td>12.39<sup>*</sup></td></tr><tr><td colspan=\"8\">Laboratory & examination, USD</td></tr><tr><td> Laboratory</td><td>3713.34</td><td>543.02</td><td>3170.31<sup>*</sup></td><td>1111.61</td><td>2601.72<sup>*</sup></td><td>1327.96</td><td>2385.37<sup>*</sup></td></tr><tr><td> Radiology</td><td>610.32</td><td>242.84</td><td>367.48<sup>*</sup></td><td>295.55</td><td>314.76<sup>*</sup></td><td>353.69</td><td>256.62<sup>*</sup></td></tr><tr><td colspan=\"8\">Medicine & material, USD</td></tr><tr><td> Western medicine</td><td>13,322.52</td><td>1573.54</td><td>11,748.98<sup>*</sup></td><td>3477.33</td><td>9845.19<sup>*</sup></td><td>4747.36</td><td>8575.16<sup>*</sup></td></tr><tr><td> Medical material</td><td>4629.44</td><td>1961.10</td><td>2668.34<sup>*</sup></td><td>2404.98</td><td>2224.46<sup>*</sup></td><td>2845.10</td><td>1784.34<sup>*</sup></td></tr><tr><td> Blood</td><td>976.72</td><td>221.17</td><td>755.54<sup>*</sup></td><td>298.85</td><td>680.86<sup>*</sup></td><td>547.96</td><td>428.75<sup>*</sup></td></tr><tr><td> Chinese medicine</td><td>62.44</td><td>70.36</td><td>−7.91</td><td>44.04</td><td>18.41</td><td>60.69</td><td>1.75</td></tr><tr><td>Bed & meal, USD</td><td>696.74</td><td>149.77</td><td>546.97<sup>*</sup></td><td>215.15</td><td>481.59<sup>*</sup></td><td>328.49</td><td>368.25<sup>*</sup></td></tr><tr><td>Other, USD</td><td>47.94</td><td>14.63</td><td>33.31<sup>*</sup></td><td>20.62</td><td>27.32<sup>*</sup></td><td>25.78</td><td>22.16<sup>*</sup></td></tr></tbody></table><table-wrap-foot><p>Model 1, no matching; Model 2, conventional propensity score matching; Model 3, risk set matching. <italic>BSI</italic> Bloodstream infection, <italic>LOS</italic> Length of stay. T test was applied for mean cost and LOS. *<italic>P</italic>-value< 0.05</p></table-wrap-foot></table-wrap></p><p id=\"Par20\">Results were similar for the sensitivity analysis that defined infection time as 2 days before the original infection date. For conventional matching (model 2), the attributable LOS due to BSI was 21.65 days, and the attributable costs were $19,530.87. These estimates were also much larger than estimates using risk-set matching (model 3), with additional 15.97 days in LOS and $15,914.98 in hospital charges due to BSI.</p></sec><sec id=\"Sec11\"><title>Microorganisms and antimicrobial resistance bacteria</title><p id=\"Par21\">The isolated microorganisms and multi-drug resistance microorganisms of BSI are summarized in Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref>. Six hundred fourteen microorganisms were cultured from 557 episodes of BSI, most of the isolated pathogens (82.76%) were monomicrobial. The proportion of gram-negative versus gram-positive bacteria was 56.18% versus 31.92%. <italic>Escherichia coli</italic> was the most common bacteria (106, 17.26%). Among the 109 multi-drug resistant bacteria, carbapenem-resistant <italic>Acinetobacter baumannii</italic> (CRAB) was the most common (42, 38.53%).\n<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Isolated microorganisms and multi-drug resistance microorganisms involved in 557 episodes of bloodstream infection (BSI)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Microorganism</th><th>No. (%) of isolations</th><th>No. (%) of episodes with monomicrobial</th><th>No. (%) of isolations of specific antimicrobial-resistant pathogen</th></tr></thead><tbody><tr><td><bold>Gram-negative bacteria</bold></td><td><bold>345 (56.18)</bold></td><td><bold>290 (84.06)</bold></td><td><bold>90 (26.09)</bold></td></tr><tr><td> <italic>Escherichia coli</italic></td><td>106 (30.72)</td><td>90 (84.91)</td><td>3 (2.83)<sup>a</sup></td></tr><tr><td> <italic>Klebsiella pneumoniae</italic></td><td>88 (25.51)</td><td>80 (90.91)</td><td>30 (34.09)<sup>a</sup></td></tr><tr><td> <italic>Acinetobacter baumannii</italic></td><td>52 (15.07)</td><td>41 (78.85)</td><td>42 (80.77)<sup>b</sup></td></tr><tr><td> <italic>Pseudomonas aeruginosa</italic></td><td>24 (6.96)</td><td>19 (79.17)</td><td>13 (54.17)<sup>c</sup></td></tr><tr><td> <italic>Enterobacter cloacae</italic></td><td>23 (6.67)</td><td>15 (65.22)</td><td>2 (8.70)<sup>a</sup></td></tr><tr><td> Other</td><td>52 (15.07)</td><td>45 (86.54)</td><td>–</td></tr><tr><td><bold>Gram-positive bacteria</bold></td><td><bold>196 (31.92)</bold></td><td><bold>162 (82.65)</bold></td><td><bold>19 (9.74)</bold></td></tr><tr><td> <italic>Enterococcus faecium</italic></td><td>42 (21.43)</td><td>33 (78.57)</td><td>1 (2.38)<sup>d</sup></td></tr><tr><td> <italic>Staphylococcus epidermidis</italic></td><td>26 (13.27)</td><td>25 (96.15)</td><td>14 (53.85)<sup>e</sup></td></tr><tr><td> <italic>Staphylococcus aureus</italic></td><td>23 (11.73)</td><td>23 (100)</td><td>4 (17.39)<sup>f</sup></td></tr><tr><td> <italic>Staphylococcus hominis</italic></td><td>16 (8.16)</td><td>12 (75)</td><td>–</td></tr><tr><td> <italic>Enterococcus faecalis</italic></td><td>13 (6.63)</td><td>8 (61.54)</td><td>–</td></tr><tr><td> Other</td><td>76 (38.78)</td><td>61 (80.26)</td><td>–</td></tr><tr><td><bold>Fungus</bold></td><td><bold>68 (11.07)</bold></td><td><bold>51 (75)</bold></td><td>–</td></tr><tr><td> <italic>Candida species</italic></td><td>65 (95.59)</td><td>48 (73.85)</td><td>–</td></tr><tr><td> Other</td><td>3 (4.41)</td><td>3 (100)</td><td>–</td></tr><tr><td><bold>Anaerobic bacteria</bold></td><td><bold>5 (0.81)</bold></td><td><bold>5 (100)</bold></td><td>–</td></tr><tr><td><bold>Total</bold></td><td><bold>614 (100)</bold></td><td><bold>508 (82.74)</bold></td><td><bold>109 (17.75)</bold></td></tr></tbody></table><table-wrap-foot><p><sup>a</sup>carbapenem-resistant <italic>Enterobacteriaceae</italic></p><p><sup>b</sup>carbapenem-resistant <italic>Acinetobacter baumannii</italic></p><p><sup>c</sup>carbapenem-resistant <italic>Pseudomonas aeruginosa</italic></p><p><sup>d</sup>vancomycin-resistant <italic>Enterococcus faecium</italic></p><p><sup>e</sup>methicillin-resistant <italic>Staphylococcus epidermidis</italic></p><p><sup>f</sup>methicillin-resistant <italic>Staphylococcus aureus</italic></p></table-wrap-foot></table-wrap></p></sec></sec><sec id=\"Sec12\"><title>Discussion</title><p id=\"Par22\">To our knowledge, this is the first analysis of LOS and charges attributable to HAIs in China to address confounding and time-dependent bias. Few international databases related to HAI collect infection time data [<xref ref-type=\"bibr\" rid=\"CR11\">11</xref>]. Even studies that include infection time usually lack information about time-varying covariates, such as catheter insertion and removal time [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>]. In this study, the real-time surveillance system collected and updated all HAI-related variables daily, allowing for time-dependent analysis based on dynamic information.</p><p id=\"Par23\">Baseline differences in age, sex, Charlson score, ICU admission, and procedures with device (central line catheter, urinary catheter, ventilator) between BSI and non-BSI patients were similar to a Belgian national surveillance study, however, the BSI patients’ average age (57.02 VS 66.9), Charlson comorbidity index (2.20 VS 3.10), and proportion of ICU admissions (16.2% VS 21.9%) were lower than the Belgian study [<xref ref-type=\"bibr\" rid=\"CR4\">4</xref>].</p><p id=\"Par24\">The most common BSI pathogen was <italic>Escherichia coli</italic> (17.26%), consistent with results from the European antimicrobial resistance surveillance study [<xref ref-type=\"bibr\" rid=\"CR21\">21</xref>]. However, other studies also report high BSI incidence due to <italic>Staphylococcus aureus</italic>, which was less common in our study (3.75%) [<xref ref-type=\"bibr\" rid=\"CR22\">22</xref>, <xref ref-type=\"bibr\" rid=\"CR23\">23</xref>]<italic>.</italic></p><p id=\"Par25\">Our results indicate LOS and charges attributable to HAI ranging from 16.86 to 25.06 days and $15,909.21 to $22,041.73, respectively. A systematic review found that LOS attributable to BSI ranged from 1.2–26.4 days, hence, our results were at the higher end relative to studies included in the review [<xref ref-type=\"bibr\" rid=\"CR6\">6</xref>]. The published studies showed that attributable BSI cost ranged from $1430 (Brazil) [<xref ref-type=\"bibr\" rid=\"CR24\">24</xref>] to $95,440 (US) [<xref ref-type=\"bibr\" rid=\"CR25\">25</xref>]. Compared to the international study, the attributable BSI charges in our study is relatively low. However, it’s much higher than the study about CLABSIs in China (US$3528.6) [<xref ref-type=\"bibr\" rid=\"CR15\">15</xref>].</p><p id=\"Par26\">Excess LOS is the main driver of hospital charges attributable to HAI. LOS and charges can be reduced through two potential approaches: BSI prevention and reductions in LOS for patients with BSI. A meta-analysis found that 57.78% of CLABSIs are preventable through intervention, such as insertion and maintenance bundle, which includes maximal barrier precautions, site selection, and better aseptic technique [<xref ref-type=\"bibr\" rid=\"CR26\">26</xref>].<italic>.</italic> Some studies indicate long-term sustainability of zero CLABSIs is possible through high compliance with the bundles and multidisciplinary team interventions [<xref ref-type=\"bibr\" rid=\"CR27\">27</xref>]. Some novel interventions introduced to shorten the LOS include real-time active alerts of positive blood cultures and antimicrobial stewardship intervention in patients with gram-negative bacteremia [<xref ref-type=\"bibr\" rid=\"CR28\">28</xref>].</p><p id=\"Par27\">To reduce time-dependent bias, we applied risk-set matching with time-varying Cox regression [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>], taking into consideration infection time as well as time-varying covariates (central venous catheter, urinary catheter, ventilator). The magnitude of time-dependent bias may vary depending on a series of factors, including infection rate, discharge rate, and confounders [<xref ref-type=\"bibr\" rid=\"CR11\">11</xref>]. Sensitivity analysis was used in this study to assess the impact of unobserved confounding and determine to what degree unobserved variable might explain the results [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>]. Findings from this study indicate future HAI surveillance should consider infection time (or specimen culture time). Time-varying procedures and treatments, such as device operation, surgery, and antibiotic administration should be recorded dynamically if possible.</p><p id=\"Par28\">This study was conducted from the hospital perspective, only calculating in-hospital medical charges. Owing to the non-profit nature of public hospitals in China, the charges were almost equal to the costs. In 2017, revenue and expenditure data from 44 top tertiary hospitals indicated that the charge-to-cost ratio was around 1.04 in China, compared to the value 2.0 in the U.S. [<xref ref-type=\"bibr\" rid=\"CR29\">29</xref>, <xref ref-type=\"bibr\" rid=\"CR30\">30</xref>]. Hence, public hospitals in China may have less motivation to actively prevent HAI and reduce patients’ cost than in other settings.</p><p id=\"Par29\">Moreover, less than 30% of patients had public insurance in this study. Public insurance is not reimbursable outside patients’ city of permanent residence, hence, 7660 (6.46%) non-local patients with public insurance were not eligible to use their insurance. Thus, much of the HAI economic burden was borne by patients’ out-of-pocket instead of the hospital, perhaps providing less incentive to prevent HAIs [<xref ref-type=\"bibr\" rid=\"CR31\">31</xref>]. However, actual economic savings include not only “cash savings” (variable cost), but also depend on the fixed costs, including buildings and equipment. While preventing HAI may not lead to “cash savings”, it could free up the limited medical resources for other revenue-generating activities [<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>].</p><p id=\"Par30\">In addition, payment reform was launched in Mainland China in 2017, gradually changing medical insurance payment methods from fee-for service (FFS) to Diagnosis Related Groups (DRGs) [<xref ref-type=\"bibr\" rid=\"CR32\">32</xref>]. The prospective payment method will shift more of the economic burden of HAI to hospitals. Therefore, hospital administrators may prioritize reducing LOS and costs attributable to HAIs going forward.</p><p id=\"Par31\">This study has several limitations. First, single center studies limit generalization. The economic level and healthcare service prices vary in different provinces in China. Second, selection bias cannot be avoided in observational cohort studies with matched samples. We used logistic regression and Cox regression to create propensity scores and conduct the matching, since the confounders included in the study would change the results of matching. Third, our study evaluated cost only from the hospital perspective, neither including indirect cost from societal perspectives, nor collected patients’ cost after hospital discharge.</p></sec><sec id=\"Sec13\"><title>Conclusions</title><p id=\"Par32\">In summary, ignoring time of infection will overestimate length of hospital stay and hospital charges attributable to BSI. This study can serve as a useful reference for future cost-effectiveness analyses of BSI interventions.</p></sec></body><back><fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><ack><title>Acknowledgements</title><p>We thank the clinicians and infection preventionists in the hospital, who contribute their effort to the electronic surveillance program and data collection. We also thank Dr. Lau Ho Yin Eric and Dr. Ho Lai Ming, both of the University of Hong Kong, for statistical analysis suggestions.</p></ack><notes notes-type=\"author-contribution\"><title>Authors’ contributions</title><p>YZZ drafted the first manuscript. QF and YXL together with YZZ designed the study. MMD provided medical suggestions about the manuscript. EBA and JMJ provide suggestions on manuscript revision. MMD, JJS, HWY, and RH worked on data collection and computer programming of the electronic surveillance system. All authors reviewed and revised the manuscript and approved the final version.</p></notes><notes notes-type=\"funding-information\"><title>Funding</title><p>None.</p></notes><notes notes-type=\"data-availability\"><title>Availability of data and materials</title><p>The datasets generated during the current study are not publicly available, to avoid disclosure of the individual privacy of the patients. However, they are available from the corresponding author (LIU Yunxi: liuyunxi301@qq.com) on reasonable request.</p></notes><notes id=\"FPar1\"><title>Ethics approval and consent to participate</title><p id=\"Par33\">This study was approved by the studied hospital institutional review board. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Cell Dev Biol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Cell Dev. Biol.</journal-id><journal-title-group><journal-title>Frontiers in Cell and Developmental Biology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-634X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850871</article-id><article-id pub-id-type=\"pmc\">PMC7431753</article-id><article-id pub-id-type=\"doi\">10.3389/fcell.2020.00782</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cell and Developmental Biology</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Roles of N6-Methyladenosine (m<sup>6</sup>A) in Stem Cell Fate Decisions and Early Embryonic Development in Mammals</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Meng</given-names></name></contrib><contrib contrib-type=\"author\"><name><surname>Zhai</surname><given-names>Yanhui</given-names></name></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Sheng</given-names></name></contrib><contrib contrib-type=\"author\"><name><surname>Dai</surname><given-names>Xiangpeng</given-names></name></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Ziyi</given-names></name><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/634830/overview\"/></contrib></contrib-group><aff><institution>Key Laboratory of Organ Regeneration and Transplantation of Ministry of Education, First Hospital, Jilin University</institution>, <addr-line>Changchun</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Louis Lefebvre, University of British Columbia, Canada</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Ye Fu, Harvard University, United States; Constance Ciaudo, ETH Zürich, Switzerland</p></fn><corresp id=\"c001\">*Correspondence: Ziyi Li, <email>ziyi@jlu.edu.cn</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Developmental Epigenetics, a section of the journal Frontiers in Cell and Developmental Biology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>782</elocation-id><history><date date-type=\"received\"><day>28</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>27</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Zhang, Zhai, Zhang, Dai and Li.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Zhang, Zhai, Zhang, Dai and Li</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>N6-methyladenosine (m<sup>6</sup>A) is one of the most abundant internal mRNA modifications, and it affects multiple biological processes related to eukaryotic mRNA. The majority of m<sup>6</sup>A sites are located in stop codons and 3′UTR regions of mRNAs. m<sup>6</sup>A regulates RNA metabolism, including alternative splicing (AS), alternative polyadenylation (APA), mRNA export, decay, stabilization, and translation. The m<sup>6</sup>A metabolic pathway is regulated by a series of m<sup>6</sup>A writers, erasers and readers. Recent studies indicate that m<sup>6</sup>A is essential for the regulation of gene expression, tumor formation, stem cell fate, gametogenesis, and animal development. In this systematic review, we summarized the recent advances in newly identified m<sup>6</sup>A effectors and the effects of m<sup>6</sup>A on RNA metabolism. Subsequently, we reviewed the functional roles of RNA m<sup>6</sup>A modification in diverse cellular bioprocesses, such as stem cell fate decisions, cell reprogramming and early embryonic development, and we discussed the potential of m<sup>6</sup>A modification to be applied to regenerative medicine, disease treatment, organ transplantation, and animal reproduction.</p></abstract><kwd-group><kwd>N6-methyladenosine</kwd><kwd>RNA metabolism</kwd><kwd>stem cell fate</kwd><kwd>cell reprogramming</kwd><kwd>embryonic development</kwd></kwd-group><counts><fig-count count=\"5\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"123\"/><page-count count=\"15\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Since the discovery of the first structurally modified nucleoside, pseudouridine, in the 1950s (<xref rid=\"B17\" ref-type=\"bibr\">Cohn and Volkin, 1951</xref>), more than 150 kinds of chemical modifications have been found on cellular RNA (<xref rid=\"B11\" ref-type=\"bibr\">Boccaletto et al., 2018</xref>). Due to the recognition of the prevalence and functional significance of N6-methyladenosine (m<sup>6</sup>A) modification on mRNA as well as the development of high-throughput sequencing technologies, there has been recent widespread interest in the biological phenomena of RNA modification (<xref rid=\"B20\" ref-type=\"bibr\">Dominissini et al., 2012</xref>; <xref rid=\"B64\" ref-type=\"bibr\">Meyer et al., 2012</xref>). In 1974, m<sup>6</sup>A was first discovered as the major form of internal methylation of mammalian mRNA (<xref rid=\"B18\" ref-type=\"bibr\">Desrosiers et al., 1974</xref>; <xref rid=\"B70\" ref-type=\"bibr\">Perry and Kelley, 1974</xref>). Early studies indicated that m<sup>6</sup>A occurred in the (G/A; m<sup>6</sup>A) C sequences of RNA and was predominately enriched in the stop codons and 3′ untranslated regions (3′UTRs; <xref rid=\"B76\" ref-type=\"bibr\">Schibler et al., 1977</xref>; <xref rid=\"B90\" ref-type=\"bibr\">Wei and Moss, 1977</xref>). m<sup>6</sup>A modifications are added or removed by a series of methyltransferases (also known as writers) and demethylases (also known as erasers; <xref rid=\"B37\" ref-type=\"bibr\">Jia et al., 2011</xref>; <xref rid=\"B51\" ref-type=\"bibr\">Liu et al., 2013</xref>; <xref rid=\"B120\" ref-type=\"bibr\">Zhen et al., 2013</xref>; <xref rid=\"B71\" ref-type=\"bibr\">Ping et al., 2014</xref>). In addition, the m<sup>6</sup>A site is recognized by binding proteins (also known as readers; <xref rid=\"B85\" ref-type=\"bibr\">Wang X. et al., 2014</xref>; <xref rid=\"B86\" ref-type=\"bibr\">Wang et al., 2015</xref>; <xref rid=\"B102\" ref-type=\"bibr\">Xiao et al., 2016</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Li et al., 2017</xref>; <xref rid=\"B58\" ref-type=\"bibr\">Mao et al., 2019</xref>). With the development of global-wide m<sup>6</sup>A detection technology, epitranscriptome data have revealed large amounts of transcripts across various species and tissues in normal and pathological processes (<xref rid=\"B20\" ref-type=\"bibr\">Dominissini et al., 2012</xref>; <xref rid=\"B64\" ref-type=\"bibr\">Meyer et al., 2012</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Huang et al., 2019</xref>). It is widely believed that DNA and histone modifications play crucial roles in gene expression regulation (<xref rid=\"B25\" ref-type=\"bibr\">Egger et al., 2004</xref>). However, a series of recent studies have shown that m<sup>6</sup>A has notable effects on the regulation of gene expression at the post-transcriptional level, animal development, and human diseases (<xref rid=\"B87\" ref-type=\"bibr\">Wang Y. et al., 2014</xref>; <xref rid=\"B86\" ref-type=\"bibr\">Wang et al., 2015</xref>; <xref rid=\"B74\" ref-type=\"bibr\">Roundtree et al., 2017a</xref>).</p><p>In this review, we summarized the latest progress regarding the molecular basis of m<sup>6</sup>A effectors and discussed the functional roles of m<sup>6</sup>A modification in the regulation of RNA metabolism. In addition, we focused on the molecular regulation mechanism of m<sup>6</sup>A modification in stem cell fate decisions and early embryonic development. Our review may contribute to a better understanding of RNA modification and its mechanism in the life sciences area.</p></sec><sec id=\"S2\"><title>Overview of RNA m<sup>6</sup>A Modification</title><sec id=\"S2.SS1\"><title>Characteristics of m<sup>6</sup>A Modification and Related Enzymes</title><p>N6-methyladenosine modifications are highly species-conserved between yeast, plants, fruit flies, and mammals. Recent transcriptome-wide m<sup>6</sup>A site positioning has provided more details about its location and prominence, revealing its universality among thousands of transcripts in humans and mice (<xref rid=\"B20\" ref-type=\"bibr\">Dominissini et al., 2012</xref>). m<sup>6</sup>A modifications mainly occur on adenine (A) in the RRACH (R = G or A, G > A, H = A or C or U, and U > A > C) sequence, and is mainly located near the stop codons and 3′UTR of mRNAs (<xref rid=\"B20\" ref-type=\"bibr\">Dominissini et al., 2012</xref>; <xref rid=\"B64\" ref-type=\"bibr\">Meyer et al., 2012</xref>; <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). In mammals, approximately 0.1 to 0.6% of adenines undergo m<sup>6</sup>A modification, with an average of 3 to 5 methylated sites in each mRNA. Notably, m<sup>6</sup>A modifications can be deposited onto the transcripts in tissue- and cell-type-specific manners (<xref rid=\"B4\" ref-type=\"bibr\">An et al., 2020</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>The characteristics of RNA m<sup>6</sup>A modification. The m<sup>6</sup>A writers, erasers, and readers in eukaryotic cells (<xref rid=\"B79\" ref-type=\"bibr\">Shi et al., 2019</xref>). The preferences and density of RNA m<sup>6</sup>A modification in different regions of mRNAs (<xref rid=\"B27\" ref-type=\"bibr\">Fitzsimmons and Batista, 2019</xref>).</p></caption><graphic xlink:href=\"fcell-08-00782-g001\"/></fig><p>Similar to DNA methylation, the m<sup>6</sup>A levels in RNA are dynamic and reversible. A series of m<sup>6</sup>A readers make up the methyltransferase complex (MTC), including the core component methyltransferase-like 3 (METTL3), and methyltransferase-like 14 (METTL14; <xref rid=\"B51\" ref-type=\"bibr\">Liu et al., 2013</xref>), and other regulatory factors: Wilms tumor 1-associating protein (WTAP; <xref rid=\"B71\" ref-type=\"bibr\">Ping et al., 2014</xref>), Vir like m<sup>6</sup>A methyltransferase associated (VIRMA; <xref rid=\"B110\" ref-type=\"bibr\">Yue et al., 2018</xref>), zinc finger CCCH-type containing 13 (ZC3H13; <xref rid=\"B92\" ref-type=\"bibr\">Wen et al., 2018</xref>), Cas-Br-M (murine) ecotropic retroviral transforming sequence-like 1 (CBLL1), and RNA binding motif protein (RBM15/15B; <xref rid=\"B68\" ref-type=\"bibr\">Patil et al., 2016</xref>). METTL3 is the main catalytic subunit of MTC, METTL14 mainly promotes binding to RNA, and WTAP is the regulatory subunit, which binds to METTL3/14 and thus contributes to the catalytic activity of methyltransferase and the deposition of m<sup>6</sup>A (<xref rid=\"B71\" ref-type=\"bibr\">Ping et al., 2014</xref>). Recent studies reported that zinc finger CCHC-type-containing 4 (ZCCHC4) and methyltransferase-like 16 (METTL16) function as m<sup>6</sup>A methyltransferases of 28S rRNA and U6 snRNA, respectively (<xref rid=\"B12\" ref-type=\"bibr\">Brown et al., 2016</xref>; <xref rid=\"B69\" ref-type=\"bibr\">Pendleton et al., 2017</xref>; <xref rid=\"B56\" ref-type=\"bibr\">Ma et al., 2019</xref>). Fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5) have been identified as m<sup>6</sup>A demethylases that play function in an Fe (II) and α-ketoglutarate (αKG)-dependent manner (<xref rid=\"B37\" ref-type=\"bibr\">Jia et al., 2011</xref>; <xref rid=\"B120\" ref-type=\"bibr\">Zhen et al., 2013</xref>). FTO was the first identified demethylase, and it can remove the m<sup>6</sup>A modification in the internal (m<sup>6</sup>A) and 5′cap (m<sup>6</sup>A<sub>m</sub>) of mRNAs in different environments (<xref rid=\"B91\" ref-type=\"bibr\">Wei et al., 2018</xref>).</p><p>To date, three classes of m<sup>6</sup>A readers have been characterized: the first class, YT521-B homology (YTH)-domain containing proteins, directly bind m<sup>6</sup>A-modified mRNAs (<xref rid=\"B85\" ref-type=\"bibr\">Wang X. et al., 2014</xref>; <xref rid=\"B86\" ref-type=\"bibr\">Wang et al., 2015</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Li et al., 2017</xref>; <xref rid=\"B78\" ref-type=\"bibr\">Shi et al., 2017</xref>; <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>); the second class, RNA structure-dependent proteins, include HNRNPC/G, and HNRNPA2B1 (<xref rid=\"B52\" ref-type=\"bibr\">Liu et al., 2015</xref>, <xref rid=\"B53\" ref-type=\"bibr\">2017</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Alarcon et al., 2015b</xref>; <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>); the third class, RNA binding proteins such as insulin-like growth factor 2 mRNA-binding proteins 1–3 (IGF2BP1/2/3), and Fragile X mental retardation protein (FMRP), can bind m<sup>6</sup>A-modified mRNAs through RNA binding domains (RBDs), such as K homology (KH), RNA recognition motif (RRM), and arginine/glycine-rich (RGG) domains (<xref rid=\"B34\" ref-type=\"bibr\">Huang et al., 2018</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Edens et al., 2019</xref>; <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). A recent study reported that a neuronal cell-specific m<sup>6</sup>A reader proline rich coiled-coil 2 A (Prrc2a), which contains glycine, arginine and glutamic acid (GRE) domains, played an important role in oligodendroglial specification and myelination (<xref rid=\"B98\" ref-type=\"bibr\">Wu et al., 2019b</xref>).</p><p>Writers and erasers contribute to the establishment, maintenance, and remodeling of the global m<sup>6</sup>A modification state across various species, tissues and cells in normal, and pathological processes. The subcellular localization of m<sup>6</sup>A effectors and regulators is essential for their function. METTL3 and YTHDF2 may depend on their cellular location and expression to show multiple functions (<xref rid=\"B122\" ref-type=\"bibr\">Zhou et al., 2015</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Lin et al., 2016</xref>). The demethylase FTO shows different substrate preferences in the cytoplasm and in the nucleus (<xref rid=\"B91\" ref-type=\"bibr\">Wei et al., 2018</xref>). Notably, the m<sup>6</sup>A modification-related methylase can be regulated by various factors involved in transcriptional, posttranscriptional (miRNAs and lncRNAs), and translational (phosphorylation, ubiquitination, or SUMOylation modification) regulation (<xref rid=\"B22\" ref-type=\"bibr\">Du et al., 2018</xref>; <xref rid=\"B77\" ref-type=\"bibr\">Schöller et al., 2018</xref>; <xref rid=\"B123\" ref-type=\"bibr\">Zhu et al., 2018</xref>). In addition, several m<sup>6</sup>A modification regulators, such as ZFP217, SMAD2/3, CEBPZ, TARBP2, and TRA2A, have been found to regulate m<sup>6</sup>A modification of mRNAs by recruiting or repelling MTC in a cell-type-specific manner (<xref rid=\"B1\" ref-type=\"bibr\">Aguilo et al., 2015</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Barbieri et al., 2017</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Bertero et al., 2018</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Fish et al., 2019</xref>; <xref rid=\"B4\" ref-type=\"bibr\">An et al., 2020</xref>). In addition, a recent study reported that H3K36me3 recruited METTL14 thus guiding m<sup>6</sup>A deposition on nascent transcripts (<xref rid=\"B35\" ref-type=\"bibr\">Huang et al., 2019</xref>). <xref rid=\"B50\" ref-type=\"bibr\">Liu et al. (2020)</xref> recently revealed that m<sup>6</sup>A of chromosome-associated regulatory RNAs (carRNAs) can regulate chromatin state and transcription through increasing activate histone modifications, such as H3K4me3 and H3K27ac. In embryonic neural stem cells (eNSCs), METTL14 knockout (KO) increases H3K4me3, H3K27me3, and H3K27ac levels by affecting the mRNA stabilization of histone-modifying enzymes (<xref rid=\"B88\" ref-type=\"bibr\">Wang et al., 2018</xref>). These results suggest that there is crosstalk between these diverse epigenetic modifications, which results in the regulation of gene expression.</p></sec><sec id=\"S2.SS2\"><title>The Effects of m<sup>6</sup>A on RNA Metabolism</title><p>Gene expression is regulated by multiple processes, including transcription, post-transcriptional regulation, and translation. An increasing number of studies have shown that RNA m<sup>6</sup>A modification affects RNA processing and metabolism, including alternative splicing (AS), alternative polyadenylation (APA), mRNA stability, export, degradation, and translation (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>RNA m<sup>6</sup>A modifications regulate RNA metabolism. m<sup>6</sup>A is deposited onto nascent mRNAs by MTC. Then the m<sup>6</sup>A-modified mRNAs undergo AS by recruiting splicing factors to m<sup>6</sup>A sites or their flanking sequences. On the other hand, YTHDC1 and VIRMA can interact with the polyadenylation cleavage factors CPSF5 and CPSF6, thus regulating mRNA APA. Then, mRNAs can be recognized by YTHDC1 or FMRP and exported into the cytoplasm. Cytoplasmic m<sup>6</sup>A readers regulate mRNA stability (IGF2BPs), decay (YTHDF2), and translation (YTHDF1/3, YTHDC2, EIF3a, and METTL3) under normal and stress conditions.</p></caption><graphic xlink:href=\"fcell-08-00782-g002\"/></fig><sec id=\"S2.SS2.SSS1\"><title>m<sup>6</sup>A Affects Pre-mRNA Splicing, Polyadenylation and Export</title><p>The majority of m<sup>6</sup>A writers and erasers are located in nuclear speckles, suggesting that they may regulate RNA processing (<xref rid=\"B51\" ref-type=\"bibr\">Liu et al., 2013</xref>; <xref rid=\"B71\" ref-type=\"bibr\">Ping et al., 2014</xref>; <xref rid=\"B110\" ref-type=\"bibr\">Yue et al., 2018</xref>). Indeed, increasing evidence suggests that m<sup>6</sup>A is associated with AS and APA events (<xref rid=\"B120\" ref-type=\"bibr\">Zhen et al., 2013</xref>; <xref rid=\"B103\" ref-type=\"bibr\">Xu et al., 2017</xref>). In HeLa cells, PAR-CLIP data analysis suggested that METTL3 and WTAP affected mRNA AS and the expression of genes related to transcription and RNA processing (<xref rid=\"B71\" ref-type=\"bibr\">Ping et al., 2014</xref>). Similarly, KO of METTL3 affects exon skipping and intron retention in mouse embryonic stem cells (mESCs; <xref rid=\"B30\" ref-type=\"bibr\">Geula et al., 2015</xref>). In addition, previous studies also found that METTL3 and the eraser ALKBH5 regulate the AS of mRNAs in spermatogenesis (<xref rid=\"B120\" ref-type=\"bibr\">Zhen et al., 2013</xref>; <xref rid=\"B103\" ref-type=\"bibr\">Xu et al., 2017</xref>). The demethylase FTO promotes exon skipping by preventing the recruitment of splicing factor 2 (SRSF2) to the splicing sites in 3T3-L1 preadipocytes (<xref rid=\"B119\" ref-type=\"bibr\">Zhao et al., 2014</xref>). m<sup>6</sup>A alters the local RNA structure and therefore affects the binding of HNRNP family proteins HNRNPC/G to m<sup>6</sup>A modified mRNA, thus affecting mRNAs AS (<xref rid=\"B52\" ref-type=\"bibr\">Liu et al., 2015</xref>, <xref rid=\"B53\" ref-type=\"bibr\">2017</xref>). Additionally, a recent study reported that KO of HNRNPA2B1 and METTL3 depletion caused similar AS effects (<xref rid=\"B2\" ref-type=\"bibr\">Alarcon et al., 2015a</xref>). A latest study by <xref rid=\"B50\" ref-type=\"bibr\">Liu et al. (2020)</xref> suggested that YTHDC1 can facilitate the degradation of m<sup>6</sup>A-modified carRNAs, such as LINE1 elements, through the NEXT complex in nucleus. Notably, the m<sup>6</sup>A nuclear reader YTHDC1 regulates mRNA splicing by recruiting the serine/arginine-rich splicing factors SRSF3 (exon retention) and SRSF10 (exon excision; <xref rid=\"B102\" ref-type=\"bibr\">Xiao et al., 2016</xref>; <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>).</p><p>Several studies illustrated that transcripts with m<sup>6</sup>A modifications more often had proximal APA sites, while unmethylated transcripts tended to use distal APA sites (<xref rid=\"B39\" ref-type=\"bibr\">Ke et al., 2015</xref>; <xref rid=\"B65\" ref-type=\"bibr\">Molinie et al., 2016</xref>). VIRMA preferentially targets m<sup>6</sup>A mRNA methylation in stop codons and the 3′UTR, and it interacts with the polyadenylation cleavage factors F5 and CPSF6, thus regulating APA (<xref rid=\"B110\" ref-type=\"bibr\">Yue et al., 2018</xref>). In addition, YTHDC1 also promotes the export of m<sup>6</sup>A-modified mRNAs by interacting with pre-mRNA 3′ end processing factors CPSF6, splicing factors SRSF3/7, and nuclear RNA export factor 1 (NXF1; <xref rid=\"B75\" ref-type=\"bibr\">Roundtree et al., 2017b</xref>), suggesting a similar function of YTHDC1 with VIRMA in regulating APA. Moreover, a latest study illustrated that a novel m<sup>6</sup>A reader, FMRP, could promote the nuclear export of m<sup>6</sup>A-modified mRNAs through another nuclear RNA export factor, CRM1, during neural differentiation (<xref rid=\"B24\" ref-type=\"bibr\">Edens et al., 2019</xref>; <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>).</p></sec><sec id=\"S2.SS2.SSS2\"><title>m<sup>6</sup>A Affects mRNA Decay, Stabilization and Translation</title><p>Transcripts transported into the cytoplasm are recognized and regulated by a series of cytoplasmic m<sup>6</sup>A readers, such as YTHDFs, IGF2BPs, YTHDC2, and EIF3a. The cytoplasmic m<sup>6</sup>A reader YTHDF2 regulates mRNA decay by recruiting the CCR4-NOT deadenylase complex, which accelerates RNA deadenylation, and degradation of m<sup>6</sup>A-modified mRNAs (<xref rid=\"B21\" ref-type=\"bibr\">Du et al., 2016</xref>). In contrast to YTHDF2, the cytoplasmic m<sup>6</sup>A readers IGF2BPs enhance mRNA stability and facilitate translation of m<sup>6</sup>A-modified mRNAs (<xref rid=\"B34\" ref-type=\"bibr\">Huang et al., 2018</xref>). YTHDF1 and YTHDF3 synergistically recruit translation initiation factors to promote mRNA translation and affect YTHDF2-mediated degradation of m<sup>6</sup>A-modified mRNAs (<xref rid=\"B86\" ref-type=\"bibr\">Wang et al., 2015</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Li et al., 2017</xref>). As the only helicase-containing reader, YTHDC2 can resolve mRNA secondary structures and promote translation elongation of mRNA with m<sup>6</sup>A modification in the CDS region (<xref rid=\"B58\" ref-type=\"bibr\">Mao et al., 2019</xref>). Unexpectedly, cytoplasmic METTL3 could interact with eIF3h to enhance translation, suggesting that METTL3 might directly regulate translation in a m<sup>6</sup>A reader-independent manner (<xref rid=\"B47\" ref-type=\"bibr\">Lin et al., 2016</xref>; <xref rid=\"B16\" ref-type=\"bibr\">Choe et al., 2018</xref>). Under heat stress conditions, YTHDF2 changes subcellular localization, moving from the cytosol to the nucleus, and it preserves the level of 5′UTR m<sup>6</sup>A in mRNAs, thus promoting cap-independent translation (<xref rid=\"B122\" ref-type=\"bibr\">Zhou et al., 2015</xref>). In addition, m<sup>6</sup>A in the 5′UTR could promote cap-independent translation of mRNAs by binding with eIF3a under stress conditions (<xref rid=\"B63\" ref-type=\"bibr\">Meyer et al., 2015</xref>; <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>).</p><p>Together with the role that YTHDF1, YTHDF3, and YTHDC2 play in translation, YTHDF2 and IGF2BPs play crucial roles in maintaining the RNA metabolic balance in different physiological states. In addition, it is important to point out that non-coding RNAs are also regulated by RNA m<sup>6</sup>A modification (<xref rid=\"B2\" ref-type=\"bibr\">Alarcon et al., 2015a</xref>, <xref rid=\"B3\" ref-type=\"bibr\">b</xref>; <xref rid=\"B68\" ref-type=\"bibr\">Patil et al., 2016</xref>). The m<sup>6</sup>A modification not only affects the cleavage, transport, stability and degradation processes of non-coding RNA but also regulates the function of biological cells by affecting the expression of non-coding RNA (<xref rid=\"B2\" ref-type=\"bibr\">Alarcon et al., 2015a</xref>, <xref rid=\"B3\" ref-type=\"bibr\">b</xref>; <xref rid=\"B106\" ref-type=\"bibr\">Yang et al., 2017</xref>; <xref rid=\"B121\" ref-type=\"bibr\">Zhou et al., 2017</xref>). In some cases, these non-coding RNAs affect the RNA-RNA or RNA-protein interactions to regulate specific biological functions. The variability of m<sup>6</sup>A reader subcellular localization, the preferences across various gene regions and consensus sequences adds layers of complexity to the m<sup>6</sup>A regulation mechanism.</p></sec></sec></sec><sec id=\"S3\"><title>Role of m<sup>6</sup>A in Stem Cell Fate</title><p>Mutipotent stem cells have been widely applied in regenerative medicine, disease treatment and organ transplantation. It has been found that RNA m<sup>6</sup>A modification plays essential roles in stem cell self-renewal, differentiation, and cell reprogramming (<xref rid=\"B9\" ref-type=\"bibr\">Batista et al., 2014</xref>; <xref rid=\"B87\" ref-type=\"bibr\">Wang Y. et al., 2014</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Aguilo et al., 2015</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Chen et al., 2015</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Geula et al., 2015</xref>). It seems that m<sup>6</sup>A modifications have distinct effects on the fate of stem cells at various stages, states and types of stem cells.</p><sec id=\"S3.SS1\"><title>Role of m<sup>6</sup>A in Pluripotent Stem Cell Fate Decisions</title><p>It is believed that the establishment, maintenance and transition of the cell states are regulated by a series of molecular regulation mechanisms. Epigenetic regulation, such as DNA modification, histone modification, chromatin remodeling, and the work of non-coding RNAs, play significant roles in early embryonic development and the self-renewal and directional differentiation of stem cells (<xref rid=\"B40\" ref-type=\"bibr\">Kouzarides, 2007</xref>; <xref rid=\"B81\" ref-type=\"bibr\">Smith and Meissner, 2013</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Bao et al., 2015</xref>). Embryonic stem cells (ESCs) derived from the inner cell mass (ICM) of blastocysts are described as being in a naïve state. As a class of versatile stem cell, naïve ESCs but not primed ESCs have the ability to form chimeric embryos. Primed epiblast stem cells (EpiSCs) derived from the ICM-derived epiblast of postimplantation embryos, represent a type of ESCs in a differentiated state. Induced pluripotent stem cells (iPSCs) were originally obtained by introducing four exogenous transcription factors (KLF4, OCT4, c-MYC, and SOX2) into the somatic cells with a virus (<xref rid=\"B57\" ref-type=\"bibr\">Maherali et al., 2007</xref>), and they have morphological and epigenetic characteristics similar to those of ESCs. Scientists have studied the molecular regulatory mechanism by which ESCs maintain self-renewal and trigger differentiation for a long time. DNA methylation, histone methylation, histone acetylation modification and non-coding RNAs have been found to contribute to determining the fate of pluripotent stem cells (<xref rid=\"B5\" ref-type=\"bibr\">Azuara et al., 2006</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Doi et al., 2009</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Durruthy-Durruthy et al., 2016</xref>). Recent studies found that RNA m<sup>6</sup>A modification might play crucial roles in pluripotent stem cell self-renewal and differentiation to specific lineages (<xref rid=\"B9\" ref-type=\"bibr\">Batista et al., 2014</xref>; <xref rid=\"B87\" ref-type=\"bibr\">Wang Y. et al., 2014</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Aguilo et al., 2015</xref>).</p><p>In 2014, <xref rid=\"B9\" ref-type=\"bibr\">Batista et al. (2014)</xref> found that the RNA of core pluripotency transcription factors had m<sup>6</sup>A modifications that were conserved in mouse and human ESCs. <xref rid=\"B87\" ref-type=\"bibr\">Wang Y. et al. (2014)</xref> illustrated that knockdown of the methyltransferases METTL3 and METTL14 inhibited the expression of pluripotency genes, such as SOX2, NANOG, and DPPA3, and it promoted the expression of developmental regulators, such as FGF5, CDX2, and SOX17 in mouse ESCs. Furthermore, METTL3 and METTL14 deletion increased RNA stability in a HuR- and miRNA-dependent manner in mESCs (<xref rid=\"B87\" ref-type=\"bibr\">Wang Y. et al., 2014</xref>). Subsequently, another study reported that Zc3h13 interacts with WTAP, VIRMA, and CBLL1, forming a biochemical complex in mESCs (<xref rid=\"B92\" ref-type=\"bibr\">Wen et al., 2018</xref>). Zc3h13 deletion decreases the global m<sup>6</sup>A mRNA modification levels and the ability to self-renewal, triggering the differentiation of mESCs (<xref rid=\"B92\" ref-type=\"bibr\">Wen et al., 2018</xref>). In addition, <xref rid=\"B1\" ref-type=\"bibr\">Aguilo et al. (2015)</xref> reported that zinc finger protein 217 (ZFP217) was a direct regulator of the pluripotency genes (NANOG, SOX2, KLF4, and c-MYC) in mESCs. On the other hand, ZFP217 interacts with METTL3, thus promoting mESCs self-renewal by decreasing m<sup>6</sup>A RNA modification of pluripotency factors and maintaining their expression levels (<xref rid=\"B1\" ref-type=\"bibr\">Aguilo et al., 2015</xref>). Similarly, a recent study revealed that METTL3 depletion prevents self-renewal of porcine iPSCs and triggers differentiation by inactivating the JAK2-STAT3 pathway in an m<sup>6</sup>A-YTHDF1/YTHDF2-dependent manner (<xref rid=\"B100\" ref-type=\"bibr\">Wu et al., 2019c</xref>). Conversely, several studies suggested that METTL3 deletion or m<sup>6</sup>A loss prolonged NANOG expression, thus promoting mESCs self-renewal and blocking differentiation (<xref rid=\"B30\" ref-type=\"bibr\">Geula et al., 2015</xref>). These seemingly contradictory outcomes could be explained by the hypothesis that m<sup>6</sup>A modification affects stem cell fate decisions by regulating the predominant genes in stem cells that possess different pluripotency states. In naïve ESCs, METTL3 knockdown or m<sup>6</sup>A loss further increased the expression of high abundance-pluripotency genes thus creating hypernaïve pluripotent states, despite the weak upregulation of lineage-specific regulators. For EpiSCs, m<sup>6</sup>A loss promotes the expression of lineage-specific genes, thus triggering cell differentiation (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>m<sup>6</sup>A regulates the pluripotent stem cell fate. METTL3 or m<sup>6</sup>A deletion has divergent effects on the fate decision of pluripotent stem cells in different states. METTL3 and ZFP217 play divergent roles in somatic cell reprogramming at different stages.</p></caption><graphic xlink:href=\"fcell-08-00782-g003\"/></fig><p>Embryonic stem cell-specific lncRNAs contribute to the pluripotency maintenance (<xref rid=\"B31\" ref-type=\"bibr\">Guttman et al., 2011</xref>). Recently, <xref rid=\"B105\" ref-type=\"bibr\">Yang et al. (2018)</xref> reported that the m<sup>6</sup>A modification deposited on the mESC-specific lincRNA linc1281 affected mESC differentiation by regulating the linc1281-Let-7 family-Lin28 ceRNA pathway. Further, the TGFβ-Activin-Nodal signaling pathway plays crucial roles in pluripotent stem cell fate decisions. In human ESCs, Activin and Nodal activate SMAD2/3, which binds with NANOG, thus maintaining cell self-renewal. A recent study found that SMAD2/3 promotes m<sup>6</sup>A deposition onto nascent transcripts by recruiting the MTC, thus facilitating exit from pluripotency toward lineage-specific differentiation in human ESCs (<xref rid=\"B10\" ref-type=\"bibr\">Bertero et al., 2018</xref>).</p></sec><sec id=\"S3.SS2\"><title>Role of m<sup>6</sup>A in Cell Reprogramming</title><p>Increasing evidence has indicated that regulating epigenetic modifications could increase somatic cell reprogramming efficiency (<xref rid=\"B114\" ref-type=\"bibr\">Zhang J. et al., 2019</xref>). Recent studies suggested that RNA m<sup>6</sup>A modifications can act as a functional regulator in cell reprogramming (<xref rid=\"B7\" ref-type=\"bibr\">Bao et al., 2015</xref>; <xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). In 2015, <xref rid=\"B30\" ref-type=\"bibr\">Geula et al. (2015)</xref> reported that METTL3 deletion suppressed mouse EpiSC reprogramming efficiency toward naïve pluripotency in early stages but had the opposite effect in the later stages. In MEFs, early KO of METTL3 diminished somatic reprogramming efficiency but did not affect reprogramming efficiency after 3 days of induction with OKSM factors (<xref rid=\"B30\" ref-type=\"bibr\">Geula et al., 2015</xref>). Subsequently, <xref rid=\"B14\" ref-type=\"bibr\">Chen et al. (2015)</xref> reported that METTL3 overexpression increases m<sup>6</sup>A abundance and promoted the expression of pluripotent factors and colony numbers of iPSCs, suggesting that m<sup>6</sup>A promotes cell reprogramming. Another study suggested that the expression level of ZFP217 gradually increased during somatic reprogramming, while METTL3 exhibited the opposite trend (<xref rid=\"B1\" ref-type=\"bibr\">Aguilo et al., 2015</xref>). ZFP217 deletion diminished the number of colonies positive for alkaline phosphatase, while ZFP217 overexpression promoted the formation of iPSC colonies (<xref rid=\"B1\" ref-type=\"bibr\">Aguilo et al., 2015</xref>). Furthermore, METTL3 knockdown partially rescued somatic cell reprogramming in Zfp217-depleted cells. Mechanistically, ZFP217 transcription activates core reprogramming factors, thus maintaining cell stemness. On the other hand, ZFP217 binds with METTL3, thus diminishing m<sup>6</sup>A deposition onto the transcripts of pluripotency genes. Taken together, these studies illustrated the important and various functional regulatory roles of ZFP217 and METTL3 in somatic cell reprogramming. METTL3 is essential for somatic cell reprogramming because it arrests the cell cycle, thus affecting cell proliferation in the early stage, while ZFP217 is indispensable for activating pluripotency factors in the later stages of somatic cell reprogramming.</p></sec><sec id=\"S3.SS3\"><title>Role of m<sup>6</sup>A in the Differentiation of Other Stem and Progenitor Cells</title><p>Several studies have suggested that m<sup>6</sup>A RNA modification plays crucial roles in the hematopoietic system, neural system, fat metabolism and muscle development. Mesenchymal stem cells (MSCs) derived from different tissues may be suitable for the treatment of various diseases due to the different secretory capacities of cytokines and growth factors.</p><p>In 2017, <xref rid=\"B113\" ref-type=\"bibr\">Zhang et al. (2017)</xref> found that m<sup>6</sup>A determines the hematopoietic stem/progenitor cell (HSPC) fate by affecting the endothelial-to-hematopoietic transition (EHT) during zebrafish embryogenesis (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). Furthermore, METTL3 deletion downregulated the m<sup>6</sup>A modification level of Notch1a transcripts, thus protecting the mRNA from YTHDF2-mediated decay to block EHT during vertebrate embryogenesis (<xref rid=\"B113\" ref-type=\"bibr\">Zhang et al., 2017</xref>). Subsequently, <xref rid=\"B84\" ref-type=\"bibr\">Vu et al. (2017)</xref> reported that METTL3 deletion promotes human HSPC differentiation and inhibits cell proliferation. Mechanistically, the knockdown of METTL3 hindered C-MYC, BCL-2, and PTEN translation and stimulated AKT to be phosphorylated to trigger downstream signaling pathway reactions (<xref rid=\"B84\" ref-type=\"bibr\">Vu et al., 2017</xref>). METTL14 was shown to be highly expressed in normal HSPCs and downregulated during myeloid differentiation (<xref rid=\"B93\" ref-type=\"bibr\">Weng et al., 2018</xref>). Similarly, METTL14 knockdown promoted terminal myeloid differentiation of normal HSPCs by inactivating the MYB/MYC axis. In contrast, <xref rid=\"B42\" ref-type=\"bibr\">Lee et al. (2019)</xref> reported that METTL3 deletion inhibited the differentiation of hematopoietic stem cells (HSCs) but led to the accumulation of HSCs in adult bone marrow. <xref rid=\"B107\" ref-type=\"bibr\">Yao et al. (2018)</xref> also confirmed that METTL3/14 played essential roles in maintaining the self-renewal of HSCs in adult bone marrow. Similarly, <xref rid=\"B15\" ref-type=\"bibr\">Cheng et al. (2019)</xref> also found that METTL3 maintained the symmetric commitment and identity of HSCs by affecting MYC mRNA stability. The divergent effects of m<sup>6</sup>A on HSC fate may be because m<sup>6</sup>A plays different roles in HSC and progenitor fate determination. Notably, abnormal expression of METTL3/14 may lead to the occurrence of malignant diseases related to the hematopoietic system, such as acute myeloid leukemia (AML; <xref rid=\"B84\" ref-type=\"bibr\">Vu et al., 2017</xref>; <xref rid=\"B93\" ref-type=\"bibr\">Weng et al., 2018</xref>). In addition, two recent studies illustrated that the m<sup>6</sup>A reader YTHDF2 was required for HSC self-renewal and AML initiation by mediating mRNA degradation (<xref rid=\"B46\" ref-type=\"bibr\">Li et al., 2018</xref>; <xref rid=\"B67\" ref-type=\"bibr\">Paris et al., 2019</xref>). YTHDF2 deletion not only promoted HSC expansion but also prevented leukemia initiation, suggesting that YTHDF2 could be a potential therapeutic target in AML (<xref rid=\"B67\" ref-type=\"bibr\">Paris et al., 2019</xref>).</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>The crucial roles of m<sup>6</sup>A in stem cell and progenitor cell differentiation. m<sup>6</sup>A plays divergent functions by affecting mRNA splicing (•), stability (■), and translation (★). ECs: endothelial cells; HSPCs: hematopoietic stem/progenitor cells; NSCs: neural stem cells; NPCs: neural progenitor cells; SSCs: spermatogonial stem cells; ADSCs: adipose-derived stem cell; and BMSCs: bone marrow mesenchymal stem cells.</p></caption><graphic xlink:href=\"fcell-08-00782-g004\"/></fig><p>Spermatogonial stem cells (SSCs) are a class of stem cells that not only can self-renew but also can differentiate into spermatocytes. Spermatogenesis involves a highly regulated differentiation process that includes mitosis, meiosis, and spermiogenesis. Multiple studies have suggested that KO of the m<sup>6</sup>A effector has significant effects on male fertility and spermatogenesis. In 2017, <xref rid=\"B48\" ref-type=\"bibr\">Lin et al. (2017)</xref> first investigated the dynamic changes in m<sup>6</sup>A levels during spermatogenesis, and m<sup>6</sup>A was found to be relatively highly enriched in pachytene spermatocytes and round spermatids. Germ cell-specific METTL3 or METTL14 KO caused mRNA translation dysregulation, thus affecting SSC proliferation and differentiation (<xref rid=\"B48\" ref-type=\"bibr\">Lin et al., 2017</xref>). Similarly, another study reported that germ cell-specific METTL3 deletion prevented spermatogonial differentiation and meiosis by altering transcript expression and splicing (<xref rid=\"B103\" ref-type=\"bibr\">Xu et al., 2017</xref>). Moreover, other studies suggested that ALKBH5, YTHDC1, and YTHDC2 KO mice exhibited deficient phenotypes in spermatogenesis or male fertility (<xref rid=\"B120\" ref-type=\"bibr\">Zhen et al., 2013</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Kasowitz et al., 2018</xref>), suggesting crucial roles for m<sup>6</sup>A in spermatogenesis and animal reproduction. Further studies regarding the regulatory mechanisms of m<sup>6</sup>A on spermatogenesis or male fertility need to be performed.</p><p>RNA m<sup>6</sup>A modification is indispensable for the development and functional maintenance of the nervous system. METTL3 and METTL14 deletion prolonged the cell cycle progression of cortical neural progenitor cells (NPCs) and reduced the differentiation of radial glial cells (RGCs) during mouse embryonic cortical neurogenesis (<xref rid=\"B109\" ref-type=\"bibr\">Yoon et al., 2017</xref>). Oligodendrocyte-specific METT14 deletion resulted in myelin abnormalities and decreased oligodendrocyte numbers but did not affect oligodendrocyte precursor cell (OPC) numbers. In OPCs, METTL14 ablation prevented the differentiation of OPCs, suggesting that m<sup>6</sup>A is a crucial regulator of oligodendrocyte differentiation. The epitranscriptome analysis results suggested that a large number of transcripts related to oligodendrocyte lineage progression were marked with m<sup>6</sup>A modification. METTL14 deletion led to aberrant mRNA splicing in OPCs as well as oligodendrocytes. <xref rid=\"B88\" ref-type=\"bibr\">Wang et al. (2018)</xref> reported that METTL14 deletion decreased cell proliferation and promoted untimely differentiation of eNSCs. Moreover, METTL14 deletion increased H3K4me3, H3K27me3, and H3K27ac levels by affecting the mRNA stabilization of histone-modifying enzymes in eNSCs (<xref rid=\"B88\" ref-type=\"bibr\">Wang et al., 2018</xref>). In contrast, a recent study found that METTL3 deletion reduced the level of histone methyltransferase Ezh2, thereby decreasing H3K27me3 levels in adult neural stem cells (aNSCs; <xref rid=\"B13\" ref-type=\"bibr\">Chen et al., 2019</xref>). Mechanistically, KO of METTL3 inhibited the proliferation of aNSCs and promoted aNSC differentiation toward the glial lineage. In addition, EZH2 overexpression rescued the defects resulting from METTL3 depletion. The diverse effects of m<sup>6</sup>A may be due to differences in methyltransferase functions and cell types. Notably, recent studies have identified two new neuronal cell-specific m<sup>6</sup>A readers, FMRP and Prcc2a, that play important roles in nervous system development in mice. FMRP deletion hindered cycle progression and promoted the proliferation of NPCs, which was similar to what was observed in METTL14 conditional KO mice (<xref rid=\"B24\" ref-type=\"bibr\">Edens et al., 2019</xref>). Mechanistically, FMRP promotes m<sup>6</sup>A-modified mRNA nuclear export through CRM1 during neural differentiation (<xref rid=\"B24\" ref-type=\"bibr\">Edens et al., 2019</xref>). Another neuronal cell-specific m<sup>6</sup>A reader is Prrc2a. In mice, Prrc2a regulates OPC proliferation and oligodendrocyte fate by stabilizing Olig2 mRNA during oligodendrocyte development (<xref rid=\"B98\" ref-type=\"bibr\">Wu et al., 2019b</xref>).</p><p>Marrow MSCs are a class of stem cells derived from marrow, adipose and umbilical cord tissues, and they can be differentiated into osteoblasts, chondrocytes, and adipocytes. Among MSCs, bone marrow mesenchymal stem cells (BMSCs) have been applied to the cell-based therapy for some osteoporosis-related and human cancers. In 2018, <xref rid=\"B101\" ref-type=\"bibr\">Wu Y. et al. (2018)</xref> identified that MSC-specific METTL3 deletion inhibited osteogenic differentiation while promoting adipogenic differentiation <italic>in vivo</italic> and <italic>in vitro</italic>. The translation efficiency of parathyroid hormone receptor-1 (Pth1r) was impaired in METTL3-deficient MSCs. In contrast, overexpression of METTL3 could rescue osteoporosis in mice (<xref rid=\"B101\" ref-type=\"bibr\">Wu Y. et al., 2018</xref>). Mechanistically, METTL3 deletion regulated osteogenesis by affecting the PTH/Pth1r signaling axis in an m<sup>6</sup>A-dependent manner. Subsequently, <xref rid=\"B83\" ref-type=\"bibr\">Tian et al. (2019)</xref> found that the expression level of METTL3 was significantly increased during osteogenic differentiation of BMSCs. Knockdown of METTL3 inhibited osteogenic differentiation, reduced p-AKT levels and affected the expression of genes in PI3K-AKT signaling pathways; the knockdown also affected the AS of VEGFa (<xref rid=\"B83\" ref-type=\"bibr\">Tian et al., 2019</xref>). Similarly, a recent study demonstrated that METTL3 regulated osteoclast differentiation by protecting the Atp6v0d2 mRNA from the degradation by YTHDF2 and promoted the nuclear retention of Traf6 mRNA (<xref rid=\"B44\" ref-type=\"bibr\">Li D. et al., 2020</xref>). METTL3 deletion prevented the expression of osteoclast-specific genes, decreased the phosphorylation levels of key factors in the MAPK, NF-κB, and PI3K-AKT signaling pathways and inhibited osteoclast differentiation (<xref rid=\"B44\" ref-type=\"bibr\">Li D. et al., 2020</xref>). A recent study revealed that the expression level of METTL3 was significantly increased during adipogenesis in porcine BMSCs (<xref rid=\"B108\" ref-type=\"bibr\">Yao et al., 2019</xref>). Moreover, METTL3 deletion promoted adipogenesis by activating the JAK1/STAT5/C/EBPβ pathway in an m<sup>6</sup>A-YTHDF2-dependent manner in porcine BMSCs (<xref rid=\"B108\" ref-type=\"bibr\">Yao et al., 2019</xref>). These findings suggested that m<sup>6</sup>A could be a crucial link between adipogenic and osteogenic lineages and that METTL3 might be a potential treatment target for the osteoporosis. Increasing evidence has also found that m<sup>6</sup>A regulates adipose-derived stem cell (ADSC) or preadipocyte fate decisions (<xref rid=\"B119\" ref-type=\"bibr\">Zhao et al., 2014</xref>; <xref rid=\"B99\" ref-type=\"bibr\">Wu R. et al., 2018</xref>; <xref rid=\"B54\" ref-type=\"bibr\">Liu Q. et al., 2019</xref>; <xref rid=\"B97\" ref-type=\"bibr\">Wu et al., 2019a</xref>).</p><p>In 3T3-L1 preadipocytes, FTO regulates adipogenesis by affecting SRSF2 binding with m<sup>6</sup>A-modified mRNAs thus leading to exon skipping (<xref rid=\"B119\" ref-type=\"bibr\">Zhao et al., 2014</xref>). On the other hand, FTO regulated the transcript abundance of cell cycle-related genes in an m<sup>6</sup>A-YTHDF2-dependent manner in 3T3-L1 preadipocytes (<xref rid=\"B99\" ref-type=\"bibr\">Wu R. et al., 2018</xref>). In addition, FTO promotes adipogenesis by decreasing the m<sup>6</sup>A level of JAK2, thus protecting its mRNA from degradation by YTHDF2 and activating the JAK2-STAT3-C/EBPβ signaling pathway during adipogenic differentiation (<xref rid=\"B97\" ref-type=\"bibr\">Wu et al., 2019a</xref>). In 3T3-L1 cells, KO of ZFP217 promoted METTL3 expression and increased global RNA m<sup>6</sup>A levels, thus downregulating the expression level of CCND1 in an m<sup>6</sup>A-YTHDF2-dependent manner (<xref rid=\"B54\" ref-type=\"bibr\">Liu Q. et al., 2019</xref>). In normal 3T3-L1 cells, ZFP217 promotes adipogenesis by activating FTO and interacting with YTHDF2 (<xref rid=\"B54\" ref-type=\"bibr\">Liu Q. et al., 2019</xref>). However, KO of ZFP217 increased the global RNA m<sup>6</sup>A level by decreasing FTO expression in a m<sup>6</sup>A-YTHDF2-dependent manner (<xref rid=\"B54\" ref-type=\"bibr\">Liu Q. et al., 2019</xref>). On the other hand, ZFP217 deletion directly decreased the expression level of FTO in 3T3-L1 cells (<xref rid=\"B54\" ref-type=\"bibr\">Liu Q. et al., 2019</xref>). Taken together, ZFP217 might act as a “super” m<sup>6</sup>A regulator by interacting with the m<sup>6</sup>A writer METTL3, eraser FTO, and reader (YTHDF2), thus playing functional roles in various cellular physiological processes.</p></sec></sec><sec id=\"S4\"><title>m<sup>6</sup>A Modification and Mammalian Embryonic Development</title><p>As one of the most important molecular processes in life, early mammalian embryonic development is determined by multiple cell fate decisions that result in an overall development blueprint for organogenesis and morphogenesis. The process of early embryonic development involves the maintenance and differentiation of totipotent cells, which is determined by the order of differentiation of various pluripotent stem cells. Embryo development is known to be regulated by a series of complex regulatory mechanisms at different levels. An increasing number of studies have suggested that RNA m<sup>6</sup>A modification is associated with animal reproduction programs, such as gametogenesis, maternal-zygote transition, and early embryo development (<xref rid=\"B120\" ref-type=\"bibr\">Zhen et al., 2013</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Geula et al., 2015</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Du et al., 2016</xref>; <xref rid=\"B118\" ref-type=\"bibr\">Zhao et al., 2017</xref>; <xref rid=\"B60\" ref-type=\"bibr\">Mendel et al., 2018</xref>; <xref rid=\"B82\" ref-type=\"bibr\">Sui et al., 2020</xref>; <xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>).</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>The m<sup>6</sup>A modification-related proteins exert essential functions in early embryonic development. The highlighted color indicates that the proteins mainly play functions at this stage.</p></caption><graphic xlink:href=\"fcell-08-00782-g005\"/></fig><sec id=\"S4.SS1\"><title>The Influences of m<sup>6</sup>A on Preimplantation Embryo Development</title><p>The maternal-to-zygotic transition (MZT) is one of the most important highly ordered and regulated events during early embryo development. The MZT is accompanied by the degradation of maternal RNA and protein and zygote genome activation (ZGA). As animal embryonic development proceeds, posttranscriptional mechanisms act as critical regulators to ensure suitable gene expression and timely progression of development programs.</p><p>In 2015, <xref rid=\"B30\" ref-type=\"bibr\">Geula et al. (2015)</xref> illustrated that METTL3 KO blastocysts of mouse showed normal morphology and normal expression of pluripotency markers (OCT4 and NANOG) compared with wild type blastocysts at E3.5 (embryonic day E3.5), suggesting that METTL3 might not affect preimplantation embryonic development of mouse. Notably, a recent study first detected the RNA m<sup>6</sup>A level during mouse oocyte maturation and embryonic development by immunofluorescence (IF) staining (<xref rid=\"B82\" ref-type=\"bibr\">Sui et al., 2020</xref>). The global RNA m<sup>6</sup>A level was gradually decreased from the germinal vesicle (GV) to the two-cell stage but increased after the two-cell stage during preimplantation embryos development (<xref rid=\"B82\" ref-type=\"bibr\">Sui et al., 2020</xref>). Knockdown of METTL3 caused an approximately half of oocytes spindle abnormalities, and the proportion of aneuploid oocytes rose, thereby hindering oocytes maturation in mice (<xref rid=\"B82\" ref-type=\"bibr\">Sui et al., 2020</xref>). In addition, METTL3 deletion decreased mRNA translation efficiency in oocytes. In parthenogenetically activated embryos, METTL3 deletion impeded the mouse ZGA process by affecting maternal mRNA degradation, and the results of EU (5-Ethynyl uridine) and phosphor-pol II ser2/5 staining also suggested that METTL3 deletion reduced the global transcription level in the two-cell embryos of mouse (<xref rid=\"B82\" ref-type=\"bibr\">Sui et al., 2020</xref>). Subsequently, <xref rid=\"B41\" ref-type=\"bibr\">Kwon et al. (2019)</xref> found that the m<sup>6</sup>A reader hnRNPA2B1 was regulated by METTL3 and played a crucial role in mouse embryonic development. In mice, HNRNPA2B1 is highly expressed in embryos at the 4-cell stage (<xref rid=\"B41\" ref-type=\"bibr\">Kwon et al., 2019</xref>). Knockdown of HNRNPA2B1 downregulated the expression of pluripotency-related genes, such as OCT4, and SOX2. METTL3 deletion decreased the mean sizes of blastocysts and the proportion that reached the morula and blastocyst stage (<xref rid=\"B41\" ref-type=\"bibr\">Kwon et al., 2019</xref>), suggesting that HNRNPA2B1 contributes to blastocyst quality. Moreover, METTL3 deletion increased the mislocalization of HNRNPA2B1 in blastocysts and resulted in embryonic developmental defects, which was similar to the effect observed in blastocysts following hnRNPA2B1knockdown (<xref rid=\"B41\" ref-type=\"bibr\">Kwon et al., 2019</xref>).</p><p>In 2017, two studies illustrated that the m<sup>6</sup>A reader YTHDF2 is required for oocyte maturation and early embryonic development (<xref rid=\"B36\" ref-type=\"bibr\">Ivanova et al., 2017</xref>; <xref rid=\"B118\" ref-type=\"bibr\">Zhao et al., 2017</xref>). Mechanistically, YTHDF2 recognizes m<sup>6</sup>A-modified mRNAs and mediates the m<sup>6</sup>A-dependent RNA degradation process during mouse ZGA (<xref rid=\"B36\" ref-type=\"bibr\">Ivanova et al., 2017</xref>). YTHDF2 deletion delayed the degradation of maternal mRNAs, thus impeding zygotic genome activation in mice. These results indicate that YTHDF2-mediated m<sup>6</sup>A-dependent mRNA degradation plays an important role in transcriptome transitions and early embryonic development. Similar to the YTHDF2 knockout mouse model, a recent study found that oocyte-specific deletion of VIRMA results in female-specific infertility in mice (<xref rid=\"B110\" ref-type=\"bibr\">Yue et al., 2018</xref>). VIRMA deletion affected oocyte meiotic maturation by regulating pre-mRNA AS (<xref rid=\"B110\" ref-type=\"bibr\">Yue et al., 2018</xref>). The role of VIRMA in early embryonic development needs to be investigated in the future.</p><p>In contrast to YTHDF2, which functions in the promotion of mRNA decay, recent studies revealed that IGF2BPs could act as a new class of cytoplasmic m<sup>6</sup>A readers that promote the stability and storage of mRNAs (<xref rid=\"B34\" ref-type=\"bibr\">Huang et al., 2018</xref>). It was reported that IGF2BP1 deletion suppressed the RNA m<sup>6</sup>A modification level in mouse embryos and caused impaired early parthenogenetic (PA) embryogenesis, whereas supplementation with betaine increased the RNA m<sup>6</sup>A modification level and rescued embryonic development (<xref rid=\"B32\" ref-type=\"bibr\">Hao et al., 2020</xref>). In addition, miR-670-3p was found to regulate IGF2BP1 and affect apoptosis in PA embryos (<xref rid=\"B32\" ref-type=\"bibr\">Hao et al., 2020</xref>). Subsequently, <xref rid=\"B49\" ref-type=\"bibr\">Liu H.B. et al. (2019)</xref> reported that IGF2BP2 (also known as IMP2) is highly expressed in oocytes and early-stage embryos. IGF2BP2 deletion was not essential for oocyte maturation, while IGF2BP2-knockout mice exhibited female infertility (<xref rid=\"B49\" ref-type=\"bibr\">Liu H.B. et al., 2019</xref>). Moreover, maternal deletion of IGF2BP2 caused embryos to arrest at the 2-cell stage (<xref rid=\"B49\" ref-type=\"bibr\">Liu H.B. et al., 2019</xref>). Transcriptome and proteome analyses suggested that knockout of IGF2BP2 inhibited the transcription and translation of downstream genes related to ZGA, such as CCAR1 and RPS14 (<xref rid=\"B49\" ref-type=\"bibr\">Liu H.B. et al., 2019</xref>). However, whether IGF2BP2 participates in regulation of mRNA stability in ZGA as an m<sup>6</sup>A reader still needs to be clarified. Notably, a recent study found that IGF2BP3, but not IGF2BP1/2, stabilized maternal mRNA and regulated early embryogenesis in zebrafish (<xref rid=\"B73\" ref-type=\"bibr\">Ren et al., 2020</xref>). The majority of target mRNAs of IGF2BP3 were different from those of YTHDF2, suggesting that IGF2BPs and YTHDF2 may be different, but indispensable regulators at the MZT stage of early embryonic development in various species.</p></sec><sec id=\"S4.SS2\"><title>The Influences of m<sup>6</sup>A on Postimplantation Embryo Development</title><p>After the embryo is implanted in the uterus, cells in the ICM transition from naïve state pluripotency to primed state pluripotency, and the blastocyst differentiates into the epiblast of the embryonic region. The ectoderm, mesoderm and endoderm, which are derived from the epidermal cells through the gastrulation, constitute the basic cells required for organogenesis and individual formation.</p><p>Consistent with the observation that m<sup>6</sup>A is indispensable for ESC fate decisions, the loss of m<sup>6</sup>A causes naïve pluripotent stem cells to enter into a “hyper” naïve state, and they cannot transition from naïve state toward lineage differentiation, thus leading to embryonic lethality (<xref rid=\"B30\" ref-type=\"bibr\">Geula et al., 2015</xref>). <italic>In vivo</italic>, METTL3<sup>–/–</sup> ESCs showed a poor ability to differentiate into mature teratomas, and hematoxylin-eosin staining (H&E) staining suggested that KO teratomas could not differentiate into the three germ layers (<xref rid=\"B30\" ref-type=\"bibr\">Geula et al., 2015</xref>). The abnormal expression and location of NANOG from E5.5 (early postimplantation) to E7.5 (late gastrointestinal motility) in epiblasts led to embryonic lethality (<xref rid=\"B30\" ref-type=\"bibr\">Geula et al., 2015</xref>). MELLT3 and METTL14 knockout (KO) mice both exhibited embryonic lethality at E6.5 (<xref rid=\"B30\" ref-type=\"bibr\">Geula et al., 2015</xref>; <xref rid=\"B61\" ref-type=\"bibr\">Meng et al., 2019</xref>), suggesting that METTL3/14 are indispensable for early embryonic development in mice. A recent study reported that METTL14 deletion results in a similar effect to that of METTL3 in mouse embryogenesis. Compared with E5.5 WT embryos, Mettl14<sup>–/–</sup> embryos exhibited a large difference in gene expression and AS events, especially exon skipping (<xref rid=\"B61\" ref-type=\"bibr\">Meng et al., 2019</xref>). One possible mechanism is that METTL3/14 or m<sup>6</sup>A deposition accelerates the conversion of the epiblast from a naïve to a primed state, thus promoting differentiation. The aberrant expression levels of naïve and primed makers in METTL14<sup>–/–</sup> embryos support this inference.</p><p>A previous study found that WTAP deletion resulted in abnormal egg cylinders at the gastrulation stage and led to embryonic lethality at E10.5 in mice (<xref rid=\"B28\" ref-type=\"bibr\">Fukusumi et al., 2008</xref>). WTAP-deficient ESCs failed to differentiate into endoderm and mesoderm, which was confirmed by chimera analysis (<xref rid=\"B28\" ref-type=\"bibr\">Fukusumi et al., 2008</xref>). These results suggested that WTAP is essential for mesoderm and endoderm differentiation in the early mouse embryo. Notably, whether WTAP, as an m<sup>6</sup>A effector, plays a functional regulatory role in this process and the relevant regulatory mechanism has yet need to be determined.</p><p>In 2017, Pendleton et al. first reported that the m<sup>6</sup>A writer METTL16 regulated MAT2A expression by affecting its splicing in a hairpin (hp1) m<sup>6</sup>A-dependent manner (<xref rid=\"B69\" ref-type=\"bibr\">Pendleton et al., 2017</xref>). Subsequently, Mendel et al. created a METTL16 knockout (METTL16<sup>–/–</sup>) mouse model (<xref rid=\"B60\" ref-type=\"bibr\">Mendel et al., 2018</xref>). The E2.5 morula and E3.5 blastocysts from METTL16 KO and WT mice exhibited normal morphology and genotyping ratios. However, only a small proportion (1.9%) of KO embryos were detected at E6.5, indicating that METTL16 deletion caused embryonic lethality around implantation (<xref rid=\"B60\" ref-type=\"bibr\">Mendel et al., 2018</xref>). Interestingly, transcriptome analysis results suggested that MAT2A was the most downregulated gene in KO embryos at the E2.5 morula stage, whereas a large number of genes were dysregulated in E3.5 blastocysts of mouse (<xref rid=\"B60\" ref-type=\"bibr\">Mendel et al., 2018</xref>). Taken together, the m<sup>6</sup>A writer METTL16 regulates the methylation of the mRNA encoding SAM synthetase MAT2A and participates in the development of mouse embryos.</p><p>Several studies have demonstrated that the cytoplasmic reader YTHDC2 is essential for the transition from mitosis to meiosis in germ cells (<xref rid=\"B6\" ref-type=\"bibr\">Bailey et al., 2017</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Hsu et al., 2017</xref>; <xref rid=\"B96\" ref-type=\"bibr\">Wojtas et al., 2017</xref>). YTHDC2 is dispensable for viability, while YTHDC2 KO mice are infertile. Similar to METTL3, deletion of YTHDC1 results in embryonic lethality of mouse (<xref rid=\"B38\" ref-type=\"bibr\">Kasowitz et al., 2018</xref>). A recent study reported that YTHDC1 not only was required for gametogenesis but was also essential for viability (<xref rid=\"B38\" ref-type=\"bibr\">Kasowitz et al., 2018</xref>). Mechanistically, as the only nuclear m<sup>6</sup>A reader, YTHDC1 regulates the APA, AS and nuclear export of m<sup>6</sup>A-modified mRNAs in mouse oocytes (<xref rid=\"B38\" ref-type=\"bibr\">Kasowitz et al., 2018</xref>). The non-redundant role of YTHDC1 is also indispensable for mouse early embryonic development. In addition, outgrowth analysis found that no colonies were formed in HNRNPA2B1 KO blastocysts, unlike WT blastocysts, suggesting that HNRNPA2B1 may contribute to postimplantation development in mice (<xref rid=\"B41\" ref-type=\"bibr\">Kwon et al., 2019</xref>).</p><p>It is believed that m<sup>6</sup>A modifications are essential for the embryonic development and fertility in animals, whereas the regulatory mechanism is still largely unknown. One reason is that the majority of m<sup>6</sup>A-related enzyme deletions lead to early embryo lethality. On the other hand, m<sup>6</sup>A-seq requires a large amount of RNA sample input, which restricts the application of m<sup>6</sup>A to early embryos. The function and molecular regulation mechanism of RNA m<sup>6</sup>A modification in mouse early development need to be further investigated.</p></sec></sec><sec id=\"S5\"><title>Perspectives</title><p>The precise temporal and spatial control of gene expression is of fundamental importance for establishing cell fate and early development of complex bodies. Reversible RNA m<sup>6</sup>A methylation has many of the same characteristics as epigenetic DNA and histone modifications. Although epigenetic DNA and histone modifications mainly affect transcription events, reversible RNA methylation mainly regulates gene expression at posttranscriptional level. The dynamic regulation of m<sup>6</sup>A represents a newly identified mechanism of posttranscriptional regulation that maintains the balance between pluripotency and cell differentiation in a timely manner to ensure proper development. Different physiological states, environmental stimulates and cell signaling events may trigger different m<sup>6</sup>A methylation functions. Notably, there exist controversy on whether distinct cytoplasmic m<sup>6</sup>A-binding proteins YTHDFs recognize different sites (<xref rid=\"B86\" ref-type=\"bibr\">Wang et al., 2015</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Li et al., 2017</xref>; <xref rid=\"B111\" ref-type=\"bibr\">Zaccara and Jaffrey, 2020</xref>). The majority of current studies suggested that YTHDF1/3 regulate translation of m<sup>6</sup>A-modified mRNAs (<xref rid=\"B86\" ref-type=\"bibr\">Wang et al., 2015</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Li et al., 2017</xref>; <xref rid=\"B78\" ref-type=\"bibr\">Shi et al., 2017</xref>). However, how do these m<sup>6</sup>A readers (YTHDFs, YTHDCs, and IGF2BPs) specifically recognize their target transcripts during stem cell fate decisions and early embryonic development? In contrast to prevailing model, Zaccara and Jaffrey proposed that DF proteins do not regulate translation in HeLa cells, and YTHDFs bind the same m<sup>6</sup>A-modified mRNAs rather than different mRNAs in mRNA degradation (<xref rid=\"B111\" ref-type=\"bibr\">Zaccara and Jaffrey, 2020</xref>). Notably, it needs to be re-examined on the conclusion came only by siRNA knockdown in HeLa cells. Recently, <xref rid=\"B117\" ref-type=\"bibr\">Zhang et al. (2020)</xref> proposed that the effects of m<sup>6</sup>A on translation are heterogeneous and context dependent. Indeed, it still needs to be investigated the complex function of YTHDFs in translation. In addition, further studies and a comprehensive understanding of the functional roles of other m<sup>6</sup>A effectors during early embryonic development in mammals are still needed.</p><p>As we review in this review, RNA m<sup>6</sup>A modification plays significant functional roles in stem cell fate decisions and early embryonic development. Given that pluripotent stem cells have great prospects for application in regenerative medicine, organ transplantation and cancer treatment, a more in-depth study of the molecular regulation mechanisms by which m<sup>6</sup>A regulates the fate of pluripotent and other stem cells will promote the use of stem cells in developing and instituting stem cell treatments in the clinic. A number of studies have shown that RNA m<sup>6</sup>A modification is essential for mammalian embryo development and fertility. Although the majority of cloned animals are viable and can be produced via somatic cell nuclear transfer (SCNT), the efficiency is still extremely low (<xref rid=\"B94\" ref-type=\"bibr\">Wilmut et al., 1997</xref>; <xref rid=\"B59\" ref-type=\"bibr\">Matoba et al., 2018</xref>). One of the most important factors impeding SCNT is that abnormal epigenetic modification in donor cells, such as DNA methylation, histone modifications, and genomic imprinting (<xref rid=\"B104\" ref-type=\"bibr\">Yamagata et al., 2007</xref>; <xref rid=\"B115\" ref-type=\"bibr\">Zhang et al., 2009</xref>; <xref rid=\"B66\" ref-type=\"bibr\">Okae et al., 2014</xref>). Notably, SCNT is accompanied by significant events, including somatic cell reprogramming, ZGA and embryonic development, which have all been revealed to be regulated by RNA m<sup>6</sup>A modification. It remains to be examined whether m<sup>6</sup>A could affect SCNT embryonic development following SCNT. Moreover, it is of great significance to improve the SCNT efficiency by regulating the m<sup>6</sup>A modification level in somatic cells.</p><p>It is difficult to collect large amounts of samples from patients in the clinic and early embryos, whereas m<sup>6</sup>A-seq technological currently requires a large amount of RNA sample (>20 μg total RNA). New or refined m<sup>6</sup>A-seq technologies with low amounts of RNA samples input (>500 ng total RNA) or antibody-independent methods (MAZTER-seq, m<sup>6</sup>A-REF-seq, DART-seq, m<sup>6</sup>A-SEAL, and m<sup>6</sup>A-label-seq) have great application potential in the study of the m<sup>6</sup>A epitranscriptome in relation to disease treatment and embryonic development (<xref rid=\"B112\" ref-type=\"bibr\">Zeng et al., 2018</xref>; <xref rid=\"B62\" ref-type=\"bibr\">Meyer, 2019</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Garcia-Campos et al., 2019</xref>; <xref rid=\"B116\" ref-type=\"bibr\">Zhang Z. et al., 2019</xref>; <xref rid=\"B80\" ref-type=\"bibr\">Shu et al., 2020</xref>; <xref rid=\"B89\" ref-type=\"bibr\">Wang et al., 2020</xref>). Moreover, recent studies have developed m<sup>6</sup>A editing technologies that enable methylation or demethylation at a single site in transcripts through CRISPR-dCas9 and CRISPR-dCas13b systems (<xref rid=\"B72\" ref-type=\"bibr\">Rauch et al., 2018</xref>; <xref rid=\"B55\" ref-type=\"bibr\">Liu X. M. et al., 2019</xref>; <xref rid=\"B95\" ref-type=\"bibr\">Wilson et al., 2020</xref>; <xref rid=\"B45\" ref-type=\"bibr\">Li J. et al., 2020</xref>). We believe that m<sup>6</sup>A modification has great potential for application in regenerative and precision medicine, including cancer treatment, organ transplantation and reproductive development.</p></sec><sec id=\"S6\"><title>Author Contributions</title><p>MZ wrote the manuscript. YZ drew the pictures. SZ and XD revised the manuscript. ZL reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by the National Key R&D Program of China (No: 2017YFA0104400) and National Natural Science Foundation of China (No: 31972874).</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aguilo</surname><given-names>F.</given-names></name><name><surname>Zhang</surname><given-names>F.</given-names></name><name><surname>Sancho</surname><given-names>A.</given-names></name><name><surname>Fidalgo</surname><given-names>M.</given-names></name><name><surname>Cecilia</surname><given-names>S. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Beilstein J Nanotechnol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Beilstein J Nanotechnol</journal-id><journal-title-group><journal-title>Beilstein Journal of Nanotechnology</journal-title></journal-title-group><issn pub-type=\"epub\">2190-4286</issn><publisher><publisher-name>Beilstein-Institut</publisher-name><publisher-loc>Trakehner Str. 7-9, 60487 Frankfurt am Main, Germany</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32832317</article-id><article-id pub-id-type=\"pmc\">PMC7431754</article-id><article-id pub-id-type=\"doi\">10.3762/bjnano.11.106</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Full Research Paper</subject></subj-group><subj-group subj-group-type=\"topic\"><subject>Nanoscience</subject></subj-group><subj-group subj-group-type=\"topic\"><subject>Nanotechnology</subject></subj-group></article-categories><title-group><article-title>Gas sorption porosimetry for the evaluation of hard carbons as anodes for Li- and Na-ion batteries</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Matsukawa</surname><given-names>Yuko</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0001-5664-8410</contrib-id><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Linsenmann</surname><given-names>Fabian</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0001-8788-2584</contrib-id><xref ref-type=\"aff\" rid=\"A2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Plass</surname><given-names>Maximilian Arthur</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-6944-2136</contrib-id><xref ref-type=\"aff\" rid=\"A3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hasegawa</surname><given-names>George</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0003-4546-5197</contrib-id><xref ref-type=\"aff\" rid=\"A4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hayashi</surname><given-names>Katsuro</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-4413-6511</contrib-id><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Fellinger</surname><given-names>Tim-Patrick</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0001-6332-2347</contrib-id><email>tim.fellinger@tum.de</email><xref ref-type=\"aff\" rid=\"A2\">2</xref></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Ong</surname><given-names>Wee-Jun</given-names></name><role>Associate Editor</role></contrib></contrib-group><aff id=\"A1\"><label>1</label>Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan</aff><aff id=\"A2\"><label>2</label>Chair for Technical Electrochemistry, Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, Garching bei München 85748, Germany</aff><aff id=\"A3\"><label>3</label>Nanochemistry Department, Max Planck Institute for Solid State Research, Heisenbergstraße 1, Stuttgart 70569, Germany</aff><aff id=\"A4\"><label>4</label>Institute of Materials and Systems for Sustainability, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan</aff><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>14</day><month>8</month><year>2020</year></pub-date><volume>11</volume><fpage>1217</fpage><lpage>1229</lpage><ext-link ext-link-type=\"doi\" xlink:href=\"10.3762/bjnano.11.106\">10.3762/bjnano.11.106</ext-link><history><date date-type=\"received\"><day>18</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>21</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Matsukawa et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Matsukawa et al.</copyright-holder><ali:free_to_read xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\"/><license license-type=\"Beilstein\"><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\">https://creativecommons.org/licenses/by/4.0</ali:license_ref><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\">https://www.beilstein-journals.org/bjnano/terms</ali:license_ref><license-p>This is an Open Access article under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"https://creativecommons.org/licenses/by/4.0\">https://creativecommons.org/licenses/by/4.0</ext-link>). Please note that the reuse, redistribution and reproduction in particular requires that the authors and source are credited.</license-p><license-p>The license is subject to the Beilstein Journal of Nanotechnology terms and conditions: (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.beilstein-journals.org/bjnano/terms\">https://www.beilstein-journals.org/bjnano/terms</ext-link>)</license-p></license></permissions><abstract><p>Hard carbons are promising candidates for high-capacity anode materials in alkali metal-ion batteries, such as lithium- and sodium-ion batteries. High reversible capacities are often coming along with high irreversible capacity losses during the first cycles, limiting commercial viability. The trade-off to maximize the reversible capacities and simultaneously minimizing irreversible losses can be achieved by tuning the exact architecture of the subnanometric pore system inside the carbon particles. Since the characterization of small pores is nontrivial, we herein employ Kr, N<sub>2</sub> and CO<sub>2</sub> gas sorption porosimetry, as well as H<sub>2</sub>O vapor sorption porosimetry, to investigate eight hard carbons. Electrochemical lithium as well as sodium storage tests are compared to the obtained apparent surface areas and pore volumes. H<sub>2</sub>O, and more importantly CO<sub>2</sub>, sorption porosimetry turned out to be the preferred methods to evaluate the likelihood for excessive irreversible capacities. The methods are also useful to select the relatively most promising active materials within chemically similar materials. A quantitative relation of porosity descriptors to the obtained capacities remains a scientific challenge.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>alkaline-ion secondary battery</kwd><kwd>gas sorption porosimetry</kwd><kwd>hard carbon</kwd><kwd>irreversible capacity</kwd><kwd>ultramicroporosity</kwd></kwd-group><funding-group><funding-statement>Y.M. wishes to thank the Kyushu University Leading Program for “Molecular System for Devices” from MEXT Japan.</funding-statement></funding-group></article-meta></front><body><sec><title>Introduction</title><p>Lithium-ion battery (LIB)-based energy storage devices have been gaining high interest in the recent years in many industrial branches, ranging from electronic devices over battery electric vehicles (BEVs) to applications in grid energy storage. Since for grid energy storage a large amount of installed absolute capacity (rather than specific capacity) is required and LIB cells are still expensive, sodium-ion batteries (SIBs) become interesting [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]. Compared to Co, which is still an essential component for state-of-the-art LIB cathode materials, Li is much more abundant in the earth’s crust [<xref rid=\"R3\" ref-type=\"bibr\">3</xref>–<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]. Nevertheless, the market for mobile applications is increasing, especially for new BEVs, and thus the availability of Li and its price will most likely become an issue [<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]. Accordingly, for grid energy storage the priority is shifting from energy density to production cost and longevity [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]. Since Na is the fourth most abundant element in the earth’s crust, the production cost for typical precursors is by one order of magnitude less expensive [<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]. LIBs and SIBs have different applications. While LIBs are used as traction batteries in BEVs, in which volumetric energy density is crucial for a long mobility range, SIBs are mainly used for grid energy storage applications due to their lower cost [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>].</p><p>Historically, the first LIB introduced by Sony Corp. used a slightly disordered carbon, a so-called soft carbon (SC), which is graphitizable at temperatures of ca. 3000 °C, and later, from 1992, a more disordered hard carbon (HC), which is not graphitizable at temperatures of ca. 3000 °C as negative electrode [<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]. We herein refer to HCs as strongly disordered carbons (having a high fraction of sp<sup>3</sup>-hybridized defects or heteroatoms), independent of their graphitizability. After the introduction of the LIB, efforts in research and development on sodium-ion anodes, i.e., lithium-analogue materials, was the next logical step. Among the alkaline metal ions Na<sup>+</sup> is the second lightest and smallest ion and still possesses a relatively low standard reduction potential of −2.7 V vs SHE (compared to −3.04 V vs SHE for Li), which is crucial to allow for high energy densities [<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]. Many studies were carried out to understand the storage mechanisms of both lithium and sodium ions inside many different carbons. Due to the progress in LIB research and the implementation of stoichiometric and highly reversible graphite anodes (forming LiC<sub>6</sub>), disordered carbons were considered less. Although the volume expansion of HCs during lithium intercalation is lower than that of graphite, implying longer lifetimes, the relatively lower volumetric energy density due to their lower density and lower energy efficiency were detrimental for their commercial usage in the uprising market of portable devices. In 1997, the market share of HC and graphite was still 52% and 43%, respectively, and today graphite is almost exclusively used as negative electrode material in commercial LIBs [<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]. Graphite with an interlayer distance of 0.335 nm cannot be intercalated by sodium without solvent co-intercalation [<xref rid=\"R9\" ref-type=\"bibr\">9</xref>,<xref rid=\"R12\" ref-type=\"bibr\">12</xref>–<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]. Theoretical studies suggest a minimum distance of around 0.37 nm in order to enable reversible intercalation of sodium ions [<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]. Therefore, the use of less crystalline carbon electrodes in SIBs cannot be avoided [<xref rid=\"R15\" ref-type=\"bibr\">15</xref>]. The lower gravimetric energy density compared to LIBs (because of the higher specific weight of Na), the lower volumetric energy density (because of the need for the less dense disordered carbons), as well as the more complicated storage mechanism complicate the commercialization SIBs.</p><p>With the current strong interest in electromotive driving and the transformation of the energy grid to sustainable and decentral electricity generation and storage, the unconventional LIB and SIB technology using disordered carbons is currently having a comeback with a strongly increasing number of recent publications (<xref ref-type=\"supplementary-material\" rid=\"SD1\">Supporting Information File 1</xref>, Figure S1). Conventional LIB technology based on graphite only allows for a limited mobility range, therefore, HCs are investigated regarding the potential for higher energy densities. The seasonal and daytime-dependent character of solar and wind energy requires stationary electricity storage for times of light and wind shortage, with a focus on low cost.</p><p>To elucidate the more complex storage mechanism of both lithium and sodium ions in disordered carbonaceous materials, much effort has been spent and many different theories were debated, which are still under discussion [<xref rid=\"R16\" ref-type=\"bibr\">16</xref>–<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]. It was found that the achievable amount of sodium that could be inserted into carbon depends on the type of carbon. Both lithium and sodium are suggested to have two consecutive but very different charging mechanisms [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>,<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]. Characteristic charge–discharge curves of both SIBs and LIBs feature two regions, first, a sloping region having a hysteresis between charge and discharge, and second, a plateau appearing at low voltage. The regions may be assigned to the insertion of alkali ions between differently stacked carbon sheets in the sloping region on the one hand. On the other hand, alkali ion adsorption within nanopores in a “plating-like” adsorption process may explain the low voltage plateau due to multilayer adsorption. A direct correlation between the specific cumulative volume of pores smaller than 0.7 nm (ultramicropores) with the respective sodium storage capacity in cellulose-derived carbon showed decreasing sloping capacity and increasing plateau-like capacity with decreasing ultramicroporosity [<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]. This finding is in contrast to the results from Dahn and co-workers [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>,<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]. Apparently, the understanding of the role of specific textural features of disordered carbons for alkaline ion storage remains difficult, also leaving uncertainty about the potential of carbon materials for commercial application.</p><p>Besides the remaining questions about the reversible capacity, the reduction of the irreversible capacity for disordered carbons is another important field of research crucial for commercialization. As already mentioned above, one of the reasons why HCs were gradually substituted by graphite in commercial LIB cells, and one of the main limitations in current SIB research, is the relatively high irreversible capacity due to the formation of a solid electrolyte interface (SEI) layer. The irreversible capacity is believed to originate from electrolyte decomposition at potentials below the stability window of the electrolyte (for LIBs typically around 0.8 V<sub>Li</sub>) [<xref rid=\"R21\" ref-type=\"bibr\">21</xref>]. Since the dielectric SEI passivates the electrode, an irreversible capacity proportional to the electrochemical active surface area is expected. Accordingly, the reduction of the specific surface area (SSA) of the anode materials as well as the deposition of amorphous carbon films were shown to reduce irreversible capacity losses [<xref rid=\"R22\" ref-type=\"bibr\">22</xref>–<xref rid=\"R23\" ref-type=\"bibr\">23</xref>]. Ji et al. found that lower total pore volumes (determined by N<sub>2</sub> sorption) gave rise to increased reversible sodium storage capacities for sucrose-derived HCs [<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]. In contradiction to this study, Yang et al. showed that the pore volume of pores between 0.3 and 0.5 nm is responsible for reversible sodium storage [<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]. Yang et al. conclusively argued that ion-sieving effects, determined by a critical pore diameter, differentiate between pores contributing to either the reversible or the irreversible capacity [<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]. Pores that can be accessed by solvent molecules of the electrolyte will contribute to the irreversible capacity, while smaller ones are suitable for the adsorption of alkali metal ions protected from side reactions with solvent molecules from the electrolyte. It was further shown that additional irreversible capacity can arise from alkali metal ions reacting with surface defects or reactive surface groups and small molecules other than the electrolyte adsorbed to the walls of nanometric pores that were not removed during the cell production [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>]. These unwanted side reactions are expected to reduce the reversible capacity that can be obtained by adsorption within the pore system.</p><p>In this work, we use different gas sorption porosimetry (GSP) techniques to investigate surface areas and porosities, contributed by pores of different size, of different HCs. We critically evaluate these methods for the quantification of parameters related to the reversible and irreversible capacity of the materials as LIB and SIB anodes.</p></sec><sec><title>Results and Discussion</title><sec><title>Strategies for improving the capacity of hard carbons and revealing the pore structures</title><p>At the electrode–electrolyte interphase, HCs can act as molecular sieves. Here, Na ions are adsorbed in the pores in a metal-plating-like mechanism and separated from bigger solvent molecules that suffer from electrochemical reductive decomposition at potentials below ca. 0.8 V<sub>Li</sub> or towards the alkali metal, respectively. Since the storage mechanisms of lithium and sodium in HC anodes seem to be analogous, this is also expected to be a central effect in LIBs. We consider the determination of the following parameters crucial for the development of improved HCs towards reversible high capacity anodes for next generation LIBs and SIBs: 1) the surface area of the electrode exposed to electrolyte molecules, 2) the critical size of the effectively sieving pore, separating the pore system from the electrolyte, and 3) the quantity of the pore volume inaccessible to the electrolyte, likely being related to the reversible capacity.</p><p>The most common method to determine SSAs and nanoporosity in the battery community are nitrogen (N<sub>2</sub>) sorption porosimetry, as well as krypton (Kr) sorption porosimetry in case of very low surface areas. A precise quantification of microporosity (pore diameters smaller than 2 nm), however, cannot be unambiguously achieved by N<sub>2</sub> sorption alone and not at all using Kr sorption. The quadrupole moment of N<sub>2</sub> may lead to stronger interactions with carbon features resulting in smaller apparent pore diameters compared to Ar sorption porosimetry [<xref rid=\"R26\" ref-type=\"bibr\">26</xref>].</p><p>Furthermore, the pore size determination and the quantification of the pore volume of pores as small as the herein relevant (smaller than approx. 0.7 nm) are complicated by the limited accessibility of these pores by N<sub>2</sub> sorption porosimetry measurements at 77.4 K, due to low kinetic energy – likewise to the electrolyte molecules [<xref rid=\"R26\" ref-type=\"bibr\">26</xref>]. The lower vapor pressures of CO<sub>2</sub> and H<sub>2</sub>O compared to N<sub>2</sub> allow for measurements at higher temperatures with the respective higher kinetic energies. Because of higher measurement temperatures, CO<sub>2</sub> GSP at 273.15 K (0 °C) and water vapor sorption at 293.15 K (20 °C) are hence suggested, despite the ambiguous character of the pore size assignments [<xref rid=\"R27\" ref-type=\"bibr\">27</xref>]. Considering the expected complex interdependencies of the porosity/pore size distribution of HCs with their electrochemical properties, a combination of these sorption techniques seems reasonable.</p><p>To minimize capacity losses due to SEI formation in graphite-based LIBs, the external surface area of electrode materials is commonly minimized (i.e., small particle sizes are avoided). To improve the Brunauer–Emmett–Teller (BET) surface area determination, Kr sorption is commonly used. However, Kr sorption (performed at 77.4 K) cannot account for the apparent surface area originating from adsorption inside micropores.</p><p>Herein, two different types of HCs prepared with different procedures are compared regarding their morphological characteristics as well as their electrochemical properties. Six samples were prepared via hydrothermal carbonization (HT) followed by pyrolytic carbonization (HT carbons), and two samples were obtained from carbonized resorcinol–formaldehyde resins (RF carbons). The electrochemical sodium storage characteristics of the RF carbons were previously reported [<xref rid=\"R28\" ref-type=\"bibr\">28</xref>–<xref rid=\"R29\" ref-type=\"bibr\">29</xref>].</p></sec><sec><title>Characterization of hard carbons</title><p>The X-ray diffraction (XRD) patterns consistently point to disordered, amorphous HCs with missing sharp (002) and (101) reflections (<xref ref-type=\"supplementary-material\" rid=\"SD1\">Supporting Information File 1</xref>, Figure S2). The described features were also found for the RF samples as reported earlier by Hasegawa and co-workers (<xref ref-type=\"supplementary-material\" rid=\"SD1\">Supporting Information File 1</xref>, Figure S3) [<xref rid=\"R28\" ref-type=\"bibr\">28</xref>–<xref rid=\"R29\" ref-type=\"bibr\">29</xref>]. The position of the (002) reflection of the HTs corresponds to large interlayer distances in the range of 0.4 nm, while the RF carbons show <italic>d</italic>-spacings of 0.43 and 0.39 nm. Raman spectra of HT2 shows the G band peak at ca. 1590 cm<sup>−1</sup> and in addition the D band peak at ca. 1340 cm<sup>−1</sup>, which is further pointing to a disordered, defective graphitic structure (<xref ref-type=\"supplementary-material\" rid=\"SD1\">Supporting Information File 1</xref>, Figure S4). The D-to-G intensity ratio is 0.98, while it is 1.12 and 1.29 for, respectively, RF-1000 and RF-1600 (<xref ref-type=\"supplementary-material\" rid=\"SD1\">Supporting Information File 1</xref>, Figure S5), all indicating highly disordered carbons having a large fraction of sp<sup>3</sup> hybridization. The morphologies of the HT samples are very similar (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>), but show a different morphology compared to the RF carbons (<xref ref-type=\"supplementary-material\" rid=\"SD1\">Supporting Information File 1</xref>, Figure S6). Scanning electron microscopy (SEM) imaging reveals spherical particles, morphologically similar to previously reported HCs derived from the hydrothermal carbonization of saccharides [<xref rid=\"R30\" ref-type=\"bibr\">30</xref>–<xref rid=\"R31\" ref-type=\"bibr\">31</xref>]. Despite the slightly different preparation protocols, morphological differences between the hydrothermally obtained HCs are not significant (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). The two RF carbon samples showed well-defined monolithic macrostructures obtained by spinodal phase separation during the preparation [<xref rid=\"R28\" ref-type=\"bibr\">28</xref>–<xref rid=\"R29\" ref-type=\"bibr\">29</xref>]. After grinding for preparation of the electrodes, however, the monolithic structure was lost (<xref ref-type=\"supplementary-material\" rid=\"SD1\">Supporting Information File 1</xref>, Figure S6). The reason why HT6 (<xref ref-type=\"fig\" rid=\"F1\">Figure 1f</xref>) shows smaller particle diameters than the other carbons (<xref ref-type=\"fig\" rid=\"F1\">Figure 1a</xref>–e) can be explained by the secondary catalytic effect of borax during the hydrothermal carbonization of sugars [<xref rid=\"R32\" ref-type=\"bibr\">32</xref>].</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>SEM images of HT carbons: a) HT1, b) HT2, c) HT3, d) HT4, e) HT5 and f) HT6.</p></caption><graphic xlink:href=\"Beilstein_J_Nanotechnol-11-1217-g002\"/></fig></sec><sec><title>Gas sorption porosimetry of hard carbons</title><p>N<sub>2</sub> sorption porosimetry of the HT samples turned out to be problematic, since the equilibrium conditions were not reached (exemplarily shown in <xref ref-type=\"supplementary-material\" rid=\"SD1\">Supporting Information File 1</xref>, Figure S7). We expect the combination of large, micrometer-sized pore systems with pore necks of a few angstroms and kinetic limitations of the N<sub>2</sub> molecules to penetrate the pore system. Kr sorption at 77.4 K as well as CO<sub>2</sub> and H<sub>2</sub>O sorption measurements at higher temperatures were therefore performed and the resulting isotherms, pore size distributions, and cumulative pore volumes for CO<sub>2</sub> and H<sub>2</sub>O are shown in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Gas adsorption isotherms (a, d), calculated pore size distributions (b, e), and cumulative pore volumes (c, f) for CO<sub>2</sub> (a–c) and for H<sub>2</sub>O (d–f) adsorption (STP-standard pressure).</p></caption><graphic xlink:href=\"Beilstein_J_Nanotechnol-11-1217-g003\"/></fig><p>The CO<sub>2</sub> isotherms for the HT samples show relatively high CO<sub>2</sub> sorption capacities with similar isotherm shapes (characteristic Langmuir isotherms). The CO<sub>2</sub> uptake values of the HTs were in the range of 60 to 80 cm<sup>3</sup> g<sup>−1</sup> (roughly corresponding to 2.7 to 3.6 mmol g<sup>−1</sup>) reflecting relatively similar porosities (<xref ref-type=\"fig\" rid=\"F2\">Figure 2a</xref>). The isotherm for RF-1000 is also very similar to those of the HT samples; however, for RF-1600 the CO<sub>2</sub> uptake is much lower (11.5 cm<sup>3</sup> g<sup>−1</sup> or approx. 0.51 mmol g<sup>−1</sup>).</p><p>As indicated by the high CO<sub>2</sub> uptakes, the apparent SSAs derived from CO<sub>2</sub> sorption are also relatively high with a deviation of 14% for the HT samples ranging from 468 m<sup>2</sup> g<sup>−1</sup> (HT5) to 544 m<sup>2</sup> g<sup>−1</sup> (HT3) (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). The SSAs for RF-1000 and RF-1600 are 546 and 270 m<sup>2</sup> g<sup>−1</sup>, respectively. Importantly, the obtained values for the SSA from CO<sub>2</sub> sorption (SSA<sub>CO2</sub>), for all samples, strongly differ from values determined by Kr sorption (SSA<sub>Kr</sub>). <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> summarizes the SSAs calculated from CO<sub>2</sub> and Kr sorption measurements using the BET theory.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Properties of the carbon samples from Kr, CO<sub>2</sub> and H<sub>2</sub>O sorption.</p></caption><table frame=\"hsides\" rules=\"groups\"><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">sample</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">SSA<sub>Kr</sub><sup>a</sup><break/>(m<sup>2</sup> g<sup>−1</sup>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">SSA<sub>CO2</sub><sup>b</sup>/SSA<sub>N2</sub><sup>c</sup><break/>(m<sup>2</sup> g<sup>−1</sup>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CPV<sub>CO2</sub><sup>d</sup>/CPV<sub>N2</sub><sup>e</sup><break/>(cm<sup>3</sup> g<sup>−1</sup>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CPV<sub>H2O</sub><sup>f</sup><break/>(cm<sup>3</sup> g<sup>−1</sup>)</td></tr><tr><td align=\"left\" colspan=\"5\" valign=\"middle\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">HT1</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.60</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">492<sup>b</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.138<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.00584</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">HT2</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.41</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">486<sup>b</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.141<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.00332</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">HT3</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2.66</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">544<sup>b</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.152<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.00376</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">HT4</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.29</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">535<sup>b</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.155<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.00633</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">HT5</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.89</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">468<sup>b</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.134<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.00523</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">HT6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">8.11</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">471<sup>b</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.138<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.00336</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">RF-1000</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">8.56</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">546<sup>b</sup>/630<sup>c</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.145<sup>d</sup>/0.188<sup>e</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.00574</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">RF-1600</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">14.69</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">270<sup>b</sup>/3.8<sup>c</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.014<sup>d</sup>/0.0002<sup>e</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.00047</td></tr></table><table-wrap-foot><fn id=\"TFN1\"><p><sup>a,b,c</sup>Specific surface area obtained by the BET method from Kr, CO<sub>2</sub>, and N<sub>2</sub> isotherms, respectively. <sup>d,e,f</sup>Cumulative pore volume (CPV) obtained from Monte Carlo, density functional theory, and Mahle method for CO<sub>2</sub>, N<sub>2</sub> and H<sub>2</sub>O in the pore size range below 0.7 nm, respectively.</p></fn></table-wrap-foot></table-wrap><p>The absolute values for the SSAs derived from Kr and CO<sub>2</sub> sorption differ by two to three orders of magnitude indicating a very different physical meaning of surface depending on the employed method. Since the kinematic diameters of the battery electrolyte molecules are of the same order of magnitude as the gas molecules used (and at the same time large compared to Li and Na ions), a careful examination of the validity of the respective methods is a sine qua non. While the SSA<sub>Kr</sub> of HT6 (8.11 m<sup>2</sup> g<sup>−1</sup>) is at least three times larger than those of other HT samples (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>), this trend is absent in case of CO<sub>2</sub> sorption. The results for Kr reflect the observed higher geometrical surface-to-volume ratio of HT6 compared to the other HC samples, as mentioned above (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Obviously, Kr adsorbs at the outer (external) surface of the particles, but does not penetrate the small pores of the particles at the measurement temperature of 77.4 K. For the CO<sub>2</sub> sorption, this outer surface is much smaller and therefore negligible compared to the large internal surface area that is accessible with CO<sub>2</sub>. However, the SSA<sub>Kr</sub> of HT6 is still smaller than that of both RF carbons, with RF-1600 showing by far the highest SSA<sub>Kr</sub>. From the perspective of graphite anodes, considering the external surface area to be directly related to SEI losses, RF-1600 would be expected to have 2–20 times higher irreversible capacities than the other samples.</p><p>In contrast to the HT samples, N<sub>2</sub> sorption was successfully carried out on the RF samples. Comparison of the values obtained from CO<sub>2</sub> versus N<sub>2</sub> shows a 13% lower SSA<sub>CO2</sub> for RF-1000 (546 m<sup>2</sup> g<sup>−1</sup><sub>CO2</sub> compared to 630 m<sup>2</sup> g<sup>−1</sup><sub>N2</sub>), while for RF-1600 the SSA<sub>CO2</sub> is 71.1 times higher than the SSA<sub>N2</sub> (270 m<sup>2</sup> g<sup>−1</sup><sub>CO2</sub> compared to approx. 4 m<sup>2</sup> g<sup>−1</sup><sub>N2</sub>). Obviously, at 77.4 K small structural differences in the pore systems decide whether or not N<sub>2</sub> molecules can penetrate.</p><p>It is important to highlight that the deviations are not artefacts from the applied models, but are real effects resulting from the unequal accessibility of the small pores for the different gas molecules. In HC anode research the use of CO<sub>2</sub> sorption rather than N<sub>2</sub> sorption thus appears to be more reasonable.</p><p>Pore size distributions of the HT carbons derived from CO<sub>2</sub> sorption data using the Monte Carlo method were centered around 0.5 nm with less abundant larger pores between 0.7 and 1.5 nm (<xref ref-type=\"fig\" rid=\"F2\">Figure 2b</xref>). RF-1600 does not have pores smaller than 0.5 nm with main porosity contributions between 0.6 and 1.1 nm. The corresponding cumulative pore volumes of the HT samples as well as of RF-1000 show that roughly two thirds of the porosity are originating from pores smaller than 0.7 nm, the so-called ultramicropores (<xref ref-type=\"fig\" rid=\"F2\">Figure 2c</xref>). Porosity contributions for RF-1600 are more homogeneously distributed, however, at much lower absolute numbers. Despite having the highest geometrical surface-to-volume ratio, RF-1600 is likely to have a much smaller electrode–electrolyte interface than the other samples.</p><p>The H<sub>2</sub>O sorption measurements also reveal similar isotherm shapes for the HT samples and for RF-1000, but not for RF-1600. The characteristic sigmoidal shapes for the HT samples, however, show different relative adsorption onset pressures, indicating differences in hydrophilicity or average pore size as well as slightly different slopes for the gas uptake (<xref ref-type=\"fig\" rid=\"F2\">Figure 2d</xref>). Similar to the results of CO<sub>2</sub> sorption, RF-1600 shows a relatively low uptake. The adsorption branch is furthermore shifted to higher relative pressures compared to the other samples, clearly pointing to a less accessible pore system. According to the Mahle model [<xref rid=\"R33\" ref-type=\"bibr\">33</xref>] and the Wang equation [<xref rid=\"R34\" ref-type=\"bibr\">34</xref>], the center of the pore diameter distribution of the HT samples is at around 1.0 nm for HT4 and HT5, while it is around 1.1 nm for HT2, HT3, and HT6 (<xref ref-type=\"fig\" rid=\"F2\">Figure 2e</xref>). HT1 and RF-1000 lie in between. The very similar synthetic protocols for the different HT materials make hydrophilicity differences appear unreasonable compared to differences in the pore system. Because of the different preparation procedures, it remains speculative to assign differences in the water sorption results between RF-1000 and HT samples to different pore characteristics only. However, it is to mention that the isotherm characteristics are generally rather similar, like in the case of CO<sub>2</sub> sorption. The results illustrate the ambiguous assignment of pore sizes for CO<sub>2</sub> and H<sub>2</sub>O sorption measurements. However, the Monte Carlo method is an advanced model compared to the Mahle model, resulting in higher reliability in reflecting the pore system for CO<sub>2</sub> sorption measurements compared to H<sub>2</sub>O sorption. Interestingly, the trends revealed by adsorption of the different gases are not identical. The determined porosity trends are RF-1000 ≈ HT3 ≈ HT4 > HT1 ≈ HT2 > HT5 ≈ HT6 ≫ RF-1600 for CO<sub>2</sub> (<xref rid=\"T1\" ref-type=\"table\">Table 1d</xref>) and HT3 ≈ HT1 > HT2 ≈ RF-1000 ≈ HT4 ≈ HT6 > HT5 ≫ RF-1600 in case for H<sub>2</sub>O (<xref rid=\"T1\" ref-type=\"table\">Table 1f</xref>). While for CO<sub>2</sub> sorption, the different uptakes are assigned to more abundant pores smaller than 0.7 nm (ultramicropores) in case of HT3 and HT4, H<sub>2</sub>O sorption reveals different characteristic pore sizes. Independent of the total porosity HT1, HT4, and HT5 have smaller characteristic pore diameters than the other HT samples. Again, this observation is not an artefact of the pore size calculation, but can also be clearly observed in the isotherms by the shift in the H<sub>2</sub>O uptake to lower relative pressure values for these samples. A possible explanation could relate the steep gas uptake to different sizes of pore necks, reflecting a barrier to penetrate the whole pore system.</p><p>Similar to the case of SSA determination by N<sub>2</sub> and CO<sub>2</sub> sorption, it is interesting to compare absolute values and relative deviations in the quantification of pore volumes of the RF carbons using N<sub>2</sub>, CO<sub>2,</sub> and H<sub>2</sub>O sorption. The total pore volume (TPV) is a simple porosity estimation derived from the total gas uptake at maximum relative pressure in a certain GSP measurement. The TPVs of RF-1000 quantified by CO<sub>2</sub> and H<sub>2</sub>O sorption are 36.0% and 47.8% smaller than those obtained by N<sub>2</sub> sorption. Apparently, the absolute quantification of porosity is questionable and the most accurate results may depend on how well the gas fits the studied material. Relative differences for similarly prepared materials may be helpful for the evaluation of HCs for application as anode electrodes in LIBs or SIBs, though. For RF-1000, it can be stated that the total porosity is 6.32, 27.40, and 6.79 times larger than that of RF-1600 according to CO<sub>2</sub>, N<sub>2</sub>, and, H<sub>2</sub>O sorption, respectively.</p></sec><sec><title>Reversible/irreversible capacities of hard carbons and the relationship with the surface structures</title><p>The mechanistic similarity of gas adsorption experiments to the lithium or sodium discharge process suggests that effects in GSP measurements may be correlated with phenomena in the corresponding battery tests [<xref rid=\"R35\" ref-type=\"bibr\">35</xref>]. Electrochemical cycling experiments were carried out in ethylene carbonate (EC)/ethyl methyl carbonate (EMC) = 3:7 (v/v) for LIB tests and in EC/diethyl carbonate (DEC) = 1:1 (v/v) for SIB tests. In both cases, the smaller solvent molecule was EC, which we thought might determine a critical ion-sieving pore size for both LIB and SIB tests. With the aim to relate the investigated morphological features of the HCs to their electrochemical properties as LIB (<xref ref-type=\"fig\" rid=\"F3\">Figure 3a</xref>,b) and SIB (<xref ref-type=\"fig\" rid=\"F3\">Figure 3c</xref>,d) anodes, irreversible capacity and reversible capacity were defined as follows: the irreversible capacity is the difference between the 1st charge and the 3rd discharge capacity. The reversible capacity is defined by the 3rd discharge capacity. The values are obtained from charge–discharge curves, which are plotted as a function of quantities of different morphological features obtained from the GSP measurements (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref> and <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). Since the charge–discharge tests vs lithium were performed in multiple cells for each of the HTs, their capacities and Coulombic efficiencies are average values. We herein regard cumulative pore volumes (CPVs) of pores smaller than 0.7 nm, i.e., pore sizes that could be considered inaccessible to solvent molecules, as well as of pores smaller than 3.0 nm<italic>.</italic> We related the capacities also to other cumulative pore volumes as well as the TPVs in search for direct relations.</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Capacities (reversible: red circle, irreversible: blue diamond, <italic>Y</italic>-axis) and Coulombic efficiencies (gray asterisk, <italic>R</italic>-axis) against SSA obtained from Kr (a, c) and CO<sub>2</sub> (b,d) adsorption isotherms of lithium (a, b) and sodium (c, d).</p></caption><graphic xlink:href=\"Beilstein_J_Nanotechnol-11-1217-g004\"/></fig><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Capacities (reversible: red circle, irreversible: blue diamond, <italic>Y</italic>-axis) and Coulombic efficiencies (gray asterisk, <italic>R</italic>-axis) against cumulative pore volumes (obtained from CO<sub>2</sub> adsorption by Monte Carlo method (a, c) and from H<sub>2</sub>O adsorption by Mahle method (b, d)) of lithium (a, b) and sodium (c, d). The inset in (a) is an enlargement of the irreversible capacity trend of HT carbons (blue line: approximate straight line of irreversible capacity of HT carbons. Slope: 3334 mAh cm<sup>−3</sup>, coefficient of determination, <italic>R</italic><sup>2</sup> = 0.67795).</p></caption><graphic xlink:href=\"Beilstein_J_Nanotechnol-11-1217-g005\"/></fig><p>The correlation of the SSA<sub>Kr</sub> with the capacities in LIB (<xref ref-type=\"fig\" rid=\"F3\">Figure 3a</xref>) or SIB (<xref ref-type=\"fig\" rid=\"F3\">Figure 3c</xref>) anodes, as well as with the respective Coulombic efficiencies, revealed no clear trend. In fact, the material with the highest SSA<sub>Kr</sub> (RF-1600) turned out to have the smallest irreversible capacity, clearly showing that the Kr sorption data are not at all related to the electrode–electrolyte interface in the final battery. HTs with higher SSA<sub>CO2</sub> tended to show higher irreversible capacities, except HT1, in the case of lithium, it was not seen in the case of sodium (<xref ref-type=\"fig\" rid=\"F3\">Figure 3b</xref>,d). It is to mention that the absolute numbers of the capacity-per-surface-area for the HT samples largely deviate from the RF samples. This indicates that the quantity of the electrochemical reactions, may it be reversible or irreversible, are not only related to the measurable surface area for amorphous electrode materials. One may consider different deposition mechanisms (e.g., van der Merwe layer-by-layer or Volmer–Weber island growth) to explain this. However, it is more likely related to different contributions of porosity that is even inaccessible to CO<sub>2</sub>, i.e., pseudo-graphitic interlayer voids or larger pores that are surrounded by smaller pores.</p><p>The same trend was also found for correlations of CPVs of pores smaller than 0.7 nm obtained from CO<sub>2</sub> sorption (CPV<sub>CO2</sub>) with irreversible capacities, however, not quite for pores smaller than 1.5 nm from H<sub>2</sub>O sorption (CPV<sub>H2O</sub>) (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). The HT samples with higher CPVs basically showed larger irreversible capacities, again only in the case of LIB tests (<xref ref-type=\"fig\" rid=\"F4\">Figure 4a</xref>,b), while such a trend speculatively remains for SIB tests on the RF carbons, although only based on two samples. Differences between the trends for the irreversible capacities of lithium (<xref ref-type=\"fig\" rid=\"F4\">Figure 4a</xref>,b) and sodium (<xref ref-type=\"fig\" rid=\"F4\">Figure 4c</xref>,d) for HT samples, may be explained with a better utilization of the HT carbons by lithium, as will be further discussed below. The fact that increased irreversible capacities relate to both increasing SSA<sub>CO2</sub> (<xref ref-type=\"fig\" rid=\"F3\">Figure 3b</xref>) and increasing CPV<sub>CO2</sub> (<xref ref-type=\"fig\" rid=\"F4\">Figure 4a</xref>,b) points to the fractal nature of HCs, meaning that at such small pore sizes a strict definition of surface area cannot be applied, as reflected in the preferred use of the term “apparent surface area” [<xref rid=\"R36\" ref-type=\"bibr\">36</xref>–<xref rid=\"R37\" ref-type=\"bibr\">37</xref>]. Another interesting observation is that the pore volumes obtained by CO<sub>2</sub> and H<sub>2</sub>O sorption, both show no direct relation to the reversible capacity although apparent pore sizes down to approx. 0.35 nm were quantified. This experimental result, in principle, contradicts both Yang et al. [<xref rid=\"R25\" ref-type=\"bibr\">25</xref>] as well as Ji et al. [<xref rid=\"R24\" ref-type=\"bibr\">24</xref>], since neither the porosity of small pores explains the capacity, nor is a trend of increasing reversible capacity with decreasing pore volumes observed. We rather find that measurable porosity in micrometer-sized hard carbons may cause irreversible capacity (especially in LIBs) and that the characterization of this porosity should rather be carried out with CO<sub>2</sub> or H<sub>2</sub>O sorption instead of N<sub>2</sub> or Kr sorption.</p><p>Of course, from the results of two RF carbons a trend cannot be concluded. However, it stands out that the slopes of assumed trends for pore descriptors derived from CO<sub>2</sub> sorption (<xref ref-type=\"fig\" rid=\"F3\">Figure 3b</xref> and <xref ref-type=\"fig\" rid=\"F4\">Figure 4a</xref>) match the ones for the HT carbons relatively well (3334 mAh cm<sup>−3</sup> for the HC carbons according to the linear fit). This indicates that CO<sub>2</sub> sorption is indeed the preferred method over N<sub>2</sub> sorption to estimate irreversible capacity losses by means of SEI formation. Relatively more promising samples may be selected based on this technique, which potentially even allows for the quantification of certain HC anodes. For the RF carbons, at least the ratios RF-1000/RF-1600 of SSA<sub>CO2</sub>, CPV<sub>CO2</sub>, and CPV<sub>H2O</sub> correspond well to the irreversible capacity ratio, all having a value of ca. 6. This suggests that in case of the RF samples, the penetration of both H<sub>2</sub>O and CO<sub>2</sub> into the pore system is similar and therefore can be used to mimic the penetration of electrolyte molecules. Furthermore, an obvious difference between LIB and SIB tests is observed for the HT samples. While linear trends are obtained for LIB tests, the SIB tests seem to have no correlation between the irreversible capacity and any porosity descriptor. Thus, it is assumed that the measured porosity of HT carbon particles cannot be fully penetrated by the solvent molecules in SIB tests, potentially because of clogging caused by the larger DEC solvent molecules. Hence, our assumption of EC determining a critical ion-sieving pore size for both LIB and SIB seems to be wrong.</p><p>As already mentioned above, the very small pores that are accessible by GSP do not relate to the obtained reversible capacities. This seems to contradict the size range of pores in which sodium can be adsorbed according to Yang et al. [<xref rid=\"R25\" ref-type=\"bibr\">25</xref>], since we can fairly well quantify porosities between 0.3 and 0.5 nm. Are these pores not contributing or is it possible that different penetration depths of solvent molecules and, hence, SEI deposits smear out the relation of pore volume to reversible capacity? A simple relation of the porosity that can be filled with sodium or lithium to the corresponding theoretical capacity may be helpful for discussion (<italic>V</italic><sup>spec</sup><sub>pore</sub> is the specific pore volume):</p><disp-formula id=\"FD1\"><label>[1]</label><alternatives><mml:math id=\"M1\"><mml:mrow><mml:msup><mml:mrow/><mml:mrow><mml:mtext>Na</mml:mtext></mml:mrow></mml:msup><mml:msub><mml:mtext>C</mml:mtext><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msup><mml:mrow><mml:mtext>mAh g</mml:mtext></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>1129</mml:mn><mml:msup><mml:mrow><mml:mtext> mAh cm</mml:mtext></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>3</mml:mn></mml:mrow></mml:msup><mml:mo>⋅</mml:mo><mml:msup><mml:mi>V</mml:mi><mml:mrow><mml:mtext>spec</mml:mtext></mml:mrow></mml:msup><mml:msub><mml:mrow/><mml:mrow><mml:mtext>pore</mml:mtext></mml:mrow></mml:msub><mml:mo>,</mml:mo></mml:mrow></mml:math><graphic xlink:href=\"Beilstein_J_Nanotechnol-11-1217-e001.jpg\" position=\"anchor\"/></alternatives></disp-formula><disp-formula id=\"FD2\"><label>[2]</label><alternatives><mml:math id=\"M2\"><mml:mrow><mml:msup><mml:mrow/><mml:mrow><mml:mtext>Li</mml:mtext></mml:mrow></mml:msup><mml:msub><mml:mtext>C</mml:mtext><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msup><mml:mrow><mml:mtext>mAh g</mml:mtext></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>2066</mml:mn><mml:msup><mml:mrow><mml:mtext> mAh cm</mml:mtext></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>3</mml:mn></mml:mrow></mml:msup><mml:mo>⋅</mml:mo><mml:msup><mml:mi>V</mml:mi><mml:mrow><mml:mtext>spec</mml:mtext></mml:mrow></mml:msup><mml:msub><mml:mrow/><mml:mrow><mml:mtext>pore</mml:mtext></mml:mrow></mml:msub><mml:mo>.</mml:mo></mml:mrow></mml:math><graphic xlink:href=\"Beilstein_J_Nanotechnol-11-1217-e002.jpg\" position=\"anchor\"/></alternatives></disp-formula><p><xref ref-type=\"disp-formula\" rid=\"FD1\">Equation 1</xref> and 2 are based on the simple assumption that sodium and lithium are being plated with their bulk densities on the inside of the pores in the HC anode. When using the CO<sub>2</sub> sorption data (CPVs) of HT4 (highest porosity) and HT5 (lowest porosity) <xref ref-type=\"disp-formula\" rid=\"FD1\">Equation 1</xref> and <xref ref-type=\"disp-formula\" rid=\"FD2\">Equation 2</xref> reveal expected capacities of, respectively, 175 and 151 mAh g<sup>−1</sup> for SIB tests and of, respectively, 320 and 277 mAh g<sup>−1</sup> for LIB tests. Comparison with the third discharge capacities (reversible capacities) shows an underestimation of the capacity of the HT carbons by 25 and 34%, respectively, for SIBs and by 11 and 22%, respectively, for LIBs. As the porosities obtained by H<sub>2</sub>O sorption are consistently smaller than those obtained by CO<sub>2</sub> sorption the underestimation is even larger with these data.</p><p>Since the characteristic sloping as well as the plateau region in the charge–discharge curves are often expected to represent, respectively, intercalation and adsorption, we also approached the correlation of the GSP data with the separated contributions of those two regions. Although, the accessible porosity could now theoretically (according to <xref ref-type=\"disp-formula\" rid=\"FD1\">Equation 1</xref> or <xref ref-type=\"disp-formula\" rid=\"FD2\">Equation 2</xref>) explain each of the contributions, a direct relation of the porosity descriptor to either one or the other branch was again not obtained (<xref ref-type=\"supplementary-material\" rid=\"SD1\">Supporting Information File 1</xref>, Figure S8). Accordingly, it is apparent that the underlying capacity is not related to the measurable porosity. Furthermore, a limited utilization of the carbon anode materials by gas molecules as well as alkaline earth metal ions is assumed. Both will depend on the actual morphology of the hard carbons indicating the complicated reality of HC anodes in SIBs as well as in LIBs.</p><p>To improve the understanding of the porosity accessible by GSP techniques more sophisticated techniques are necessary. Especially the connectivity of pores affecting the penetration depth and electrode utilization will play a crucial role in understanding and predicting the sodium and lithium storage capacities of HC anodes, and thereby elucidate the full potential of those materials for LIB and SIB technology.</p></sec></sec><sec><title>Conclusion</title><p>We carried out a comparative study of porosity descriptors obtained by different gas sorption porosimetry techniques (Kr, N<sub>2</sub>, CO<sub>2</sub>, and H<sub>2</sub>O) for the relation with the electrochemical performance of HC anodes in lithium and sodium ion battery tests. Different to the case of graphite anodes, the geometry of the particles does not allow for conclusions on capacity losses through SEI formation. The use of Kr sorption to estimate the accessible surface for the used electrolyte solvent molecules by BET surface area measurements leads to a strong underestimation of capacity losses. The surface area values obtained from Kr sorption are two orders of magnitude lower than those obtained from CO<sub>2</sub> sorption, and can give misleading results regarding expected irreversible losses. In the present study, the sample with the highest relative Kr sorption-based surface area even exhibited the lowest capacity losses amongst the tested samples. The CO<sub>2</sub> sorption-derived surface area values turned out to be more reliable than the values obtained from the most commonly used N<sub>2</sub> sorption analysis. N<sub>2</sub> sorption porosimetry measurements can also dramatically underestimate the pore accessibility for electrolyte molecules and, hence, the SEI formation. Furthermore, the porosity contributed by ultramicropores (0.3–0.7 nm) could not be related directly to the obtained reversible capacities, neither for LIB nor for SIB tests. Instead, it was found that for LIBs even the porosity from pores below 0.7 nm (determined with CO<sub>2</sub> and/or H<sub>2</sub>O sorption) was proportional to capacity losses due to SEI formation. For SIBs the trends are debatable. However, they point to a limited utilization of ultramicropores of mesoscopic hard carbons in both gas sorption experiments and battery cycling. Finally, it is recommended to employ H<sub>2</sub>O and especially CO<sub>2</sub> sorption porosimetry for the research on HC anodes to support progress in the understanding of the SEI formation and reversible alkali metal storage.</p></sec><sec><title>Experimental</title><sec><title>Material synthesis</title><sec><title>Preparation of carbons derived from ᴅ-fructose</title><p>The carbons synthesized via hydrothermal method were produced by a step synthesis similar to the reported preparation according to Väli et al. [<xref rid=\"R30\" ref-type=\"bibr\">30</xref>] and Fellinger and co-workers [<xref rid=\"R32\" ref-type=\"bibr\">32</xref>]<italic>.</italic> The first step was a hydrothermal synthesis of a precursor, followed by a pyrolytic carbonization to get the final HC product. At first, a 25 wt % solution of ᴅ-fructose (Sigma-Aldrich, Germany) in deionized water (Millipore, Merck, Germany) was prepared. Similar to a synthesis reported by Fellinger et al. [<xref rid=\"R32\" ref-type=\"bibr\">32</xref>], Na<sub>2</sub>B<sub>4</sub>O<sub>7</sub>·10H<sub>2</sub>O (Borax) (Sigma-Aldrich, Germany) was used as a structure directing agent for the preparation of one sample. The concentration in the ᴅ-fructose solution was 0.06 wt %. 20 g of the solution with or without borax was poured into a 25 mL quartz tube and was heated in a Teflon-lined autoclave for 24 h at different temperatures from 190 to 220 °C. After cooling down to room temperature, the autoclaves were opened and the black precursors were washed with around 2 L of water and then 1 L of ethanol to remove residuals of the polymerization by vacuum filtration. After drying the precursor overnight at 70 °C under vacuum, it was carbonized in a tube furnace with argon flow at a rate of higher than 0.5 L min<sup>−1</sup> at 1000 °C for 1, 5, or 10 h. The carbons produced by this hydrothermal method (HT carbons) will be called HT<italic>x</italic>, where <italic>x</italic> is an integer. The synthesis conditions (temperature of hydrothermal treatment and carbonization time under argon flow) are listed in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>.</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Synthesis conditions for the six HT carbons.</p></caption><table frame=\"hsides\" rules=\"groups\"><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">sample</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">temperature of HT treatment (°C)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">carbonization time (h)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">addition of borax during HT treatment</td></tr><tr><td align=\"left\" colspan=\"4\" valign=\"middle\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">HT1</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">190</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">HT2</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">200</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">HT3</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">220</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">HT4</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">200</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">HT5</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">200</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">HT6</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">200</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">0.06 wt %</td></tr></table></table-wrap></sec><sec><title>Preparation of carbons from resorcinol-formaldehyde gels</title><p>Porous carbon monoliths were obtained by carbonization of resorcinol–formaldehyde (RF) gels obtained via a sol–gel process as previously reported [<xref rid=\"R28\" ref-type=\"bibr\">28</xref>–<xref rid=\"R29\" ref-type=\"bibr\">29</xref>]. In short, 2.20 g of resorcinol (Sigma-Aldrich Co.) was added to a mixture of 4.0 mL of 10 mM HCl aq. (Kishida Chemical Co., Ltd.) and 0.80 mL of ethanol (Kishida Chemical Co., Ltd.) to obtain a homogeneous solution. The solution was cooled to 0 °C and then a formaldehyde solution (37 wt % in H<sub>2</sub>O containing 10–15 wt % methanol) (Sigma-Aldrich, Germany) was added under vigorous stirring. After mixing for 5 min, the solution was kept at 40 °C for 24 h for gelation and then aged at 80 °C for 24 h. The gel was washed with ethanol at 60 °C for 4 h three times and dried at 60 °C. The dried gel was subsequently carbonized at 1000 or 1600 °C for 2 h in a stream of argon gas (1 L min<sup>−1</sup>). The carbon samples derived from RF gels (RF carbons) were labeled with the carbonization temperature, namely RF-1000 and RF-1600.</p></sec></sec><sec><title>Characterization</title><p>Powder X-ray diffraction (XRD) measurements and Raman spectroscopy were employed to confirm that the samples had an amorphous carbon structure, using an X-ray diffractometer (X'Pert Pro, PANalytical, Netherlands, using Cu Kα radiation with a generator voltage of 45 kV and a tube current of 40 mA) and a Raman spectrometer (Jubin-Yvon iHR550, HORIBA, Japan, equipped with a Laser Quantum Ventus 532 nm laser using a power of 25 mW at an exposure time of 10 s), respectively. To investigate the morphology of the samples, scanning electron microscopy (SEM) measurements were performed on a scanning electron microscope (NEOSCOPE JCM-6000, JEOL, Japan, equipped with a tungsten filament at 15 kV).</p><p>Gas and vapor sorption analyses were performed to examine the surface area, pore size distribution, and pore volume of the HC samples by a gas/vapor adsorption measurement instrument (autosorb iQ, Quantachrome Instruments, USA). All samples were outgassed under vacuum at 350 °C for more than 4 h before measurement. Nitrogen (N<sub>2</sub>) and krypton (Kr) sorption measurements were carried out at 77.4 K (−196.75 °C). Unfortunately, the adsorption measurements of N<sub>2</sub> on the HT samples could not reach equilibrium and it was therefore not possible to obtain reliable isotherms. This might be due to the large particle size and the abundant micropores in the samples as confirmed by carbon dioxide (CO<sub>2</sub>) sorption measurements. CO<sub>2</sub> and water vapor (H<sub>2</sub>O) sorption were carried out at 273.15 K (0 °C) and 293.15 K (20 °C), respectively. From the results of Kr and CO<sub>2</sub> adsorption, the specific surface area (SSA) was calculated using the Brunauer–Emmett–Teller (BET) method. The pore size distribution was obtained from CO<sub>2</sub> adsorption measurements using the Monte Carlo method. The isotherms of H<sub>2</sub>O adsorption were used to estimate the pore size distribution using the equation reported by Wang et al. [<xref rid=\"R34\" ref-type=\"bibr\">34</xref>], which is based on the Mahle model [<xref rid=\"R33\" ref-type=\"bibr\">33</xref>]. Since our system could measure the adsorption amounts only up to <italic>p</italic>/<italic>p</italic><sub>0</sub> = 0.9, the measured value at this point was assumed to be the end point of the simulation curve at <italic>p</italic>/<italic>p</italic><sub>0</sub> = 1. The parameters were changed to get the simulation curves close to the actual adsorption isotherm curves. The TPVs were calculated from the last adsorption measurement point of CO<sub>2</sub> (at <italic>p</italic>/<italic>p</italic><sub>0</sub> = 0.029) and H<sub>2</sub>O adsorption (at <italic>p</italic>/<italic>p</italic><sub>0</sub> = 0.9).</p></sec><sec><title>Electrode preparation</title><p>The hard carbon electrodes were prepared by mixing HC powder (95 wt %) and polyvinylidene difluoride (PVdF, Kynar HSV 900, Arkema, France) (5 wt %) in <italic>N</italic>-methyl-2-pyrrolidinone (NMP, Sigma-Aldrich, Germany) for 10 min at 2000 rpm using a planetary centrifugal mixer (ARV-310, Thinky, Japan), such that the resulting slurry had a solid content of 0.8 mg mL<sup>−1</sup>. The viscous slurry was coated on a copper foil current collector with a thickness of 10 μm (MTI corporation, USA) on an automatic table-top coating machine (Coatema, Germany) using the doctor blade method, resulting in a wet film thickness of 90 µm. The HC loading of the electrodes was 3 ± 0.1 mg cm<sup>−2</sup>. After drying the films in an oven at 50 °C for 3 h, electrodes with a diameter of 10.95 mm were punched out. The electrodes were then dried in a glass oven (Büchi, Switzerland) under dynamic vacuum at 120 °C overnight and transferred into an argon-filled glovebox (H<sub>2</sub>O and O<sub>2</sub> content <0.1 ppm, MBraun, Germany) without exposure to ambient air.</p></sec><sec><title>Electrochemical characterization</title><p>In order to investigate the Li-ion capacity of the hard carbon active materials, three-electrode Swagelok T-cells were assembled in an argon-filled glovebox using four glass fiber separators (glass microfiber filter, 691, VWR, Germany, with a diameter of 11 mm), a lithium counter electrode (450 µm thick Li foil, Rockwood Lithium) and a Li reference electrode. The cells were then filled with 120 µL of electrolyte (1 M LiPF<sub>6</sub> in EC/EMC = 3:7 (v/v), <20 ppm H<sub>2</sub>O, BASF, Germany). All electrochemical tests were carried out in a climatic chamber (Binder, Germany) at 25 °C using a battery cycler (Series 400, Maccor, USA). After connecting the cells to the potentiostat, the cells were rested for 2 h in order to assure proper wetting of the electrodes. Three charge-discharge cycles were performed at a rate of C/10 (35.5 mA g<sup>−1</sup>). The lower cut-off potential was 10 mV and the upper cut-off potential 1.5 V vs Li<sup>+</sup>/Li whereby the potential was controlled vs a Li reference electrode. Afterwards, the cells were cycled for 20 times with a rate of 1C (355 mA g<sup>−1</sup>). The Na-ion capacity of the hard carbon samples was also investigated in a three-electrode Swagelok T-cell using sodium metal (99.95%, Sigma-Aldrich, Germany) as counter and reference electrodes and 1 M NaPF<sub>6</sub> (99%, Sigma-Aldrich, Germany) in EC/DEC = 1:1 (v/v) (<20 ppm H<sub>2</sub>O, BASF, Germany) as electrolyte. The same cut-off potentials and cycling procedure as for the Li-ion cells were used.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supporting Information</title><supplementary-material content-type=\"local-data\" id=\"SD1\"><label>File 1</label><caption><p>Additional figures.</p></caption><media mime-subtype=\"pdf\" mimetype=\"application\" xlink:href=\"Beilstein_J_Nanotechnol-11-1217-s001.pdf\" xlink:type=\"simple\" id=\"d39e1732\" position=\"anchor\"/></supplementary-material></sec></body><back><ref-list><ref id=\"R1\"><label>1</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dunn</surname><given-names>B</given-names></name><name><surname>Kamath</surname><given-names>H</given-names></name><name><surname>Tarascon</surname><given-names>J-M</given-names></name></person-group><source>Science</source><year>2011</year><volume>334</volume><fpage>928</fpage><lpage>935</lpage><pub-id 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Beilstein J Org Chem</journal-id><journal-id journal-id-type=\"iso-abbrev\">Beilstein J Org Chem</journal-id><journal-title-group><journal-title>Beilstein Journal of Organic Chemistry</journal-title></journal-title-group><issn pub-type=\"epub\">1860-5397</issn><publisher><publisher-name>Beilstein-Institut</publisher-name><publisher-loc>Trakehner Str. 7-9, 60487 Frankfurt am Main, Germany</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32831957</article-id><article-id pub-id-type=\"pmc\">PMC7431755</article-id><article-id pub-id-type=\"doi\">10.3762/bjoc.16.167</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Full Research Paper</subject></subj-group><subj-group subj-group-type=\"topic\"><subject>Chemistry</subject><subj-group subj-group-type=\"topic\"><subject>Organic Chemistry</subject></subj-group></subj-group></article-categories><title-group><article-title>Automated high-content imaging for cellular uptake, from the Schmuck cation to the latest cyclic oligochalcogenides</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Martinent</surname><given-names>Rémi</given-names></name><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>López-Andarias</surname><given-names>Javier</given-names></name><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Moreau</surname><given-names>Dimitri</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-9282-3324</contrib-id><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Cheng</surname><given-names>Yangyang</given-names></name><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sakai</surname><given-names>Naomi</given-names></name><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Matile</surname><given-names>Stefan</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-8537-8349</contrib-id><email>stefan.matile@unige.ch</email><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Niemeyer</surname><given-names>Jochen</given-names></name><role>Guest Editor</role></contrib></contrib-group><aff id=\"A1\"><label>1</label>School of Chemistry and Biochemistry, National Centre of Competence in Research (NCCR) Chemical Biology, University of Geneva, Geneva, Switzerland</aff><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>14</day><month>8</month><year>2020</year></pub-date><volume>16</volume><fpage>2007</fpage><lpage>2016</lpage><ext-link ext-link-type=\"doi\" xlink:href=\"10.3762/bjoc.16.167\">10.3762/bjoc.16.167</ext-link><history><date date-type=\"received\"><day>21</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>14</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Martinent et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Martinent et al.</copyright-holder><ali:free_to_read xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\"/><license license-type=\"Beilstein\"><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\">https://creativecommons.org/licenses/by/4.0</ali:license_ref><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\">https://www.beilstein-journals.org/bjoc/terms</ali:license_ref><license-p>This is an Open Access article under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"https://creativecommons.org/licenses/by/4.0\">https://creativecommons.org/licenses/by/4.0</ext-link>). Please note that the reuse, redistribution and reproduction in particular requires that the authors and source are credited.</license-p><license-p>The license is subject to the Beilstein Journal of Organic Chemistry terms and conditions: (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.beilstein-journals.org/bjoc/terms\">https://www.beilstein-journals.org/bjoc/terms</ext-link>)</license-p></license></permissions><abstract><p>Recent progress with chemistry tools to deliver into living cells has seen a shift of attention from counterion-mediated uptake of cell-penetrating peptides (CPPs) and their mimics, particularly the Schmuck cation, toward thiol-mediated uptake with cell-penetrating poly(disulfide)s (CPDs) and cyclic oligochalcogenides (COCs), here exemplified by asparagusic acid. A persistent challenge in this evolution is the simultaneous and quantitative detection of cytosolic delivery and cytotoxicity in a high-throughput format. Here, we show that the combination of the HaloTag-based chloroalkane penetration assay (CAPA) with automated high-content (HC) microscopy can satisfy this need. The automated imaging of thousands of cells per condition in multiwell plates allows us to obtain quantitative data on not only the fluorescence intensity but also on the localization in a very short time. Quantitative and statistically relevant results can be obtained from dose–response curves of the targeted delivery to selected cells and the cytotoxicity in the same experiment, even with poorly optimized cellular systems.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>automation</kwd><kwd>cell-penetrating peptides</kwd><kwd>cellular uptake</kwd><kwd>cytosolic delivery</kwd><kwd>cytotoxicity</kwd><kwd>high-content imaging</kwd><kwd>thiol-mediated uptake</kwd></kwd-group><funding-group><funding-statement>We thank the University of Geneva, the NCCR Chemical Biology, the NCCR Molecular Systems Engineering, and the Swiss NSF for financial support.</funding-statement></funding-group></article-meta><notes><p>This article is part of the thematic issue \"Molecular recognition\" and is dedicated to the memory of Carsten Schmuck.</p></notes></front><body><sec><title>Introduction</title><p>The effective delivery of substrates of free choice into cells with minimal endosomal capture on the one hand and a minimal toxicity on the other remains one of the grand challenges in science [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R17\" ref-type=\"bibr\">17</xref>]. This challenge is most pronounced with large substrates, such as proteins, oligonucleotides, or nanoparticles, due to the permeability barriers formed by the lipophilic core of the cell membrane [<xref rid=\"R18\" ref-type=\"bibr\">18</xref>–<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]. In recent decades, the use of arginine-rich cell-penetrating peptides (CPPs) as carriers has emerged as an attractive approach to tackle this central challenge [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R3\" ref-type=\"bibr\">3</xref><xref rid=\"R11\" ref-type=\"bibr\">11</xref>–<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]. The noncovalent interaction between the guanidinium cations from CPPs and cell membrane-associated anions, such as phospholipids, proteoglycans, or sialic acids, is considered to enhance the cell surface accumulation of substrates, and thus fulfilling the first prerequisite of all internalization (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>) [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>,<xref rid=\"R16\" ref-type=\"bibr\">16</xref>–<xref rid=\"R17\" ref-type=\"bibr\">17</xref><xref rid=\"R20\" ref-type=\"bibr\">20</xref>–<xref rid=\"R21\" ref-type=\"bibr\">21</xref>]. However, this ion-pair interaction weakens significantly in polar solvents. The presence of competing anions in physiological solutions often disturbs the binding, and thus restricting the intracellular delivery to a certain extent.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Schematic representation of binding models between organic cations (simple ammonium, guanidinium, Schmuck cation) and oxoanions.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-2007-g002\"/></fig><p>In recent years, the development of artificial guanidinium systems with improved binding affinity and stability towards oxoanions has emerged as an important topic in this field [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R22\" ref-type=\"bibr\">22</xref>–<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]. In this context, Carsten Schmuck has created the 2-(guanidiniocarbonyl)pyrrole (GCP) cation <bold>1</bold> as a synthetic analogue of the guanidinium cations, somehow a “super-guanidinium” conceived to drive “arginine magic” [<xref rid=\"R20\" ref-type=\"bibr\">20</xref>–<xref rid=\"R21\" ref-type=\"bibr\">21</xref>] to the extreme (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>) [<xref rid=\"R23\" ref-type=\"bibr\">23</xref>]. The power of the Schmuck cation to bind carboxylate and phosphate anions in competitive water has several origins [<xref rid=\"R22\" ref-type=\"bibr\">22</xref>]. Firstly, in comparison to simple guanidinium cations <bold>2</bold> (p<italic>K</italic><sub>a</sub> 12.5) and ammonium cations <bold>3</bold> (p<italic>K</italic><sub>a</sub> 10.5), the Schmuck cation has a lower p<italic>K</italic><sub>a</sub> value of 7 to 8 due to the increased acidity of acylguanidiniums, which favors the formation of stronger hydrogen-bonded ion pairs (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>, magenta part). Secondly, the binding is further enhanced by the addition of hydrogen-bonding interactions between the amide NH moiety in position 5 of the pyrrole ring or the pyrrole NH group and the oxoanion (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>, blue part). Thirdly, the rigid and planar conformation of the GCP moiety is beneficial to bind planar anions such as carboxylate (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>, red part). Finally, the selectivity and specificity for different substrates can be achieved through the additional secondary interactions between the GCP side chain and the anionic substrates (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>, green part).</p><p>The binding of the Schmuck cation with carboxylates in aqueous solvents was evaluated by a series of experimental studies, such as NMR, UV, CD, and fluorescence titrations [<xref rid=\"R23\" ref-type=\"bibr\">23</xref>,<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]. The Schmuck cation indeed showed a much higher affinity towards carboxylates, with dissociation constants of <italic>K</italic><sub>D</sub> ≈ 1 mM (<bold>4</bold>: 620 µM; <bold>5</bold>: 1.3 mM) compared to simple acylguanidinium cations (<bold>6</bold>: <italic>K</italic><sub>D</sub> = 20 mM, <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). The amide NH unit in position 5 of the pyrrole ring is crucial for this binding affinity, which even exceeds the effect of the pyrrole NH moiety (<bold>4</bold> and <bold>5</bold> vs <bold>7</bold>: <italic>K</italic><sub>D</sub> = 7.7 mM). In addition, the size and electronic structure of the pyrrole core are also important for this ion-pair interaction. For example, the replacement of the pyrrole core to pyridine in <bold>8</bold> or to furan results in a much weaker binding because of the repulsion force between the lone pair on the heteroatom and the oxoanion.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>From Schmuck cations to cell-penetrating dipeptides, with schematic representation of the binding model between GCPs and GAGs on the cell surface, and with DNA.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-2007-g003\"/></fig><p>The superior oxoanion binding of the Schmuck cation makes GCP-based peptides attractive candidates for intracellular delivery. Already a small dipeptide <bold>9</bold> with two Schmuck amino acids shows a strong binding (<italic>K</italic><sub>D</sub> = 100 nM) and clustering ability towards heparin due to the strong noncovalent interaction between GCP and sulfate anions, such as glycosaminoglycans (GAGs) in heparin [<xref rid=\"R26\" ref-type=\"bibr\">26</xref>]. This binding model <bold>10</bold> can be applied to cell-surface GAGs to enhance the cellular uptake efficiency. The peptide <bold>9</bold>, with rhodamine B attached, successfully enters into the living cells while the control peptide <bold>11</bold> with a simple guanidinium group shows a negligible uptake efficiency. In addition, the efficient delivery of a model protein (avidin, around 67 kDa) into cells through biotin–avidin technology could be achieved in the presence of this strikingly small peptide. However, the uptake of peptide <bold>9</bold>-labeled avidin was dramatically reduced into cells that express less GAGs on the cell surfaces. These results further support the importance of GAG binding to the uptake of Schmuck cations.</p><p>Arginine-rich CPPs are of general interest in gene delivery. However, a long linear CPP sequence with at least eight to nine arginine residues is necessary. In comparison to arginine-rich CPPs, Schmuck peptides form more stable complexes <bold>12</bold> with the phosphodiesters in the DNA backbone, and thus making it possible to transfect cells with shorter peptides. In 2015, the Schmuck group reported the first example of a small peptide with only four amino acids for gene transfection [<xref rid=\"R27\" ref-type=\"bibr\">27</xref>]. The binding affinity of <bold>13</bold> to DNA far exceeds the related tetrapeptide analogues with arginine or lysine residues. As a result, the gene transfection efficiency of <bold>13</bold> is better than that of polyethylenimine (PEI) with a large number of charges, which is one of the current standards in gene transfection. The uptake takes place through an endosomal pathway. The low p<italic>K</italic><sub>a</sub> value of the four GCP moieties could result in an improved buffering capacity, which could facilitate endosomal escape by the proton-sponge effect [<xref rid=\"R28\" ref-type=\"bibr\">28</xref>]. Significant inhibition of DNA transfection by bafilomycin (a macrolide antibiotic that can block the endosomal acidification process) was observed, which further supports the endosomal uptake mechanism. Except for this small peptide, GCP was also integrated into larger peptides, including branched [<xref rid=\"R29\" ref-type=\"bibr\">29</xref>], three-armed [<xref rid=\"R30\" ref-type=\"bibr\">30</xref>–<xref rid=\"R31\" ref-type=\"bibr\">31</xref>], dendritic [<xref rid=\"R32\" ref-type=\"bibr\">32</xref>], and self-assembled oligomers [<xref rid=\"R33\" ref-type=\"bibr\">33</xref>–<xref rid=\"R34\" ref-type=\"bibr\">34</xref>] for gene delivery and transfection based on the endosomal uptake and release.</p><p>However, a better binding affinity to DNA does not necessarily mean a higher DNA transfection efficiency. As an example, two GCP-modified peptide tweezers <bold>14</bold> with nanomolar dissociation constants, identified by the high-throughput screening of a combinatorial library of 259 molecular tweezers through an ethidium bromide (EB) displacement assay, show a negligible DNA transfection efficiency (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). However, the derivative <bold>15</bold>, with two lipophilic hydrocarbon chains, results in a remarkable DNA delivery and transfection [<xref rid=\"R35\" ref-type=\"bibr\">35</xref>]. These two hydrocarbon chains attached to the tweezers are used to facilitate endosomal escape. These results indicate that the balance between the number of GCP, the binding affinity, and the buffering capacity of Schmuck peptides plays a key role in the gene transfection process.</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Peptide tweezers and cyclic peptides with Schmuck cations for gene transfection.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-2007-g004\"/></fig><p>In 2016, the Schmuck group developed the first cyclic peptides that can be used as gene transfection vectors [<xref rid=\"R36\" ref-type=\"bibr\">36</xref>]. Unlike linear Schmuck peptides, the cyclic peptide <bold>16</bold> can self-assemble into nanofibers due to the binding between the GCP moiety and the backbone of an adjacent peptide. This binding interaction could stabilize the stacking of peptides by offsetting a charge repulsion of the extra lysine residues, and thus allowing the formation of stable cationic nanofibers. These nanoaggregates, assembled from the monomer <bold>16</bold>, are efficient gene transfection vectors. However, the control peptide <bold>17</bold>, which cannot self-assemble into nanotubes, shows negative transfection results. In contrast to the linear Schmuck peptides, the inactivity of a bafilomycin treatment in gene transfection processes indicates the nonendocytic cellular uptake pathway.</p><p>Among the central challenges with CPPs in general are the cytotoxicity and the endosomal capture, particularly with an increasing number of charges and substrate size [<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]. To address both problems, the peptide backbone has been replaced by poly(disulfide)s [<xref rid=\"R18\" ref-type=\"bibr\">18</xref>,<xref rid=\"R37\" ref-type=\"bibr\">37</xref>]. The resulting cell-penetrating poly(disulfide)s (CPDs) <bold>18</bold> are at least as efficient as CPPs but less toxic because they are degraded by reductive depolymerization as soon as they reach the cytosol, and their endosomal capture is minimal because they enter cells by thiol-mediated uptake (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>) [<xref rid=\"R38\" ref-type=\"bibr\">38</xref>–<xref rid=\"R48\" ref-type=\"bibr\">48</xref>]. This mechanism operates by dynamic covalent disulfide exchange on the cell surface and on the way into the cell (<xref ref-type=\"fig\" rid=\"F5\">Figure 5b</xref>) [<xref rid=\"R49\" ref-type=\"bibr\">49</xref>–<xref rid=\"R50\" ref-type=\"bibr\">50</xref>]. The cyclic oligochalcogenides (COCs) <bold>19</bold>–<bold>22</bold> were introduced to maximize the speed and selectivity of this dynamic covalent exchange chemistry for an efficient cytosolic delivery (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>) [<xref rid=\"R51\" ref-type=\"bibr\">51</xref>]. Moving from the original emphasis on the disulfide ring tension into an increasing unorthodox COC chemistry covering ETPs [<xref rid=\"R52\" ref-type=\"bibr\">52</xref>], diselenolanes [<xref rid=\"R53\" ref-type=\"bibr\">53</xref>], and benzopolysulfanes [<xref rid=\"R54\" ref-type=\"bibr\">54</xref>], the activity gradually increased.</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Evolution from CPPs to CPDs and COCs.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-2007-g005\"/></fig><p>The Achilles heel of much research on new transporters for cellular uptake, from CPPs and Schmuck cations to CPDs and COCs, is the quantitative detection of the delivery in the intact functional form to the cytosol, firmly excluding possible false positives from endosomal capture on the one hand and cytotoxicity on the other [<xref rid=\"R5\" ref-type=\"bibr\">5</xref>,<xref rid=\"R55\" ref-type=\"bibr\">55</xref>]. Recently, the CAPA has been introduced to quantify cytosolic delivery [<xref rid=\"R55\" ref-type=\"bibr\">55</xref>]. The combination with image-based high-content (HC) screening [<xref rid=\"R56\" ref-type=\"bibr\">56</xref>–<xref rid=\"R58\" ref-type=\"bibr\">58</xref>] has been suggested to further improve the standards set by the CAPA [<xref rid=\"R59\" ref-type=\"bibr\">59</xref>]. Standard high-throughput (HT) screening has been used regularly to facilitate studies on cellular uptake [<xref rid=\"R60\" ref-type=\"bibr\">60</xref>]. In standard assays, a single macroscopic parameter, usually the fluorescence intensity, is automatically recorded for microtiter plates with hundreds to thousands of wells. This standard HT screening provides access to quantitatively reliable curves for the dependence on the concentration, incubation time, activators, inhibitors, and so on within a reasonable time. High-content screening (HCS) combines this automated HT format with image-based information.</p><p>To summarize briefly what has been outlined previously in more detail [<xref rid=\"R56\" ref-type=\"bibr\">56</xref>], HCS requires automated high-speed microscopy, including robotic liquid handlers and plate washers, automated data analysis tools, and large data storage systems for the terabits of data produced per day. The high number of images generated by the automated microscope cannot be analysed manually, and therefore need to go through a completely automated analysis pipeline. For this purpose, dedicated software is used to first create specific masks that detect the objects of interest in the image (e.g., cells, cellular organelles, etc.) This can be achieved by using several steps of image curation and modification (e.g., deconvolution) and using several analysis modules adapted to the shape, size, and intensity of the object of interest. Once all masks are properly detected and associated to the master object (the cell), a set of meaningful data can be extracted from each object in the different fluorescent channels. The data set generated for each cell can later be used for a deep phenotypic analysis, using an advanced unsupervised statistical analysis method (e.g., principal component analysis, diverse clustering methods, etc.), or for directly plotting specific parameters of interest. This fully automated workflow allows then to extract deep complex information from thousands of cells extremely rapidly and in a very consistent manner, allowing a very efficient comparison of the cell treatment conditions and sequential experiments. For cellular uptake, this promises the recording of statistically relevant dose–response curves for a targeted delivery, together with other characteristics of interest. This can include the incubation time or a cytotoxicity that is precisely defined on the changes in the appearance of interest. In the following, these expectations are evaluated explicitly, with a particular emphasis on quantifying the eventual contributions from off-target fluorescence as well as extracting quantitative information on the cytosolic delivery and cell viability in one and the same automated HC screen.</p></sec><sec><title>Results and Discussion</title><p>The new, concise, at least trifunctional COC transporter <bold>23</bold> was designed and synthesized to explore the usefulness of a HC screening to study the cellular uptake (<xref ref-type=\"fig\" rid=\"F5\">Figure 5a</xref>). Asparagusic acid was chosen as the arguably best explored COC for the thiol-mediated uptake, beginning with a dynamic covalent disulfide exchange with exofacial thiols, followed by either endocytosis or the direct crossing of membranes into cytosols through successive thiolate–disulfide exchange reactions or micellar pores (<xref ref-type=\"fig\" rid=\"F5\">Figure 5b</xref>) [<xref rid=\"R61\" ref-type=\"bibr\">61</xref>]. Anionic glutamate was added to minimize the passive diffusion across the membrane and to maximize the solubility in water. Biotin was used to interface the transporter and streptavidin to probe the COC-mediated protein delivery. A chloroalkane, finally, was needed for the HC CAPA.</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>Structure of a) the trifunctional transporter <bold>23</bold> and c) the HaloTag reporter <bold>26</bold>. b) Schematic mechanism of the thiol-mediated uptake of COCs with dynamic covalent disulfide exchange with exofacial thiols (left), preceding walking along the disulfide tracks, and micellar pores (right).</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-2007-g006\"/></fig><p>The trifunctional transporter <bold>23</bold> was first complexed with streptavidin <bold>24</bold> (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>). The resulting complex <bold>25</bold> was then incubated with HGM cells. These are stable cell lines, expressing the self-labeling HaloTag protein and GFP in the outer mitochondrial membrane (<xref ref-type=\"fig\" rid=\"F6\">Figure 6a</xref>) [<xref rid=\"R55\" ref-type=\"bibr\">55</xref>]. If the complex <bold>25</bold> indeed reaches the cytosol, the chloroalkane will react with carboxylate in the active site to produce an ester, and thus covalently attach the transporter to the fusion protein (<xref ref-type=\"fig\" rid=\"F6\">Figure 6b</xref>). The subsequently added reporter <bold>26</bold> passively diffuses into the cells and labels all free HaloTags. The fluorescence signal from the HaloTag reporter <bold>26</bold> is then employed to generate dose–response curves and to calculate the CP<sub>50</sub> value of the transporters, described as the half-maximal cell penetration.</p><fig id=\"F6\" position=\"float\"><label>Figure 6</label><caption><p>CAPA assay for the complex <bold>25</bold>, composed of three transporters <bold>23</bold> bound to one streptavidin <bold>24</bold> (with the structure of the homotetramer loaded with four biotins). Cytosolic delivery into HMG cells (a) has to precede the reaction of <bold>25</bold> with HaloTags on mitochondria (b). Afterwards, the unreacted HaloTags are labeled with the reporter <bold>26</bold> for quantification (c).</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-2007-g007\"/></fig><p>In a CAPA, the final fluorescence response is usually recorded by flow cytometry [<xref rid=\"R55\" ref-type=\"bibr\">55</xref>,<xref rid=\"R62\" ref-type=\"bibr\">62</xref>]. In a HC CAPA, flow cytometry is replaced by HC automated microscopy, using multiwell plates and registering data on the fluorescence intensity and fluorescence localization in thousands of cells per condition, at HT [<xref rid=\"R56\" ref-type=\"bibr\">56</xref>–<xref rid=\"R59\" ref-type=\"bibr\">59</xref>]. To properly assess its potential, automated HC imaging was optimized first. The fluorescence of the Hoechst dye and the GFP of the fusion protein were used to segment whole cells and mitochondria, respectively (blue and yellow areas, <xref ref-type=\"fig\" rid=\"F7\">Figure 7a</xref>). The structural characteristics of the cells were extracted from both masks.</p><fig id=\"F7\" position=\"float\"><label>Figure 7</label><caption><p>Examples from the automated HC imaging of stable HGM cells with HaloTag–GFP on mitochondria, labeled with <bold>26</bold> and Hoechst dye. a) GFP channel (left) and cell selection (right), showing the cell body mask (light blue areas) and the mitochondrial mask (yellow areas). b) Overlay image of the GFP (green), <bold>26</bold> (red), and the Hoechst dye (blue) channels, showing off-target fluorescence from <bold>26</bold> (white arrows). Scale bar: 10 µm.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-2007-g008\"/></fig><p>Possible damaged cells with early signs of apoptosis or abnormal mitochondrial networks were excluded (<xref ref-type=\"fig\" rid=\"F7\">Figure 7a</xref>, right). With these criteria, on average, 15% of the cells were discarded. This possibility to simultaneously quantify the cytotoxicity and the cytosolic delivery in the same HT experiment is one of the key advantages of the HC CAPA (vide infra).</p><p>After the addition of the reporter <bold>26</bold>, a large-scale analysis was carried out, correlating its fluorescence intensity with that of the GFP, proportional to the concentration of the HaloTag, cell by cell. A linear regression of the correlations using the whole-cell body as a mask gave a very good r<sup>2</sup> of 0.977 (<xref ref-type=\"fig\" rid=\"F8\">Figure 8a</xref>). The same linear regression of correlations, masking exclusively the mitochondria region, gave a small but significant increase to r<sup>2</sup> = 0.983 (<xref ref-type=\"fig\" rid=\"F8\">Figure 8b</xref>). Although optimized toward perfection with stable HGM cells, this increase demonstrated that the HC CAPA adds a precision that is overlooked with flow cytometry. The off-target staining of <bold>26</bold> in regions outside mitochondria accounted for this source of error in the CAPA, which is corrected in the HC CAPA (<xref ref-type=\"fig\" rid=\"F7\">Figure 7b</xref>, arrows).</p><fig id=\"F8\" position=\"float\"><label>Figure 8</label><caption><p>Evaluation of the automated HC imaging of stable HGM cells with HaloTag–GFP on mitochondria, labeled with <bold>26</bold>, showing the fluorescence intensity of GFP versus <bold>26</bold> in a) selected cells inside the cell body mask (light blue and yellow areas in <xref ref-type=\"fig\" rid=\"F7\">Figure 7a</xref>) and in b) selected regions with a high GFP signal, considered as the mitochondrial network (yellow areas only in <xref ref-type=\"fig\" rid=\"F7\">Figure 7a</xref>).</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-2007-g009\"/></fig><p>The optimized HC CAPA system was then explored with the complex <bold>25</bold>, recording the cytosolic delivery and the cytotoxicity quantitatively in one HT experiment. A nearly constant number of selected cells based on the above criteria, with an increasing concentration, revealed that complex <bold>25</bold> is not toxic up to 20 µM (<xref ref-type=\"fig\" rid=\"F9\">Figure 9a</xref>, black). The effective concentration of <bold>25</bold> obtained with the mitochondria mask was CP<sub>50</sub> = 7.3 ± 0.5 μM (<xref ref-type=\"fig\" rid=\"F9\">Figure 9a</xref> and <xref ref-type=\"fig\" rid=\"F9\">Figure 9b</xref>, yellow). With the whole-cell mask, a CP<sub>50</sub> value of 8.0 ± 0.6 μM was obtained (<xref ref-type=\"fig\" rid=\"F9\">Figure 9a</xref> and <xref ref-type=\"fig\" rid=\"F9\">Figure 9b</xref>, blue). According to <italic>p</italic> < 0.05, the underestimate made with the whole-cell mask was significant (<xref ref-type=\"fig\" rid=\"F9\">Figure 9b</xref>). Namely, the cytosolic delivery of <bold>25</bold> measured with the mitochondria mask was i) 10% better and ii) also 10% more accurate. These small but significant differences demonstrated that the HC CAPA improves even on a cytometric CAPA, although the optimization of this assay with stable HGM cells is near perfection.</p><fig id=\"F9\" position=\"float\"><label>Figure 9</label><caption><p>a) Automated HC imaging of the cellular uptake of <bold>25</bold>, covering the concentration dependence for the HC CAPA with the cell body mask (blue), the HC CAPA with the mitochondrial mask (yellow), and the toxicity defined as the percentage of damage-free cells selected for the HC CAPA (black), all recorded in one automated experiment. (b) The CP<sub>50</sub> value of <bold>25</bold> with standard errors using the cell body mask (blue) and the mitochondrial mask (yellow). The statistical significance was determined using the one-tailed paired Student’s t-test: *<italic>p</italic> < 0.05.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-2007-g010\"/></fig><p>The CP<sub>50</sub> value of 7.3 ± 0.5 µM obtained for <bold>25</bold> outperformed the CP<sub>50</sub> value of 14.9 ± 0.5 μM of the previously reported cell-penetrating streptavidin, in which four asparagusic acids are covalently bound to the protein through irreversible triazole linkages [<xref rid=\"R59\" ref-type=\"bibr\">59</xref>]. The CP<sub>50</sub> value of 7.3 ± 0.5 µM of <bold>25</bold> was not far above the CP<sub>50</sub> value of 3.1 ± 0.5 µM of HIV Tat, the original CPP [<xref rid=\"R55\" ref-type=\"bibr\">55</xref>]. As the uptake efficiency generally decreases with the size [<xref rid=\"R18\" ref-type=\"bibr\">18</xref>], this similarity is particularly impressive, considering that the COC carrying a 52 kDa protein is compared to a small undecapeptide.</p><p>The compatibility of the assay with other fusion proteins and more adverse conditions, such as transient transfection, has never been assessed for the standard cytometric CAPA, but a low precision and reproducibility were anticipated [<xref rid=\"R62\" ref-type=\"bibr\">62</xref>]. The high accuracy and selectivity promised that our image-based HC CAPA could expand the scope of the CAPA beyond HGM cells. To elaborate on this attractive perspective, HeLa cells were transiently transfected with a plasmid expressing a fusion protein of HaloTag–GFP–Golgi localization sequence [<xref rid=\"R63\" ref-type=\"bibr\">63</xref>]. The HaloTags installed in the Golgi were then labeled with the reporter <bold>26</bold>.</p><p>Under these more challenging conditions, obvious problems arose from the toxicity of the transfecting agent, over- and underexpression of the fusion protein, and undesired localization out of the selected organelle. To explore how automated HC imaging can handle these problems, a whole-cell body mask was applied first (<xref ref-type=\"fig\" rid=\"F10\">Figure 10a</xref>, cyan, including yellow). With this mask, the fluorescence of GFP and <bold>26</bold> correlated with r<sup>2</sup> = 0.626 (<xref ref-type=\"fig\" rid=\"F11\">Figure 11a</xref>). This very poor correlation for the transient transfection of the HaloTags on the Golgi contrasted sharply with an r<sup>2</sup> value of 0.983 obtained with the stable HGM cells with the HaloTags expressed on the mitochondria. The difference between the two plots in <xref ref-type=\"fig\" rid=\"F11\">Figure 11a</xref> and <xref ref-type=\"fig\" rid=\"F8\">Figure 8a</xref> was striking even for the naked eye. A comparison of the respective images confirmed a massive off-target emission of <bold>26</bold> in the former, with r<sup>2</sup> = 0.626 (<xref ref-type=\"fig\" rid=\"F10\">Figure 10b</xref>, red, arrows) and little off-target emission in the latter, with r<sup>2</sup> = 0.983 (<xref ref-type=\"fig\" rid=\"F7\">Figure 7b</xref>, red, arrows).</p><fig id=\"F10\" position=\"float\"><label>Figure 10</label><caption><p>Examples of automated HC imaging of transiently transfected HeLa cells with HaloTag–GFP on Golgi, labeled with <bold>26</bold>. a) GFP channel showing the applied cell-body mask (light blue), Golgi mask (yellow), and the mask of the cell regions with low or no GFP signal (dark blue) in all cells. b) Same image as in a) with an overlay (yellow) of the GFP (green) and <bold>26</bold> (red) channels, showing off-target fluorescence from <bold>26</bold> in some cells (white arrows). c) GFP channel after the selection of the cells with an adequate level of expression of the fusion protein. The applied masks for the cell body (light blue), the Golgi (yellow), and the cell regions with low or no GFP signal (dark blue) are shown only in the selected cells. Over-transfected cells (without dark blue regions) are not masked and excluded from the analysis. A very intense GFP signal (green) can be seen in the excluded cells. d) The same image as in c) with an overlay (yellow) of the GFP (green) and <bold>26</bold> (red) channels, showing off-target punctate fluorescence from <bold>26</bold> in some specific locations inside the selected cells (white arrows). Scale bar: 10 µm.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-2007-g011\"/></fig><fig id=\"F11\" position=\"float\"><label>Figure 11</label><caption><p>Evaluation of the automated HC imaging of the transiently transfected HeLa cells with HaloTag–GFP on Golgi, labeled with <bold>26</bold>, showing the fluorescence intensity of GFP versus <bold>26</bold> in a) all cells inside the cell body mask (yellow and blue areas in <xref ref-type=\"fig\" rid=\"F10\">Figure 10a</xref>), b) all cells inside the punctate regions with a high GFP signal, considered as the Golgi apparatus (yellow areas in <xref ref-type=\"fig\" rid=\"F10\">Figure 10a</xref>), c) selected cells inside the cell body mask (light and dark blue areas in <xref ref-type=\"fig\" rid=\"F10\">Figure 10c</xref>), and d) punctate regions with a high GFP signal in selected cells, considered as the Golgi apparatus (yellow areas in <xref ref-type=\"fig\" rid=\"F10\">Figure 10c</xref>).</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-2007-g012\"/></fig><p>Masking of the regions with the intense GFP signal from the Golgi apparatus without an initial cell selection was tested next (<xref ref-type=\"fig\" rid=\"F10\">Figure 10a</xref>, yellow only). With this Golgi mask without cell selection, the correlation of the fluorescence of the GFP and <bold>26</bold>, with an r<sup>2</sup> value of 0.549, was even worse than the r<sup>2</sup> value of 0.626 for the whole-cell mask (<xref ref-type=\"fig\" rid=\"F11\">Figure 11b</xref>).</p><p>The initial cell selection aimed to narrow the polydispersity of expression and to exclude the overtransfected and damaged cells. For this purpose, cells with GFP fluorescence in all cell bodies were removed, and only those cells with nonfluorescent regions were kept (dark blue regions in <xref ref-type=\"fig\" rid=\"F10\">Figure 10c</xref>). This selection removed about 80% of all cells, and only the top 20% were kept for further analysis. However, applying a cell body mask to this remaining 20%, the fitting of r<sup>2</sup> = 0.621 of the GFP and the <bold>26</bold> emission did not improve either (<xref ref-type=\"fig\" rid=\"F10\">Figure 10c</xref>, cyan, including yellow and <xref ref-type=\"fig\" rid=\"F11\">Figure 11c</xref>). The inspection of the images confirmed that even the selected cells showed a significant off-target emission of <bold>26</bold> (<xref ref-type=\"fig\" rid=\"F10\">Figure 10d</xref>, red, arrows).</p><p>The masking of the Golgi apparatus, based on the GFP emission in the selected cells, was applied next to remove this off-target staining from <bold>26</bold> (<xref ref-type=\"fig\" rid=\"F11\">Figure 11c</xref>, yellow only). The application of both the cell selection and the Golgi mask to the analysis finally improved the correlation of the GFP and the <bold>26</bold> emission to r<sup>2</sup> = 0.816 (<xref ref-type=\"fig\" rid=\"F11\">Figure 11d</xref>). This value was slowly approaching the precision realized without further effort using the stable cell line HGM (<xref ref-type=\"fig\" rid=\"F8\">Figure 8b</xref>). While the error from the whole-cell analyses with HGM cells is not catastrophic (r<sup>2</sup> = 0.977 vs r<sup>2</sup> = 0.983), these results show that automated high-content imaging is absolutely necessary with less optimized systems (r<sup>2</sup> = 0.626 vs r<sup>2</sup> = 0.816).</p><p>The difference between the optimized HGM cells and the unoptimized transfected cells is again best appreciated with the naked eye: Whereas the improvement of the automated selection from <xref ref-type=\"fig\" rid=\"F8\">Figure 8a</xref> to <xref ref-type=\"fig\" rid=\"F8\">Figure 8b</xref> is not visible on the first view, the improvement from <xref ref-type=\"fig\" rid=\"F11\">Figure 11a</xref> to <xref ref-type=\"fig\" rid=\"F11\">Figure 11d</xref> is massive, and <xref ref-type=\"fig\" rid=\"F11\">Figure 11d</xref> starts to resemble more the quasiideal <xref ref-type=\"fig\" rid=\"F8\">Figure 8b</xref>. The automated removal of 80% of all cells to obtain <xref ref-type=\"fig\" rid=\"F11\">Figure 11d</xref> (compared to 15% to produce <xref ref-type=\"fig\" rid=\"F8\">Figure 8b</xref>) is not problematic because HC microscopy registers, screens, and evaluates the data after the optimization of the ideal conditions for the analysis [<xref rid=\"R56\" ref-type=\"bibr\">56</xref>–<xref rid=\"R59\" ref-type=\"bibr\">59</xref>].</p></sec><sec><title>Conclusion</title><p>The specific objective of this study was to assess the power and uniqueness of HC imaging for cellular uptake. For this, the HC CAPA, which combines automated microscopy and precise localized quantification, has been first improved using a cell selection process, aiming at removing damaged or abnormal cells (around 15%). The correlation of the GFP fluorescence (proportional to the HaloTag expression) and the fluorescence of the CAPA assay reporter <bold>26</bold> is identified as a practical method to assess the accuracy of the mask analysis. After a linear regression, a slight increase in the goodness of fit from r<sup>2</sup> = 0.977 to 0.983 quantified the increase in accuracy achieved moving from a cell-body mask to a mitochondrial mask. This difference revealed that even with fully optimized stable HGM cell lines, whole-cell analyses, such as flow cytometry, of the CAPA contain a small but nonnegligible error that can be removed with the HC CAPA.</p><p>To quantify the impact of the HC CAPA on the detection of the cytosolic delivery, the newly introduced peptide-based COC <bold>23</bold> was evaluated for the transport of a model protein with the new analytical improvements. With a CP<sub>50</sub> value of 7.3 ± 0.5 μM with the mitochondrial mask, complex <bold>25</bold> showed an excellent transport efficiency. A slightly larger CP<sub>50</sub> value of 8.0 ± 0.6 μM obtained with the whole-cell mask confirmed that the error from the whole-cell analyses, such as flow cytometry, in the CAPA is small but not negligible (i.e., 10% for the activity, 10% for the accuracy). The CP<sub>50</sub> value obtained for the COC–protein complex was in the range of the protein-free HIV Tat, and thus confirming the excellent activity of the COCs. Most importantly, with our refined HC CAPA, quantitative dose–response curves for the cytotoxicity could be obtained with the same HT screening experiment, quantifying the cytosolic delivery, and the cytotoxicity can be defined according to the cellular defects of interest. The COC–protein complex <bold>25</bold> was nontoxic up to at least 20 µM.</p><p>Finally, the HC CAPA is not limited to optimized stable cell lines to generate precise and reproducible data. The compatibility with transient transfection is exemplified with HeLa cells with a GFP–HaloTag construct in the Golgi apparatus, after the application of the cell selection process and a Golgi mask to the analysis. As a result, over- and underexpressing cells, around 80% of the total cells, could be efficiently removed from the analysis, and the localized fluorescence in the selected cells could be quantified with the Golgi mask. The goodness-of-fit after a linear regression (r<sup>2</sup> = 0.816) of the correlation between the GFP fluorescence and the reporter <bold>26</bold> in transiently transfected cells was increasingly close to the one obtained with the HGM stable cell line (r<sup>2</sup> = 0.983).</p><p>These results imply that automated HC microscopy in general and the HC CAPA in particular can be used as a universal tool to quantify the cellular uptake, including transiently transfected cells and other less robust systems. Quantitative, statistically relevant data on the targeted delivery and a precisely defined cytotoxicity are obtained in the same HT experiment. We hope that automated HC microscopy will be useful for the community to routinely assess synthetic transport systems, from the Schmuck cation to the latest CPPs, CPDs, and COCs.</p></sec><sec sec-type=\"supplementary-material\"><title>Supporting Information</title><supplementary-material content-type=\"local-data\" id=\"SD1\"><label>File 1</label><caption><p>Experimental details.</p></caption><media mime-subtype=\"pdf\" mimetype=\"application\" xlink:href=\"Beilstein_J_Org_Chem-16-2007-s001.pdf\" xlink:type=\"simple\" id=\"d39e1079\" position=\"anchor\"/></supplementary-material></sec></body><back><ack><p>We thank S. Vossio for help with the high-content microscopy and analysis, J. A. Kritzer (Tufts University) and M. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Beilstein J Org Chem</journal-id><journal-id journal-id-type=\"iso-abbrev\">Beilstein J Org Chem</journal-id><journal-title-group><journal-title>Beilstein Journal of Organic Chemistry</journal-title></journal-title-group><issn pub-type=\"epub\">1860-5397</issn><publisher><publisher-name>Beilstein-Institut</publisher-name><publisher-loc>Trakehner Str. 7-9, 60487 Frankfurt am Main, Germany</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32831953</article-id><article-id pub-id-type=\"pmc\">PMC7431756</article-id><article-id pub-id-type=\"doi\">10.3762/bjoc.16.163</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Full Research Paper</subject></subj-group><subj-group subj-group-type=\"topic\"><subject>Chemistry</subject><subj-group subj-group-type=\"topic\"><subject>Organic Chemistry</subject></subj-group></subj-group></article-categories><title-group><article-title>Controlling the stereochemistry in 2-oxo-aldehyde-derived Ugi adducts through the cinchona alkaloid-promoted electrophilic fluorination</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Yuqing</given-names></name><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Gaigai</given-names></name><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Peshkov</surname><given-names>Anatoly A</given-names></name><xref ref-type=\"aff\" rid=\"A2\">2</xref><xref ref-type=\"aff\" rid=\"A3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yao</surname><given-names>Ruwei</given-names></name><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hasan</surname><given-names>Muhammad</given-names></name><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zaman</surname><given-names>Manzoor</given-names></name><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Liu</surname><given-names>Chao</given-names></name><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kashtanov</surname><given-names>Stepan</given-names></name><xref ref-type=\"aff\" rid=\"A4\">4</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Pereshivko</surname><given-names>Olga P</given-names></name><email>olga.pereshivko@nu.edu.kz</email><xref ref-type=\"aff\" rid=\"A1\">1</xref><xref ref-type=\"aff\" rid=\"A2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Peshkov</surname><given-names>Vsevolod A</given-names></name><email>vsevolod.peshkov@nu.edu.kz</email><xref ref-type=\"aff\" rid=\"A1\">1</xref><xref ref-type=\"aff\" rid=\"A2\">2</xref><xref ref-type=\"aff\" rid=\"A5\">5</xref></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>O'Hagan</surname><given-names>David</given-names></name><role>Guest Editor</role></contrib></contrib-group><aff id=\"A1\"><label>1</label>College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Dushu Lake Campus, Suzhou, 215123, P.R. China</aff><aff id=\"A2\"><label>2</label>Department of Chemistry, School of Science and Technology, Nazarbayev University, 53 Kabanbay Batyr Ave, Block 7, Nur-Sultan 010000, Republic of Kazakhstan</aff><aff id=\"A3\"><label>3</label>Saint Petersburg State University, Saint Petersburg 199034, Russian Federation</aff><aff id=\"A4\"><label>4</label>Department of Chemistry, Xi’an Jiaotong-Liverpool University, Suzhou, 215123, P.R. China</aff><aff id=\"A5\"><label>5</label>The Environment and Resource Efficiency Cluster (EREC), Nazarbayev University, Nur-Sultan, Republic of Kazakhstan</aff><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><volume>16</volume><fpage>1963</fpage><lpage>1973</lpage><ext-link ext-link-type=\"doi\" xlink:href=\"10.3762/bjoc.16.163\">10.3762/bjoc.16.163</ext-link><history><date date-type=\"received\"><day>6</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>30</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Wang et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Wang et al.</copyright-holder><ali:free_to_read xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\"/><license license-type=\"Beilstein\"><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\">https://creativecommons.org/licenses/by/4.0</ali:license_ref><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\">https://www.beilstein-journals.org/bjoc/terms</ali:license_ref><license-p>This is an Open Access article under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"https://creativecommons.org/licenses/by/4.0\">https://creativecommons.org/licenses/by/4.0</ext-link>). Please note that the reuse, redistribution and reproduction in particular requires that the authors and source are credited.</license-p><license-p>The license is subject to the Beilstein Journal of Organic Chemistry terms and conditions: (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.beilstein-journals.org/bjoc/terms\">https://www.beilstein-journals.org/bjoc/terms</ext-link>)</license-p></license></permissions><abstract><p>In this report, we introduce a new strategy for controlling the stereochemistry in Ugi adducts. Instead of controlling stereochemistry directly during the Ugi reaction we have attempted to stereodefine the chiral center at the peptidyl position through the post-Ugi functionalization. In order to achieve this, we chose to study 2-oxo-aldehyde-derived Ugi adducts many of which partially or fully exist in the enol form that lacks the aforementioned chiral center. This in turn led to their increased nucleophilicity as compared to the standard Ugi adducts. As such, the stereocenter at the peptidyl position could be installed and stereodefined through the reaction with a suitable electrophile. Towards this end, we were able to deploy an asymmetric cinchona alkaloid-promoted electrophilic fluorination producing enantioenriched post-Ugi adducts fluorinated at the peptidyl position.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>cinchona alkaloids</kwd><kwd>electrophilic fluorination</kwd><kwd>enantioselective synthesis</kwd><kwd>2-oxo-aldehydes</kwd><kwd>Ugi reaction</kwd></kwd-group><funding-group><funding-statement>This work was supported by the start-up fund from Soochow University (grant Q410900714), National Natural Science Foundation of China (grant 21650110445), Natural Science Foundation of Jiangsu Province of China (grant BK20160310), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the project of scientific and technologic infrastructure of Suzhou (grant SZS201708). M.H. and M.Z. are grateful to the Chinese Scholarship Council (CSC) for providing doctoral scholarships.</funding-statement></funding-group></article-meta><notes><p>This article is part of the thematic issue \"Organo-fluorine chemistry V\".</p></notes></front><body><sec><title>Introduction</title><p>Multicomponent reactions (MCRs) [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R13\" ref-type=\"bibr\">13</xref>] in general and isocyanide-based MCRs [<xref rid=\"R14\" ref-type=\"bibr\">14</xref>–<xref rid=\"R18\" ref-type=\"bibr\">18</xref>] in particular represent powerful tools of modern synthetic chemistry that allow to generate structurally diverse and complex products in a step and atom-economic manner from simple and accessible precursors. Three-component Passerini [<xref rid=\"R19\" ref-type=\"bibr\">19</xref>–<xref rid=\"R22\" ref-type=\"bibr\">22</xref>] and four-component Ugi [<xref rid=\"R23\" ref-type=\"bibr\">23</xref>–<xref rid=\"R24\" ref-type=\"bibr\">24</xref>] reactions that rely on the ability of organic isocyanides to participate in the nucleophilic attack onto the carbonyl or imine group are among the most studied MCRs. Accordingly, a wide range of post-MCR transformations have been elaborated allowing to upgrade the Passerini and Ugi adducts into potentially bioactive heterocycles [<xref rid=\"R25\" ref-type=\"bibr\">25</xref>–<xref rid=\"R32\" ref-type=\"bibr\">32</xref>]. While both Passerini and Ugi reactions proved to be quite robust towards different classes of substrates and possess broad functional group tolerance, the difficulties associated with controlling their stereochemical outcome [<xref rid=\"R33\" ref-type=\"bibr\">33</xref>–<xref rid=\"R35\" ref-type=\"bibr\">35</xref>] severely restrict their applicability in medicinal chemistry [<xref rid=\"R36\" ref-type=\"bibr\">36</xref>].</p><p>The typical Passerini three-component reaction (P-3CR) of a carboxylic acid <bold>1</bold>, an aldehyde <bold>2</bold>, and an isocyanide <bold>3</bold> being conducted in a polar-aprotic solvent such as THF or dichloromethane results in the assembly of an α-acyloxyamide adduct <bold>4</bold>. Just a few protocols for the asymmetric P-3CR [<xref rid=\"R37\" ref-type=\"bibr\">37</xref>–<xref rid=\"R40\" ref-type=\"bibr\">40</xref>] and its modifications [<xref rid=\"R41\" ref-type=\"bibr\">41</xref>–<xref rid=\"R47\" ref-type=\"bibr\">47</xref>] have been reported over the course of the last two decades (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). In the majority of those protocols [<xref rid=\"R37\" ref-type=\"bibr\">37</xref>–<xref rid=\"R39\" ref-type=\"bibr\">39</xref>], the stereocontrol was achieved with the aid of different Lewis acid promoters bearing chiral ligands, while the most recent and at the same time the most general procedure developed by Liu, Tan and co-workers relied on the stereoinduction by a chiral phosphoric acid (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>) [<xref rid=\"R40\" ref-type=\"bibr\">40</xref>].</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Asymmetric protocols for Passerini three-component reaction (P-3CR).</p></caption><table frame=\"hsides\" rules=\"groups\"><tr><td align=\"center\" colspan=\"5\" valign=\"middle\" rowspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i001.jpg\"/></td></tr><tr><td align=\"center\" colspan=\"5\" valign=\"middle\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Asymmetric protocol</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Catalytic system</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Conditions</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Yield range, %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ee range, %</td></tr><tr><td align=\"left\" colspan=\"5\" valign=\"top\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Dömling and co-workers, 2003 [<xref rid=\"R37\" ref-type=\"bibr\">37</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i002.jpg\"/><break/>(1 equiv)<break/>Ti(OiPr)<sub>4</sub> (1.35 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">THF (0.5 M), overnight</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">12–48</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">32–42</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Schreiber and co-workers, 2004 [<xref rid=\"R38\" ref-type=\"bibr\">38</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i003.jpg\"/><break/>(20 mol %)<break/>Cu(OTf)<sub>2</sub> (20 mol %)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DCM (0.04 M), molecular sieves, 18–48 h</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">75–98</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">62–98</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Wang, Zhu and co-workers, 2008 [<xref rid=\"R39\" ref-type=\"bibr\">39</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i004.jpg\"/><break/>(10 mol %)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">toluene (0.33 M), argon, −40 °C, 48 h</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">51–70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">63 to >99</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Liu, Tan and co-workers, 2015 [<xref rid=\"R40\" ref-type=\"bibr\">40</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i005.jpg\"/><break/>(10–20 mol %)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CHCl<sub>3</sub> (0.033 M or 0.05 M), argon, MgSO<sub>4</sub>, rt or −20 °C or −35 °C, 36 h</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">41–99</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">84–99</td></tr></table></table-wrap><p>In the standard Ugi four-component reaction (U-4CR) a carboxylic acid <bold>1</bold>, an aldehyde <bold>2</bold>, and an isocyanide <bold>3</bold> are complemented by a primary amine <bold>5</bold> that altogether undergo a condensation into a peptide-like adduct <bold>6</bold>. These reactions are typically conducted in polar protic solvents such as methanol or water. Several examples of diastereoselective U-4CR have been described [<xref rid=\"R48\" ref-type=\"bibr\">48</xref>–<xref rid=\"R54\" ref-type=\"bibr\">54</xref>]. Yet, the Ugi reaction turned out to be a rather challenging chemical transformation in terms of establishing the asymmetric version [<xref rid=\"R33\" ref-type=\"bibr\">33</xref>–<xref rid=\"R35\" ref-type=\"bibr\">35</xref>]. Apart from the two asymmetric three-component modifications lacking the acid component [<xref rid=\"R55\" ref-type=\"bibr\">55</xref>–<xref rid=\"R56\" ref-type=\"bibr\">56</xref>], no general asymmetric protocol for the four-component mode has been developed until very recently. In 2018, Houk, Tan and co-workers described an efficient enantioselective procedure operating in the presence of catalytic amounts of chiral phosphoric acids (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>) [<xref rid=\"R57\" ref-type=\"bibr\">57</xref>]. Two synthetic protocols have been elaborated allowing the engagement of different classes of substrates (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). In addition, the group of Tan adapted their findings to the chiral phosphoric acid-catalyzed three-component Ugi reaction of an aldehyde <bold>2</bold>, an isocyanide <bold>3</bold>, and a primary amine <bold>5</bold> [<xref rid=\"R58\" ref-type=\"bibr\">58</xref>].</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Asymmetric protocols for Ugi four-component reaction (U-4CR) by Houk, Tan and co-workers.</p></caption><table frame=\"hsides\" rules=\"groups\"><tr><td align=\"center\" colspan=\"5\" valign=\"middle\" rowspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i006.jpg\"/></td></tr><tr><td align=\"center\" colspan=\"5\" valign=\"middle\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Substrate scope</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Catalytic system</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Conditions</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Yield range, %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ee range, %</td></tr><tr><td align=\"center\" colspan=\"5\" valign=\"top\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">R<sup>2</sup> = Alk; R<sup>3</sup> = Ar</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i007.jpg\"/><break/>(5 mol %)<break/>Cy = cyclohexyl</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DCM (0.05 M), argon, 5 Å MS, −20 °C, 12 h</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">43–96</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">75–97</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">R<sup>2</sup> = Ar; R<sup>3</sup> = Alk</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i008.jpg\"/><break/>(10 mol %)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">cyclohexane (0.025 M), argon, 5 Å MS, 20 °C, 36 h–7 d</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">60–96</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">80–94</td></tr></table></table-wrap><p>As a part of our ongoing research, we have been involved in the synthesis and utilization of 2-oxo-aldehyde-derived Ugi adducts <bold>8</bold> (<xref ref-type=\"fig\" rid=\"C1\">Scheme 1</xref>). These products possess an increased nucleophilicity of the peptidyl position compared to the standard Ugi adducts owing to an additional electron-withdrawing group introduced with the 2-oxo-aldehyde <bold>7</bold>. For example, we have explored the potential of the peptidyl reactive site in a number of enolization-driven post-Ugi cyclizations leading to the assembly of pyrrol-2-ones <bold>9</bold> and <bold>10</bold> [<xref rid=\"R59\" ref-type=\"bibr\">59</xref>–<xref rid=\"R60\" ref-type=\"bibr\">60</xref>]. We have also taken advantage of a 1,3-dicarbonyl moiety present in <bold>8</bold> by engaging them in an enolization-triggered complexation with boron trifluoride diethyl etherate. The resulting <italic>O</italic>,<italic>O</italic>-chelated boron complexes <bold>11</bold> turned out to be strong solid-state emitters featuring clear aggregation-induced emission (AIE) characteristics [<xref rid=\"R61\" ref-type=\"bibr\">61</xref>].</p><fig id=\"C1\" position=\"float\"><label>Scheme 1</label><caption><p>Post-transformations of 2-oxo-aldehyde-derived Ugi adducts <bold>8</bold>.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-g003\"/></fig><p>Encouraged by these results, we decided to attempt the reaction of 2-oxo-aldehyde-derived Ugi adducts <bold>8</bold> with suitable electrophiles in the presence of chiral catalyst or promotor. In this way, the peptidyl position of <bold>8</bold> could be simultaneously functionalized and stereodefined leading to the formation of enantioenriched products <bold>12</bold> (<xref ref-type=\"fig\" rid=\"C1\">Scheme 1</xref>). Towards this goal, we have previously tested the reactivity of <bold>8</bold> in the intermolecular aldol addition to ethyl glyoxalate [<xref rid=\"R62\" ref-type=\"bibr\">62</xref>]. As these attempts met with failure, we have turned our attention to enantioselective fluorination reactions [<xref rid=\"R63\" ref-type=\"bibr\">63</xref>–<xref rid=\"R65\" ref-type=\"bibr\">65</xref>]. Specifically, we decided to explore the enantioselective electrophilic fluorination of carbonyl compounds promoted by cinchona alkaloid derivatives developed independently by Shibata, Takeuchi and co-workers [<xref rid=\"R65\" ref-type=\"bibr\">65</xref>–<xref rid=\"R66\" ref-type=\"bibr\">66</xref>] and by the group of Cahard [<xref rid=\"R67\" ref-type=\"bibr\">67</xref>–<xref rid=\"R70\" ref-type=\"bibr\">70</xref>]. The method proved to be applicable to a broad range of substrates under a variety of conditions [<xref rid=\"R71\" ref-type=\"bibr\">71</xref>–<xref rid=\"R78\" ref-type=\"bibr\">78</xref>] and was successfully utilized for the synthesis of several bioactive molecules [<xref rid=\"R79\" ref-type=\"bibr\">79</xref>–<xref rid=\"R81\" ref-type=\"bibr\">81</xref>]. Herein we present its adaptation for the enantioselective fluorination of 2-oxo-aldehyde-derived Ugi adducts <bold>8</bold> leading to the installment of a fluorine-bound quaternary stereocenter at the peptidyl position. It should be stressed that the exploration of methods for the enantioselective fluorination continues to be an important topic considering the ever-growing role of fluorine derivatives in drug design and development [<xref rid=\"R82\" ref-type=\"bibr\">82</xref>–<xref rid=\"R83\" ref-type=\"bibr\">83</xref>]. An alternative strategy to prepare related quaternary carbon-containing adducts bearing fluorine and nitrogen atoms involves the asymmetric addition of α-fluoro-β-ketoesters or α-fluoro-α-nitro esters to appropriate electrophiles [<xref rid=\"R84\" ref-type=\"bibr\">84</xref>–<xref rid=\"R89\" ref-type=\"bibr\">89</xref>].</p></sec><sec><title>Results and Discussion</title><p>Initially, we prepared a series of Ugi adducts <bold>8</bold> by varying acid, 2-oxo-aldehyde, amine, and isocyanide components (<xref ref-type=\"fig\" rid=\"C2\">Scheme 2</xref>). The product <bold>8a</bold> obtained from benzoic acid, phenylglyoxal, benzylamine, and <italic>tert</italic>-butyl isocyanide was selected for the screening of the reaction parameters (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). The overall process was conducted in a two-step one-pot fashion as was originally designed by Shibata. In the first step, a cinchona alkaloid derivative was allowed to react with an electrophilic fluorinating agent to afford an <italic>N</italic>-fluoroammonium salt of the cinchona alkaloid via transfer fluorination. In the second step, the Ugi adduct <bold>8a</bold> was added to this in situ generated <italic>N</italic>-fluorocinchona intermediate to afford the desired fluorinated product <bold>12a</bold>. Thus, according to this synthetic and mechanistic path, the <italic>N</italic>-fluorocinchona intermediate acted as an asymmetric electrophilic fluorinating agent.</p><fig id=\"C2\" position=\"float\"><label>Scheme 2</label><caption><p>Synthesis of 2-oxo-aldehyde-derived Ugi adducts.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-g004\"/></fig><table-wrap id=\"T3\" position=\"float\"><label>Table 3</label><caption><p>Screening of the conditions for enantioselective fluorination of Ugi adduct <bold>8a</bold>.<sup>a</sup></p></caption><table frame=\"hsides\" rules=\"groups\"><tr><td align=\"center\" colspan=\"7\" valign=\"middle\" rowspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i009.jpg\"/></td></tr><tr><td align=\"center\" colspan=\"7\" valign=\"middle\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Entry</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cinchona alkaloid derivative</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Fluorinating agent</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Solvent</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Time, h</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Yield,<sup>b</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ee (major enantiomer),<sup>c</sup> %</td></tr><tr><td align=\"left\" colspan=\"7\" valign=\"middle\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Q</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MeCN</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">88</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">19 (F)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Q</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">THF</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">19</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">94</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21 (F)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Q</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DCM</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">74</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">26 (F)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Q</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DCM</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">42</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">20 (F)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Q</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CHCl<sub>3</sub></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">71–79</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">31–44 (F)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CPN</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CHCl<sub>3</sub></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>e</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>e</sup></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DHQ</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CHCl<sub>3</sub></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">16-21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">85–90</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">46–57 (F)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DHQ</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">THF</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">76</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">43 (F)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Q-Bn</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CHCl<sub>3</sub></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">78–83</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">39–41 (F)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Q-Ac</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CHCl<sub>3</sub></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21–24</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">35–59</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">42–51 (F)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Q-Bz</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CHCl<sub>3</sub></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">62</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">32 (F)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DHQ-Bn</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor or NFSI</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CHCl<sub>3</sub></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">16–22</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">60–81</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">57–73 (F)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">QD</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor or NFSI</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CHCl<sub>3</sub></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21–23</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">83–94</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">51–69 (S)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DHQD</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CHCl<sub>3</sub></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">86–92</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">15–21 (S)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">QD-Me</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CHCl<sub>3</sub></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">75</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">14 (S)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">QD-Ac</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CHCl<sub>3</sub></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">27 (S)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">QD-Bn</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CHCl<sub>3</sub></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">80</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">29 (S)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DHQD-Bn</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Selectfluor</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">CHCl<sub>3</sub></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">87</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23 (S)</td></tr></table><table-wrap-foot><fn id=\"TFN1\"><p><sup>a</sup>The reactions were run on a 0.15–0.2 mmol scale; <sup>b</sup>isolated yield; <sup>с</sup>determined by chiral HPLC analysis, S: slower enantiomer, F: faster enantiomer; <sup>d</sup>the reaction was conducted with 0.2 equiv of Q; <sup>e</sup>no product <bold>12a</bold> was formed.</p></fn></table-wrap-foot></table-wrap><p>Conducting the fluorination of <bold>8a</bold> with a quinine (Q)/Selectfluor combination in different solvents revealed chloroform as a preferred reaction medium for this process with acetonitrile, tetrahydrofuran, and dichloromethane being less efficient in terms of ee of the obtained product <bold>12a</bold> (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>, entries 1–5). Using catalytic quantities of quinine led to a prolonged reaction time and diminished yield and ee of <bold>12a</bold> compared to the analogous reaction with stoichiometric amounts of quinine (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>, entry 4 versus entry 3). Thus, we focused on the exploration of a stoichiometric version considering that most of cinchona alkaloid derivatives are readily accessible and recoverable. The original Shibata’s protocol also relied on the use of stoichiometric amounts of the chiral promotor that was in an agreement with a two-step reaction mode requiring the initial formation of the asymmetric fluorinating agent. An attempt to use cupreine, a member of the cinchona family featuring free phenolic hydroxy group, met with failure producing no desired product <bold>12a</bold> (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>, entry 6). Using dihydroquinine (DHQ) led to an improved ee of <bold>12a</bold> compared to the analogous reactions with quinine (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>, entries 7 and 8 versus entries 5 and 2, respectively). Testing ether and ester derivatives of Q and DHQ (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>, entries 9–12) led to further improvement allowing to obtain <bold>12a</bold> with up to 73% ee through the reaction with dihydroquinine benzyl ether (DHQ-Bn, <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>, entry 12). Switching to quinidine (QD), dihydroquinidine (DHQD), and their derivatives allowed to alter the stereoselectivity of the process leading to the preferred formation of another enantiomer of <bold>12a</bold> (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>, entries 13–18) with the QD-promoted reaction affording the best ee value (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>, entry 13). Additionally, we tested <italic>N</italic>-fluorobenzenesulfonimide (NFSI) as an alternative fluorinating agent in a combination with DHQ-Bn and QD with the outcome being indistinguishable with the analogous reactions with Selectfluor.</p><p>Having these results in hand, we moved to investigating the scope and limitation of this procedure with the Ugi adducts <bold>8</bold> obtained previously (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). In order to have a more balanced representation we decided to test most of these substrates <bold>8</bold> with eight different cinchona alkaloid derivatives including Q, DHQ, QD, DHQD, and their benzyl ether derivatives. To assure reproducibility, some of these reactions were repeated two to five times. In all such cases a range for the yield and ee value is reported in <xref rid=\"T4\" ref-type=\"table\">Table 4</xref>. Testing Ugi adducts <bold>8b</bold> and <bold>8c</bold> that similarly to <bold>8a</bold> were obtained from benzylamine and <italic>tert</italic>-butyl isocyanide by varying either acid or 2-oxo-aldehyde components revealed that it was difficult to achieve high enantioselectivity for such substrates. Specifically, none of the studied chiral promoters could afford the desired products <bold>12b</bold> and <bold>12c</bold> with the ee value consistently higher than 50%. Turning to the substrates <bold>8d–g</bold> derived from aromatic amines we were able to achieve improved enantioselectivity. The best results were obtained with Q-Bn, DHQ-Bn, DHQD, and QD-Bn that performed consistently well with each of the substrates <bold>8d</bold>–<bold>g</bold> delivering the corresponding fluorinated products <bold>12d</bold>–<bold>g</bold> with the ee values higher than 50% and in some cases close or even over 70%. Furthermore, it was possible to divert the selectivity of these reactions towards the preferred formation of either of the enantiomers by choosing an appropriate chiral promotor. Testing Ugi adduct <bold>8h</bold> allowed us to highlight the possibility of simultaneous variation of acid, 2-oxo-aldehyde, and amine components. Overall, four cinchona derivatives were screened with <bold>8h</bold> producing the fluorinated product <bold>12h</bold> with moderate to good enantioselectivity, which was consistent with the performance of other arylamine-derived Ugi adducts <bold>8d</bold>–<bold>g</bold>. The last investigated Ugi adduct <bold>8i</bold> featured the variation of the isocyanide component. Conducting fluorinations with this <italic>n</italic>-butyl isocyanide-derived Ugi adduct <bold>8i</bold>, the desired product <bold>12i</bold> could be successfully obtained. However, the degree of enantioselective induction was lower than in the reactions with the analogous <italic>tert</italic>-butyl isocyanide-derived substrate <bold>8d</bold>. This could probably be ascribed to a reduced rigidity and bulkiness of the <italic>n</italic>-butyl group as compared to <italic>tert</italic>-butyl.</p><table-wrap id=\"T4\" position=\"float\"><label>Table 4</label><caption><p>Scope and limitations of the asymmetric electrophilic fluorination of 2-oxo-aldehyde-derived Ugi adducts <bold>8</bold>.<sup>a</sup></p></caption><table frame=\"hsides\" rules=\"groups\"><tr><td align=\"center\" colspan=\"10\" valign=\"middle\" rowspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i010.jpg\"/></td></tr><tr><td align=\"center\" colspan=\"10\" valign=\"middle\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Q</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">DHQ</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Q-Bn</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">DHQ‑Bn</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">QD</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">DHQD</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">QD-Bn</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">DHQD-Bn</td></tr><tr><td align=\"left\" colspan=\"10\" valign=\"middle\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"center\" rowspan=\"2\" valign=\"middle\" colspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i011.jpg\"/><break/><bold>12a</bold></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">yield,<sup>b</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">71–79</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">85–90</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">78–83</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">60–81</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">83–94</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">86–92</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">80</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">87</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ee,<sup>c</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">31–44<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">46–57<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">39–41<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">57–73<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">51–69<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">15–21<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">29<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23<break/>(S)</td></tr><tr><td align=\"left\" colspan=\"10\" valign=\"top\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"center\" rowspan=\"2\" valign=\"middle\" colspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i012.jpg\"/><break/><bold>12b</bold></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">yield,<sup>b</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">78–85</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">84–97</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">79</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">76–90</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">83–89</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">95–97</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">67–80</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">81</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ee,<sup>c</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23–34<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">29–39<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">39<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">38–51<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">24–28<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">9–15<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">20–24<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21<break/>(F)</td></tr><tr><td align=\"left\" colspan=\"10\" valign=\"top\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"center\" rowspan=\"2\" valign=\"middle\" colspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i013.jpg\"/><break/><bold>12c</bold></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">yield,<sup>b</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">59–81</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">60–81</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">83</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">74–89</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">68–85</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">72–78</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">81</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">78</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ee,<sup>c</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">39–46<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">39–47<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">35<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">36<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">30–43<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23–25<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">25<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23<break/>(F)</td></tr><tr><td align=\"left\" colspan=\"10\" valign=\"top\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"center\" rowspan=\"2\" valign=\"middle\" colspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i014.jpg\"/><break/><bold>12d</bold></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">yield,<sup>b</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">91</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">81</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">78</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">96</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">71–91</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">84</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">85</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">82</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ee,<sup>c</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">24<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">22<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">66<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">71<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">47–60<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">57<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">59<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">38<break/>(F)</td></tr><tr><td align=\"left\" colspan=\"10\" valign=\"top\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"center\" rowspan=\"2\" valign=\"middle\" colspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i015.jpg\"/><break/><bold>12e</bold></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">yield,<sup>b</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">68–78</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">91–97</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">78–82</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">86–97</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">89–98</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">84–91</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">95–98</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">85</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ee,<sup>c</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">18–30<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">19–23<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">65–69<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">71–74<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">49–50<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">58–65<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">61–67<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">42<break/>(F)</td></tr><tr><td align=\"left\" colspan=\"10\" valign=\"top\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"center\" rowspan=\"2\" valign=\"middle\" colspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i016.jpg\"/><break/><bold>12f</bold></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">yield,<sup>b</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">88–89</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">91–92</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">84–88</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">85–86</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">71–78</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">89–98</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">89</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">79</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ee,<sup>c</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23–27<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">18–19<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">66–67<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">69-72<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">50–55<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">53–64<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">57–58<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40<break/>(F)</td></tr><tr><td align=\"left\" colspan=\"10\" valign=\"top\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"center\" rowspan=\"2\" valign=\"middle\" colspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i017.jpg\"/><break/><bold>12g</bold></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">yield,<sup>b</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">74–85</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">90</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">82–84</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">86–87</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">82–88</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">75–76</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">75–84</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">78–81</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ee,<sup>c</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">25–37<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">18–24<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">65–66<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">63–70<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">48–59<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">51–61<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">59–60<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">37–54<break/>(F)</td></tr><tr><td align=\"left\" colspan=\"10\" valign=\"top\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"center\" rowspan=\"2\" valign=\"middle\" colspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i018.jpg\"/><break/><bold>12h</bold></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">yield,<sup>b</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">64</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">86</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">84</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">75</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ee,<sup>c</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">71<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">73<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">54<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">55<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td></tr><tr><td align=\"left\" colspan=\"10\" valign=\"top\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"center\" rowspan=\"2\" valign=\"middle\" colspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-i019.jpg\"/><break/><bold>12i</bold></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">yield,<sup>b</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">51</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">54</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">67</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">49</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ee,<sup>c</sup> %</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">44<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">48<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">55<break/>(F)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">29<break/>(S)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–<sup>d</sup></td></tr></table><table-wrap-foot><fn id=\"TFN2\"><p><sup>a</sup>The reactions were run on a 0.15–0.2 mmol scale; <sup>b</sup>isolated yield; <sup>с</sup>determined by chiral HPLC analysis, S: slower enantiomer, F: faster enantiomer; <sup>d</sup>not conducted.</p></fn></table-wrap-foot></table-wrap><p>For the substrates <bold>8a</bold>–<bold>g</bold> tested with eight cinchona alkaloid derivatives fluorinations with QD and DHQD led to the reversed stereoselectivity as compared to the reactions with Q and DHQ. For the benzylamine-derived substrates <bold>8a</bold>–<bold>c</bold> benzyl ether derivatives of cinchona alkaloids favored the formation of the same enantiomer of <bold>12</bold> as their free alcohol counterparts (e.g., both Q-Bn and Q favored the formation of the same enantiomer of <bold>12</bold>). In contrast, for the substrates <bold>8d</bold>–<bold>g</bold> derived from aromatic amines a reversed trend was observed. In case of <bold>8d</bold>–<bold>g</bold>, benzyl ether derivatives of cinchona alkaloids favored the formation of the opposite enantiomer of <bold>12</bold> as compared to the one favored by the parent alkaloids featuring free alcohol moiety (e.g., Q-Bn favored the formation of the enantiomer of <bold>12</bold> with the absolute configuration opposite to the one favored by Q). Substrates <bold>8h</bold> and <bold>8i</bold> were tested with only four cinchona alkaloid derivatives, but the observed stereochemical trends appeared to be consistent with those established for the rest of arylamine-derived Ugi adducts <bold>8d</bold>–<bold>g</bold>.</p><p>Finally, we attempted to determine the absolute configuration of a stereocenter for the major enantiomer of the representative fluorinated product. Using a sample of product <bold>12e</bold> with the ee value of 74% we were able to obtain crystals suitable for the structure determination via X-ray crystallographic analysis. It was found that a part of the substance crystallized in a racemic form while the rest crystallized in an enantiopure form. By resolving both types of crystals [<xref rid=\"R90\" ref-type=\"bibr\">90</xref>], the configuration of the major slow enantiomer was successfully established as <italic>S</italic> (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Molecular representation of the X-ray crystal structure of (<italic>S</italic>)-<bold>12e</bold> (slow enantiomer).</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1963-g002\"/></fig></sec><sec><title>Conclusion</title><p>In summary, we have explored a new strategy for controlling the stereochemistry in the Ugi adducts through the enantioselective post-Ugi functionalization. Specifically, we have adapted the cinchona alkaloid-promoted asymmetric electrophilic fluorination for derivatizing the 2-oxo-aldehyde-derived Ugi adducts. This allowed us to obtain the post-Ugi products fluorinated at the peptidyl position with the enantiomeric excess values in several instances reaching more than 70%.</p></sec><sec sec-type=\"supplementary-material\"><title>Supporting Information</title><supplementary-material content-type=\"local-data\" id=\"SD1\"><label>File 1</label><caption><p>Experimental procedures, characterization data and copies of spectra.</p></caption><media mime-subtype=\"pdf\" mimetype=\"application\" xlink:href=\"Beilstein_J_Org_Chem-16-1963-s001.pdf\" xlink:type=\"simple\" id=\"d39e2003\" position=\"anchor\"/></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SD2\"><label>File 2</label><caption><p>Crystallographic data (CIF) for compound (<italic>S</italic>)-<bold>12e</bold>.</p></caption><media mime-subtype=\"x-cif\" mimetype=\"chemical\" xlink:href=\"Beilstein_J_Org_Chem-16-1963-s002.cif\" xlink:type=\"simple\" id=\"d39e2016\" position=\"anchor\"/></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SD3\"><label>File 3</label><caption><p>Crystallographic data (CIF) for compound (rac)-<bold>12e</bold>.</p></caption><media mime-subtype=\"x-cif\" mimetype=\"chemical\" xlink:href=\"Beilstein_J_Org_Chem-16-1963-s003.cif\" xlink:type=\"simple\" id=\"d39e2026\" position=\"anchor\"/></supplementary-material></sec></body><back><ack><p>We thank Prof. Xingwang Wang for his advice to explore the electrophilic fluorination as a tool for the enantioselective post-Ugi functionalization.</p></ack><ref-list><ref id=\"R1\"><label>1</label><element-citation publication-type=\"journal\"><person-group 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Beilstein J Org Chem</journal-id><journal-id journal-id-type=\"iso-abbrev\">Beilstein J Org Chem</journal-id><journal-title-group><journal-title>Beilstein Journal of Organic Chemistry</journal-title></journal-title-group><issn pub-type=\"epub\">1860-5397</issn><publisher><publisher-name>Beilstein-Institut</publisher-name><publisher-loc>Trakehner Str. 7-9, 60487 Frankfurt am Main, Germany</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32831956</article-id><article-id pub-id-type=\"pmc\">PMC7431757</article-id><article-id pub-id-type=\"doi\">10.3762/bjoc.16.166</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Review</subject></subj-group><subj-group subj-group-type=\"topic\"><subject>Chemistry</subject><subj-group subj-group-type=\"topic\"><subject>Organic Chemistry</subject></subj-group></subj-group></article-categories><title-group><article-title>Syntheses of spliceostatins and thailanstatins: a review</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Donaldson</surname><given-names>William A</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-0840-3731</contrib-id><email>william.donaldson@marquette.edu</email><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Bräse</surname><given-names>Stefan</given-names></name><role>Associate Editor</role></contrib></contrib-group><aff id=\"A1\"><label>1</label>Department of Chemistry, Marquette University, P. O. Box 1881, Milwaukee, WI 53201-1881, USA</aff><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>13</day><month>8</month><year>2020</year></pub-date><volume>16</volume><fpage>1991</fpage><lpage>2006</lpage><ext-link ext-link-type=\"doi\" xlink:href=\"10.3762/bjoc.16.166\">10.3762/bjoc.16.166</ext-link><history><date date-type=\"received\"><day>25</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>1</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Donaldson</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Donaldson</copyright-holder><ali:free_to_read xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\"/><license license-type=\"Beilstein\"><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\">https://creativecommons.org/licenses/by/4.0</ali:license_ref><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\">https://www.beilstein-journals.org/bjoc/terms</ali:license_ref><license-p>This is an Open Access article under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"https://creativecommons.org/licenses/by/4.0\">https://creativecommons.org/licenses/by/4.0</ext-link>). Please note that the reuse, redistribution and reproduction in particular requires that the authors and source are credited.</license-p><license-p>The license is subject to the Beilstein Journal of Organic Chemistry terms and conditions: (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.beilstein-journals.org/bjoc/terms\">https://www.beilstein-journals.org/bjoc/terms</ext-link>)</license-p></license></permissions><abstract><p>The spliceostatins/thailanstatins are a family of linear peptides/polyketides that inhibit pre-mRNA splicing, and as such act as potent cytotoxic compounds. These compounds generally contain 9 stereocenters spread over a common (2<italic>Z</italic>,4<italic>S</italic>)-4-acetoxy-2-butenamide fragment, an (all-<italic>cis</italic>)-2,3,5,6-tetrasubstituted tetrahydropyran fragment and a terminal oxane ring joined by a dienyl chain. Due to the impressive antitumor properties of these compounds, along with their complex structure, a number of total syntheses have been reported. This review will compare the synthetic strategies reported through the end of 2019.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>antiproliferative</kwd><kwd>polyketide natural products</kwd><kwd>tetrahydropyrans</kwd><kwd>total synthesis</kwd></kwd-group></article-meta></front><body><sec><title>Introduction</title><p>The spliceostatins/thailanstatins (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>) are a family of linear peptide/polyketide natural products isolated from the bacteria <italic>Burkholderia</italic> sp. FERM BP-3421 [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R3\" ref-type=\"bibr\">3</xref>] (originally identified as <italic>Pseudomonas</italic> sp. No 2663) and <italic>Burkholderia</italic> sp. MSMB 43 [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>–<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]. These compounds are of interest due to their ability to bind to a subunit of the human spliceosome, splicing factor 3b [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>], which inhibits pre-mRNA splicing, and as such act as potent cytotoxic compounds. A review of the discovery, target identification, and biological applications of the compounds that exhibit these binding characteristics has been published [<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]. These compounds all contain a common (2<italic>Z</italic>,4<italic>S</italic>)-4-acetoxy-2-butenamide fragment (in green, <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>), appended to an (all-<italic>cis</italic>)-2,3,5,6-tetrasubstituted tetrahydropyran fragment (in red, <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). The members of this family primarily differ with respect to the terminal oxane ring (in blue, <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>) which is attached to the common fragments by a dienyl chain. The exciting antitumor properties of these compounds (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>), along with their complex structure, have led to a significant synthetic activity. The present review will cover the total syntheses of the spliceostatins/thailanstatins through the end of 2019.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Structures of spliceostatins/thailanstatins.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g002\"/></fig><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Biological activities of spliceostatins/thailanstatins (IC<sub>50</sub> values in nM).</p></caption><table frame=\"hsides\" rules=\"groups\"><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" colspan=\"5\" valign=\"top\" rowspan=\"1\">tumor cell line</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" colspan=\"5\" valign=\"top\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">compound</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MCF-7</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">A549</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">HCT116</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">SW480</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">P388</td></tr><tr><td align=\"left\" colspan=\"6\" valign=\"top\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">FR901464 (<bold>1</bold>)<sup>a</sup></td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.91</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.66</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.31</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.51</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.69</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">FR901465 (<bold>2</bold>)<sup>a</sup></td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.59</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.44</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.34</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.53</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.48</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">FR901463 (<bold>3</bold>)<sup>a</sup></td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.46</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.35</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.22</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.40</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.82</td></tr><tr><td align=\"left\" colspan=\"6\" valign=\"middle\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">H1975</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">N87</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BT474</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MDA-MB-DYT2</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MDA-MB-468</td></tr><tr><td align=\"left\" colspan=\"6\" valign=\"middle\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">thailanstatin B (<bold>5</bold>)<sup>b</sup></td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n.t.<sup>c</sup></td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">>100</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">>100</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n.t.</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">>100</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">spliceostatin F (<bold>6</bold>) <sup>b</sup></td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n.t.</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.641</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.85</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n.t.</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.35</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">thailanstatin A (<bold>7</bold>) <sup>b</sup></td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">320</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">59</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">145</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">161</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">142</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">spliceostatin B (<bold>8</bold>) <sup>b</sup></td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n.t.</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n.t.</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n.t.</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n.t.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">spliceostatin D (<bold>9</bold>) <sup>b</sup></td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">950</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n.t.</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n.t.</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n.t.</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n.t.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">spliceostatin E (<bold>10</bold>)<sup>b</sup></td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n.t.</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3.67</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3.72</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4.16</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.56</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">spliceostatin G (<bold>11</bold>)<sup>b</sup></td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n.t.</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">>100</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">>100</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">>100</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">n.t.</td></tr></table><table-wrap-foot><fn id=\"TFN1\"><p><sup>a</sup>See reference [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]. <sup>b</sup>See reference [<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]. <sup>c</sup>n.t. = not tested</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Review</title><sec><title>Synthesis of the (2<italic>Z</italic>,4<italic>S</italic>)-4-acetoxy- or protected (2<italic>Z</italic>,4<italic>S</italic>)-4-hydroxy-2-butenoic acid fragment</title><p>The (2<italic>Z</italic>,4<italic>S</italic>)-4-acetoxy-2-butenoic acid fragment is common to all of the spliceostatins/thailanstatins. The various routes to this building block (<xref ref-type=\"fig\" rid=\"C1\">Scheme 1</xref>) serve as a tutorial on the methodology for the asymmetric synthesis of a (<italic>Z</italic>)-2-hydroxy-3-pentene unit, with most proceeding via the <italic>cis</italic>-reduction of 4-acetoxy-2-pentynoic acid (<bold>13</bold>). The synthesis by Kitahara et al. [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>–<xref rid=\"R9\" ref-type=\"bibr\">9</xref>] is the exception, where the chiral center is derived from relatively inexpensive (<italic>S</italic>)-ethyl lactate (<bold>14</bold>). This was transformed into the silyl ether-protected 2-hydroxypropanals <bold>15a</bold> and <bold>15b</bold> via literature procedures [<xref rid=\"R10\" ref-type=\"bibr\">10</xref>], followed by the application of the Still–Gennari <italic>Z</italic>-selective Horner–Wadsworth–Emmons olefination [<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]. Koide’s group [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>–<xref rid=\"R13\" ref-type=\"bibr\">13</xref>] reported that the asymmetric addition of the enyne <bold>16</bold> to acetaldehyde in the presence of zinc triflate and the chiral additive <italic>N</italic>-methylephedrine [<xref rid=\"R14\" ref-type=\"bibr\">14</xref>] gave the (<italic>S</italic>)-propargyl alcohol <bold>17</bold> in 72% ee; the chiral purity could be improved to 96% ee by recrystallization. Unfortunately, attempts to catalyze this reaction with Zn(OTf)<sub>2</sub>/ephedrine were unsuccessful. More recently, Ghosh and co-workers [<xref rid=\"R15\" ref-type=\"bibr\">15</xref>] used the (<italic>R,R</italic>)-ProPhenol ligand [<xref rid=\"R16\" ref-type=\"bibr\">16</xref>] to accomplish a catalytic asymmetric addition of methyl propynoate to acetaldehyde to give <bold>18</bold> in high enantiopurity (98% ee). Jacobsen’s group [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>–<xref rid=\"R18\" ref-type=\"bibr\">18</xref>] utilized the Noyori Ru-catalyzed transfer hydrogenation [<xref rid=\"R19\" ref-type=\"bibr\">19</xref>] of the 3-butyne-2-one <bold>19</bold>, which gave <bold>20</bold> with 97% ee. The removal of the silyl protecting group, followed by a carboxylation and acylation gave <bold>13</bold>. Koide’s group [<xref rid=\"R13\" ref-type=\"bibr\">13</xref>] reported a second-generation route to <bold>13</bold>, which utilized the Corey–Bakshi–Shibata chiral oxazaborolidine catalyst <bold>21</bold> [<xref rid=\"R20\" ref-type=\"bibr\">20</xref>] for the asymmetric reduction of the THP-protected 5-hydroxy-3-pentyn-2-one <bold>22</bold> to generate the secondary alcohol <bold>23</bold>. The acylation of <bold>23</bold>, followed by the treatment with Jones’ reagent effected the THP deprotection as well as an overoxidation to give <bold>13</bold>. The <italic>syn</italic>-selective reduction of <bold>13</bold> was accomplished with a balloon worth of pressure of H<sub>2</sub> in the presence of the Lindlar catalyst. Of the asymmetric strategies, Jacobsen’s and Ghosh’s routes proceeded with the highest enantioselectivity; the catalyst loading was lower for the Jacobsen route (0.5 mol %) compared to the Ghosh route (20 mol %).</p><fig id=\"C1\" position=\"float\"><label>Scheme 1</label><caption><p>Synthetic routes to protected (2<italic>Z</italic>,4<italic>S</italic>)-4-hydroxy-2-butenoic acid fragments.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g003\"/></fig></sec><sec><title>Synthesis of the (all-<italic>cis</italic>)-2,3,4,5-tetrasubstituted tetrahydropyran fragment</title><sec><title>Syntheses from ʟ-threonine-derived aldehyde</title><p>Three groups utilized 4-formyl-2,2,5-trimethyl-3-oxazolidine (<bold>24</bold>) [<xref rid=\"R21\" ref-type=\"bibr\">21</xref>], derived from relatively inexpensive ʟ-threonine (<$1/g in bulk) as a chiral pool precursor for the amine stereocenter of the (all-<italic>cis</italic>)-2,3,5,6-tetrasubstituted tetrahydropyran fragment. In Kitahara’s synthesis [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>–<xref rid=\"R9\" ref-type=\"bibr\">9</xref>], the Wittig olefination of <bold>24</bold>, followed by a catalytic hydrogenation, removal of the dimethylaminal protecting group, and lactonization gave <bold>25</bold> as a mixture of diastereomers (<xref ref-type=\"fig\" rid=\"C2\">Scheme 2</xref>). The further transformation of <bold>25</bold> afforded the dihydropyran <bold>26</bold>, which upon catalytic hydrogenation over Pt<sub>2</sub>O and then low-temperature DIBAL reduction afforded the all-<italic>cis</italic> tetrasubstituted tetrahydropyran <bold>27</bold>.</p><fig id=\"C2\" position=\"float\"><label>Scheme 2</label><caption><p>Kitahara synthesis of the (all-<italic>cis</italic>)-2,3,5,6-tetrasubstituted tetrahydropyran.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g004\"/></fig><p>In Koide et al.’s synthesis, the unsaturated lactone <bold>29</bold> was prepared via the Wittig methenylation of <bold>24</bold>, followed by aminal hydrolysis, methallylation of the 2° alcohol <bold>28</bold>, and ring-closing metathesis using Grubbs’ 2nd generation catalyst (<bold>G-II</bold>, <xref ref-type=\"fig\" rid=\"C3\">Scheme 3</xref>) [<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]. Replacing the <italic>N</italic>-Boc protecting group with an <italic>N</italic>-tosyl group and allylic oxidation gave <bold>30</bold>. The introduction of the allyl group at C-11 made use of the Kishi protocol [<xref rid=\"R22\" ref-type=\"bibr\">22</xref>] of the allyl-Grignard addition, followed by an ionic reduction. The <italic>N</italic>-tosyl group was removed and the resultant amine protected to give the <italic>N</italic>-Boc tetrahydropyran <bold>31</bold>. The Koide group had originally attempted the reduction–allylation–ionic reduction sequence on the Boc-protected amine <bold>32</bold>, however, this gave lower overall yields due to the competing formation of a pyrrolidine byproduct.</p><fig id=\"C3\" position=\"float\"><label>Scheme 3</label><caption><p>Koide synthesis of (all-<italic>cis</italic>)-2,3,5,6-tetrasubstituted tetrahydropyran.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g005\"/></fig><p>Using the Stork–Zhao conditions [<xref rid=\"R23\" ref-type=\"bibr\">23</xref>], the Nicolaou group performed an α-methyliodomethylenation of <bold>24</bold> to give <bold>33</bold> with a good stereoselectivity (<italic>Z:E</italic> ≈ 95:5, <xref ref-type=\"fig\" rid=\"C4\">Scheme 4</xref>) [<xref rid=\"R24\" ref-type=\"bibr\">24</xref>–<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]. After the hydrolysis of the dimethylaminal and the reaction of the resultant amine with phthalic anhydride, a Pd-catalyzed Stille coupling [<xref rid=\"R26\" ref-type=\"bibr\">26</xref>] of <bold>34</bold> with 3-(tributylstannyl)-2-propen-1-ol gave <bold>35</bold>. The oxidation of <bold>35</bold> with an excess of MnO<sub>2</sub> gave the (2<italic>E</italic>,4<italic>Z</italic>)-dienal <bold>36</bold>. Alternatively, the Stille coupling of <bold>34</bold> with 3-(tributylstannyl)acrolein also afforded <bold>36</bold>. After much experimentation with up to eight amine catalysts, this group found that the intramolecular oxa-Michael addition using 20 mol % of the diarylprolinol organocatalyst <bold>37</bold> in the presence of benzoic acid gave <bold>38</bold>. The use of the enantiomer of <bold>37</bold> gave the dihydropyran with the opposite configuration at C-11. The catalytic hydrogenation of <bold>38</bold> proceeded with a poor stereoselectivity, however, a similar reduction of the diethyl acetal of <bold>38</bold>, followed by an acetal hydrolysis gave the aldehyde <bold>39</bold> with a good stereocontrol (>10:1).</p><fig id=\"C4\" position=\"float\"><label>Scheme 4</label><caption><p>Nicolaou synthesis of the (all-<italic>cis</italic>)-2,3,5,6-tetrasubstituted tetrahydropyran.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g006\"/></fig><p>It is not possible to make a direct comparison of the efficiency of these three routes as they do not lead to an identical endpoint. However, Nicolaou’s route is the shortest (6 or 7 steps, 9.8–9.2% yield), while Kitahara’s synthesis is the highest-yielding and does not involve the use of expensive transition metals or organocatalysts.</p></sec><sec><title>Syntheses to generate the C-14 stereocenter via C–N bond formation</title><p>Two groups implemented strategies that rely on the generation of the C-14 stereocenter by stereoselective C–N bond formation. The Jacobsen group utilized an asymmetric Cr(III)-catalyzed cycloaddition reaction [<xref rid=\"R27\" ref-type=\"bibr\">27</xref>] between (2<italic>Z</italic>,4<italic>E</italic>)-3-(triethylsilyloxy)-2,4-hexadiene (<bold>40</bold>) and the aldehyde <bold>41</bold> to generate the 4-silyloxydihydropyran <bold>43</bold> in a high yield and enantioselectivity (<xref ref-type=\"fig\" rid=\"C5\">Scheme 5</xref>) [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>–<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]. The Rubottom oxidation [<xref rid=\"R28\" ref-type=\"bibr\">28</xref>] of <bold>43</bold> gave a separable mixture of the desired <bold>44</bold> and its C-14 epimer (≈7:1 ratio). The reductive deoxygenation of <bold>44</bold> proceeded via the tosylhydrazone to afford <bold>45</bold>, which upon desilylation and alkyne isomerization produced <bold>46</bold>. The hydrozirconation of <bold>46</bold> with Schwartz’s reagent under equilibrating conditions, followed by the reaction with I<sub>2</sub> gave the vinyl iodide <bold>47</bold>. Finally, the activation of the C-14 hydroxy group and the S<sub>N</sub>2 displacement with azide gave the C-8–C-16 fragment <bold>48</bold>.</p><fig id=\"C5\" position=\"float\"><label>Scheme 5</label><caption><p>Jacobsen synthesis of the (all-<italic>cis</italic>)-2,3,5,6-tetrasubstituted tetrahydropyran.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g007\"/></fig><p>Ghosh relied on a reductive amination of the tetrahydropyranone <bold>50</bold> to generate the (all-<italic>cis</italic>)-tetrahydropyran fragment. This group reported multiple different routes to <bold>50</bold>. In an abortive route, the addition of allyl-Grignard to 5-methylfurfural, followed by the resolution of the racemic homoallylic alcohol with Amano lipase gave (−)-<bold>51</bold> (<xref ref-type=\"fig\" rid=\"C6\">Scheme 6</xref>) [<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]. An Achmatowicz oxidation [<xref rid=\"R29\" ref-type=\"bibr\">29</xref>] with <italic>tert</italic>-butyl hydroperoxide catalyzed by VO(acac)<sub>2</sub> afforded the hemiketal <bold>52</bold>, and the ionic reduction [<xref rid=\"R22\" ref-type=\"bibr\">22</xref>] of <bold>52</bold> gave the enone (−)-<bold>53</bold>. The enone transposition of <bold>53</bold> was effected by the addition of methyl-Grignard, followed by the oxidation with PCC. Unfortunately, all attempted 1,4-reduction conditions afforded an inseparable mixture of <italic>trans</italic>-<bold>54</bold> as the major product along with minor amounts of the desired <italic>cis</italic>-<bold>55</bold>.</p><fig id=\"C6\" position=\"float\"><label>Scheme 6</label><caption><p>Unproductive attempt to generate the (all-<italic>cis</italic>)-tetrahydropyranone <bold>50</bold>.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g008\"/></fig><p>Alternatively, the asymmetric reduction of the 2-acylfuran <bold>56</bold> using the Corey–Bakshi–Shibata reagent (<bold>21</bold>) [<xref rid=\"R30\" ref-type=\"bibr\">30</xref>] gave the alcohol <bold>57</bold> in a high yield and high enantioselectivity (<xref ref-type=\"fig\" rid=\"C7\">Scheme 7</xref>) [<xref rid=\"R15\" ref-type=\"bibr\">15</xref>–<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]. The Achmatowicz oxidation and ionic reduction generated the enone <bold>58</bold>, which is regioisomeric with <bold>53</bold>. The 1,4-addition of lithium dimethylcopper gave the desired <italic>cis</italic>-<bold>55</bold> with high a diastereoselectivity (25:1). The cross-metathesis of <bold>55</bold> with 3-methyl-3-buten-1-yl tosylate in the presence of Grubbs’ 2nd generation catalyst yielded <bold>59</bold>, which, upon elimination with potassium <italic>tert</italic>-butoxide led to the diene <bold>50</bold>. The reductive amination of <bold>50</bold> afforded an inseparable mixture of the C-14 amines (6:1 ratio). However, the amidation of this mixture with <bold>12c</bold> gave a separable mixture of the desired <bold>49</bold> (52%) along with the C-14 epimer (8%).</p><fig id=\"C7\" position=\"float\"><label>Scheme 7</label><caption><p>Ghosh synthesis of the C-7–C-14 (all-<italic>cis</italic>)-tetrahydropyran segment.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g009\"/></fig><p>More recently, the Ghosh group provided an alternative synthesis from the triacetyl ᴅ-glucal <bold>60</bold> (<xref ref-type=\"fig\" rid=\"C8\">Scheme 8</xref>) [<xref rid=\"R31\" ref-type=\"bibr\">31</xref>]. The exhaustive hydrolysis and selective protection as the 4,6-<italic>O</italic>-di-<italic>tert</italic>-butylsilylene derivative <bold>61</bold> [<xref rid=\"R32\" ref-type=\"bibr\">32</xref>] was followed by a 3-<italic>O</italic>-vinylation. A thermal 3,3-sigmatropic Claisen rearrangement of <bold>62</bold> gave the <italic>cis</italic>-2,6-disubstituted dihydropyran <bold>63</bold>, which upon sequential Wittig olefination with 2-(triphenylphosporanylidene)propanal and then methylenetriphenylphosphorane yielded the diene <bold>64</bold>. The removal of the di-<italic>tert</italic>-butylsilylene protecting group and the selective arylsulfonylation of the primary alcohol was effected with the bulky 2,4,6-triisopropylsulfonyl chloride, which upon reduction with aluminum hydride, followed by the oxidation of the remaining alcohol group gave the dihydropyran-3-one <bold>65</bold>. In a fashion similar to that of the dihydropyranone <bold>58</bold> [<xref rid=\"R16\" ref-type=\"bibr\">16</xref>], the 1,4-addition of dimethyl copper lithium to <bold>65</bold> gave the desired <italic>cis</italic>-<bold>50</bold> as a single diastereomer [<xref rid=\"R31\" ref-type=\"bibr\">31</xref>], allowing for a convergence with the route in <xref ref-type=\"fig\" rid=\"C7\">Scheme 7</xref>.</p><fig id=\"C8\" position=\"float\"><label>Scheme 8</label><caption><p>Ghosh’s alternative route to the (all-<italic>cis</italic>)-tetrahydropyranone <bold>50</bold>.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g010\"/></fig><p>Alternatively, the Wittig methenylation of <bold>63</bold>, followed by a silyl ether cleavage gave <bold>66</bold> (<xref ref-type=\"fig\" rid=\"C9\">Scheme 9</xref>) [<xref rid=\"R33\" ref-type=\"bibr\">33</xref>]. A three-step sequence similar to that from <bold>64</bold> to <bold>65</bold> allowed for the transformation of <bold>66</bold> to <bold>58</bold>, and thus providing an additional pathway for a convergence.</p><fig id=\"C9\" position=\"float\"><label>Scheme 9</label><caption><p>Alternative synthesis of the dihydro-3-pyrone <bold>58</bold>.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g011\"/></fig></sec></sec><sec><title>Synthesis of the C-1–C-6 tetrahydropyran fragment of FR901464 (<bold>1</bold>)/spliceostatins/thailanstatins</title><sec><title>Syntheses of the C-1–C-6 segment of FR901464 (<bold>1</bold>) from chiral pool precursors</title><p>Kitahara’s group fashioned the C-1–C-6 tetrahydropyran fragment of FR901464 (<bold>1</bold>) from commercially available 2-deoxy-ᴅ-glucose. In the first-generation approach (<bold>66</bold>, <xref ref-type=\"fig\" rid=\"C10\">Scheme 10</xref>) [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>], a sequence of protection and oxidation steps generated the tetrahydropyrone <bold>67</bold> at which point an olefination with the Tebbe reagent and the addition of methanol afforded the cyclic ketal as a separable mixture of the diastereomers <bold>68</bold> and <bold>69</bold> (≈4.5:1 ratio). The desilylation of <bold>68</bold> gave the 2° alcohol, which after oxidation and Tebbe olefination afforded the exocyclic olefin <bold>70</bold>. The manipulation of the C-4 and C-6 protecting groups gave the secondary allylic alcohol <bold>71</bold>, which underwent an epoxidation with mCPBA to give <bold>72</bold>. A second sequence of C-4/C-6 protection, manipulation, and oxidation gave the aldehyde <bold>73</bold>. The disadvantages of this route include the overall length (13 or 14 steps), low yield (5.4%), and the relative expense of the starting material ($22.3/g).</p><fig id=\"C10\" position=\"float\"><label>Scheme 10</label><caption><p>Kitahara’s 1st-generation synthesis of the C-1–C-6 fragment of FR901464 (<bold>1</bold>).</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g012\"/></fig><p>In their second-generation approach (<xref ref-type=\"fig\" rid=\"C11\">Scheme 11</xref>) [<xref rid=\"R9\" ref-type=\"bibr\">9</xref>], the acetonide-protected dithiane was alkylated according to Horton’s procedure [<xref rid=\"R34\" ref-type=\"bibr\">34</xref>]. A deprotection and cyclic-ketal formation gave <bold>70</bold>, allowing for a convergence with <xref ref-type=\"fig\" rid=\"C10\">Scheme 10</xref>.</p><fig id=\"C11\" position=\"float\"><label>Scheme 11</label><caption><p>Kitahara 1st-generation synthesis of the C-1–C-6 fragment of FR901464 (<bold>1</bold>).</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g013\"/></fig><p>Very recently, the groups of Nimura and Arisawa reported the synthesis of a phenyl C-glucoside derivative of spliceostatin beginning from ᴅ-glucal (<xref ref-type=\"fig\" rid=\"C12\">Scheme 12</xref>) [<xref rid=\"R35\" ref-type=\"bibr\">35</xref>]. A Heck coupling of the tris(trimethylsilyl) ether of <bold>74</bold> with phenylboronic acid in the presence of Pd(OAc)<sub>2</sub> and benzoquinone (BQ) gave the <italic>C</italic>-phenyl glucoside <bold>75</bold> [<xref rid=\"R36\" ref-type=\"bibr\">36</xref>]. Notably, the use of oxidants other than BQ gave either the TMS enol ether or the 2,3-dihydro-6-phenyl-4<italic>H</italic>-pyran-4-one. The C-3 exocyclic methylene group was introduced by a Wittig olefination, and after the manipulation of the protecting groups, a VO(acac)<sub>2</sub>-catalyzed oxidation stereoselectively generated the spirocyclic epoxide <bold>76</bold>. A second-protecting group shuffle afforded the primary alcohol <bold>77</bold>. A Mitsunobu substitution of <bold>77</bold> with 2-mercaptobenzothiazole, followed by an oxidation with a large excess of mCPBA afforded the sulfone <bold>78</bold>. While not producing the identical product, this route is shorter and higher-yielding (11 steps, 11.5%) than the Kitahara synthesis. However, it suffers from repeated protection/deprotection steps.</p><fig id=\"C12\" position=\"float\"><label>Scheme 12</label><caption><p>Nimura/Arisawa synthesis of the C-1-phenyl segment.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g014\"/></fig><p>The Ghosh group utilized (<italic>R</italic>)-glyceraldehyde acetonide (<bold>79</bold>, readily available from ᴅ-mannitol) as a chiral pool precursor for the introduction of the C-6 stereocenter (<xref ref-type=\"fig\" rid=\"C13\">Scheme 13</xref>) [<xref rid=\"R15\" ref-type=\"bibr\">15</xref>–<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]. The administration of the vinyllithium reagent, generated from the addition of the vinyl bromide <bold>80</bold> to <bold>79</bold>, gave a separable 1:1 mixture of the diastereomeric alcohols <bold>81a</bold> and <bold>81b</bold>. The undesired stereoisomer <bold>81b</bold> was converted into <bold>81a</bold> by a Mitsunobu reaction/hydrolysis sequence. The use of the dithiane <bold>80</bold> protecting group was crucial for the following steps. Initial attempts of using a dioxolane protecting group for the C-1 ketone (instead of dithiane) led to an insurmountable difficulty of the selective hydrolysis of the dioxolane and acetonide ketals. The protection of the C-4 hydroxy group, hydrolysis of the acetonide, and selective tosylation of the 1° alcohol were prerequisites for the generation of the C-5–C-6 bond. To this end, the reaction of <bold>82</bold> with the ylide generated from trimethylsulfonium iodide gave the allylic alcohol <bold>83</bold>. The removal of the dithiane protecting group and the cyclic-ketal formation gave <bold>84</bold>. The oxidative hydrolysis of the PMB ether and the reaction with mCPBA afforded the epoxide <bold>85</bold>. While relatively short (9 steps), the nonstereoselective formation of <bold>81a</bold>/<bold>b</bold> led to a lower overall yield.</p><fig id=\"C13\" position=\"float\"><label>Scheme 13</label><caption><p>Ghosh synthesis of the C-1–C-6 fragment of FR901464 (<bold>1</bold>) from (<italic>R</italic>)-glyceraldehyde acetonide.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g015\"/></fig></sec><sec><title>Syntheses of the C-1–C-6 segment of FR901464 (<bold>1</bold>) via asymmetric catalysis</title><p>Similar to their preparation of the C-8–C-15 segment, Jacobsen’s group relied on a Cr(III)-catalyzed silyloxydiene/aldehyde cycloaddition for the C-1–C-7 segment (<xref ref-type=\"fig\" rid=\"C14\">Scheme 14</xref>) [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>–<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]. The reaction of the protected glycolaldehyde <bold>86</bold> with (1<italic>E</italic>,3<italic>E</italic>)-2-triethylsilyloxy-1,3-hexadien-5-yne (<bold>87</bold>) in the presence of <bold>42</bold> gave the dihydropyran <bold>88</bold> with excellent enantioselectivity. A Rubottom oxidation, protection of the C-4 alcohol, and a Wittig methenylation afforded <bold>89</bold>. The selective deprotection of the primary TBS ether, followed by an Appel iodination and the cleavage of the secondary triisopropylsilyl ether were prerequisites for a vanadium-catalyzed stereoselective epoxidation of the exocyclic double bond to give <bold>90</bold>. The C-4 hydroxy group was eventually protected as a triethylsilyl ether. Through abortive attempts, Jacobsen found that the generation of the exocyclic epoxide prior to the formation of the C-6–C-9 conjugated diene was necessary in order to avoid the unwanted epoxidation of the C-6–C-7 olefin.</p><fig id=\"C14\" position=\"float\"><label>Scheme 14</label><caption><p>Jacobsen synthesis of the C-1–C-7 segment of FR901464 (<bold>1</bold>).</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g016\"/></fig><p>Koide employed a unique strategy in which the exocyclic epoxide was generated as the initial stereocenter (<xref ref-type=\"fig\" rid=\"C15\">Scheme 15</xref>) [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>–<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]. The Sharpless asymmetric epoxidation [<xref rid=\"R37\" ref-type=\"bibr\">37</xref>] of 5-methyl-2-methylene-4-penten-1-ol gave the epoxyalcohol <bold>92</bold> in 94% ee, which was oxidized to the aldehyde <bold>93</bold>. While the addition of the lithium salt of methyl propynoate proceeded in a nondiastereoselective fashion, the use of a zirconium/silver-mediated alkynylation gave the alcohol <bold>94</bold> with a 6:1 diastereoselectivity. The Red-Al reduction of <bold>94</bold>, protection of the 2° alcohol, and reduction of the enoate to an allylic alcohol were prerequisites for the stereoselective 2,3-sigmatropic selenoxide rearrangement to generate <bold>95</bold>. Further protecting group manipulation and Johnson–Lemieux cleavage afforded the methyl ketone <bold>96</bold>. The cyclic hemiketal <bold>97</bold> was unstable at an elevated temperature (37 °C), with <italic>t</italic><sub>1/2</sub> = 48 h. Both the Jacobsen (10 steps, 24.4% yield) and the Koide syntheses (11 steps, 22.3% yield) are relatively efficient in terms of the length and overall yield.</p><fig id=\"C15\" position=\"float\"><label>Scheme 15</label><caption><p>Koide synthesis of the C-1–C-7 segment of FR901464 (<bold>1</bold>).</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g017\"/></fig></sec><sec><title>Syntheses of the C-1–C-5 segment of thailanstatin methyl esters</title><p>Both syntheses of the C-1–C-5 tetrahydropyran segment of thailanstatin A (<bold>7</bold>) utilize sugar precursors. In the Ghosh synthesis [<xref rid=\"R31\" ref-type=\"bibr\">31</xref>], the triacetyl ᴅ-glucal <bold>60</bold> is converted into the <italic>trans</italic> C-1-allylated tetrahydropyran <bold>98</bold> according to literature procedures [<xref rid=\"R38\" ref-type=\"bibr\">38</xref>–<xref rid=\"R39\" ref-type=\"bibr\">39</xref>] (<xref ref-type=\"fig\" rid=\"C16\">Scheme 16</xref>). After a protecting group manipulation, the C-1 allyl group was truncated via ozonolysis, and the C-3 hydroxy group was transformed into an exocyclic methylene group to give <bold>99</bold>. The conversion of the C-6 alcohol moiety to a vinyl group by oxidation and Wittig methenylation to give <bold>100</bold> was relatively inefficient (31%) and was the main factor in diminishing the overall yield. The transformation of the silyl-protected 1° alcohol group of <bold>100</bold> into an ester afforded <bold>101</bold>. Finally, the C-3 spirocyclic oxirane was introduced stereoselectively by a VO(acac)<sub>2</sub>-directed epoxidation. While highly stereoselective, this relatively lengthy route was somewhat inefficient (16 steps, 1.4% overall yield).</p><fig id=\"C16\" position=\"float\"><label>Scheme 16</label><caption><p>Ghosh synthesis of the C-1–C-5 segment <bold>102</bold> of thailanstatin A (<bold>7</bold>).</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g018\"/></fig><p>The Nicolaou synthesis [<xref rid=\"R24\" ref-type=\"bibr\">24</xref>–<xref rid=\"R25\" ref-type=\"bibr\">25</xref>] of the C-1–C-9 tetrahydropyranyl segment commenced with the protected dihydropyranone <bold>103</bold> (<xref ref-type=\"fig\" rid=\"C17\">Scheme 17</xref>), readily prepared from ᴅ-glucal [<xref rid=\"R38\" ref-type=\"bibr\">38</xref>]. The application of an iodine-catalyzed Mukiyama–Michael addition of the ketene silyl acetal <bold>104</bold> to <bold>103</bold> afforded the <italic>trans</italic>-1,5-disubstituted tetrahydropyranone <bold>105</bold>. After the generation of the C-3-exocyclic olefin and functional group manipulation, the Takai olefination [<xref rid=\"R40\" ref-type=\"bibr\">40</xref>] of the aldehyde <bold>106</bold> gave the <italic>trans</italic>-vinyl iodide <bold>107</bold>. The removal of the silyl protecting group and hydrolysis of the methyl ester afforded the carboxylic acid <bold>108</bold>. A subsequently attempted Suzuki–Miyaura coupling of <bold>108</bold> with vinyl boronates was described as “capricious”, and thus the acid was esterified with the <italic>tert</italic>-butyl donor reagent <bold>109</bold> to afford <bold>110</bold>. In a fashion similar to that used by Ghosh’s group, the VO(acac)<sub>2</sub>-catalyzed epoxidation of <bold>110</bold> afforded the spirocyclic oxirane <bold>111</bold>. A subsequent ring opening with LiCl gave <bold>112</bold>, which was used in the synthesis of thailanstatin B (<bold>5</bold>). Nicolaou’s route to <bold>111</bold> (9 steps, 29.9% yield) is shorter and considerably more efficient than the Ghosh synthesis of <bold>102</bold>.</p><fig id=\"C17\" position=\"float\"><label>Scheme 17</label><caption><p>Nicolaou synthesis of the C-1–C-9 segments of spliceostatin D (<bold>9</bold>) and thailanstatins A (<bold>7</bold>) and B (<bold>5</bold>).</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g019\"/></fig></sec><sec><title>Syntheses of the C-1–C-6 segment of spliceostatin E (<bold>10</bold>)</title><p>The Ghosh group’s synthesis of the C-1–C-6 segment of spliceostatin E (<bold>10</bold>) relied on a Cu-catalyzed Grignard addition to <italic>tert</italic>-butyldiphenylsilyl-protected (<italic>R</italic>)-glycidol, followed by the generation of the mixed acetal <bold>113</bold> (<xref ref-type=\"fig\" rid=\"C18\">Scheme 18</xref>) [<xref rid=\"R33\" ref-type=\"bibr\">33</xref>]. A ring-closing metathesis gave an inseparable mixture of the dihydropyranyl ethers <bold>114a</bold> and <bold>14b</bold>, which could be equilibrated under acidic conditions (<bold>114b</bold>/<bold>114a</bold> > 20:1). A standard functional group manipulation afforded the vinyldihydropyran-2-one (−)-<bold>115</bold>.</p><fig id=\"C18\" position=\"float\"><label>Scheme 18</label><caption><p>Ghosh synthesis of the C-1–C-6 segment <bold>115</bold> of spliceostatin E (<bold>10</bold>).</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g020\"/></fig></sec></sec><sec><title>Fragment coupling to complete the synthesis of FR901464 (<bold>1</bold>)/spliceostatins/thailanstatins</title><sec><title>Fragment coupling via Wittig and Julia olefinations</title><p>Kitahara’s group utilized Wittig and modified/one-pot Julia olefination [<xref rid=\"R41\" ref-type=\"bibr\">41</xref>] reactions to fashion the dienyl segment, joining the two tetrahydropyran fragments. As their 2nd-generation synthesis was more efficient, this will be described (<xref ref-type=\"fig\" rid=\"C19\">Scheme 19</xref>) [<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]. To this end, the C-9–C-15 aldehyde <bold>27</bold> underwent an olefination with (carbethoxyethylidene)triphenylphosphorane to afford <bold>116</bold>. The enoate <bold>116</bold> was elaborated into the 1,3-benzothiazolesulfone <bold>117</bold> by standard transformations, prior to the cleavage of the Boc amide protecting group. The free amine <bold>118</bold> underwent an amidation with the TBS-protected (2<italic>Z</italic>,4<italic>S</italic>)-4-hydroxy-2-butenoic acid <bold>12b</bold> to give <bold>119</bold>. The modified Julia olefination of the aldehyde <bold>73</bold> with the anion derived from <bold>119</bold> proceeded with a high <italic>E</italic>-selectivity to generate the mixed cyclic ketal <bold>120</bold>. Finally, the removal of the C-4’ silyl protecting group, acylation of the resultant alcohol, removal of the C-4 silyl protecting group, and ketal hydrolysis generated FR901464 (<bold>1</bold>).</p><fig id=\"C19\" position=\"float\"><label>Scheme 19</label><caption><p>Fragment coupling via Wittig and modified Julia olefinations by Kitahara.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g021\"/></fig></sec><sec><title>Fragment coupling via olefin cross-metathesis</title><p>Koide’s group was the first to demonstrate the use of a Ru-catalyzed olefin cross-metathesis for the generation of the dienyl segment joining the two tetrahydropyranyl segments (<xref ref-type=\"fig\" rid=\"C20\">Scheme 20</xref>) [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>–<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]. The cleavage of the Boc amide of <bold>31</bold> and the eventual amidation with (2<italic>Z</italic>,4<italic>S</italic>)-4-acetoxy-2-butenoic acid (<bold>12c</bold>) gave <bold>121</bold>. The construction of the diene <bold>49</bold> involved cross-metathesis with an excess of methacrolein, catalyzed by <bold>122</bold>, followed by a Wittig olefination. The union of the diene <bold>49</bold> with 1.8 equivalents of the vinyl tetrahydropyran <bold>97</bold> was achieved with the Ru catalyst <bold>122</bold>. One recycle of the recovered starting material from this reaction gave FR901464 (<bold>1</bold>) in a combined yield of 40%. This reaction needed to be done at room temperature due to the lability of the hemiketal <bold>97</bold>. The use of Grubbs’ 2nd generation catalyst or the Grubbs–Hoveyda catalyst also gave <bold>1</bold>, albeit in a diminished yield (12–13%).</p><fig id=\"C20\" position=\"float\"><label>Scheme 20</label><caption><p>Fragment coupling via cross-metathesis by Koide.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g022\"/></fig><p>The Ghosh synthesis of spliceostatin A (<bold>4</bold>), FR901464 (<bold>1</bold>) [<xref rid=\"R15\" ref-type=\"bibr\">15</xref>–<xref rid=\"R16\" ref-type=\"bibr\">16</xref>], spliceostatin E (<bold>10</bold>) [<xref rid=\"R41\" ref-type=\"bibr\">41</xref>], and thailanstatin A methyl ester (<bold>123</bold>) [<xref rid=\"R31\" ref-type=\"bibr\">31</xref>] used the cross-metathesis strategy for the coupling of the diene <bold>49</bold> with <bold>85</bold>, <bold>115</bold>, and <bold>102</bold>, respectively (<xref ref-type=\"fig\" rid=\"C21\">Scheme 21</xref>). In order to avoid the decomposition problems encountered by Koide, the mixed cyclic ketal <bold>85</bold> was used as a coupling partner for the preparation of spliceostatin A (<bold>4</bold>). Due to this change, Grubbs’ second-generation catalyst, at a lower loading and elevated reaction temperatures, could be used since <bold>4</bold> was not labile under these thermal conditions. Ketal <bold>4</bold> underwent a hydrolysis to <bold>1</bold> under acidic conditions. The yields for the cross-metathesis coupling were generally higher when using an excess of <bold>85</bold> or <bold>115</bold>, as compared to the cross-metathesis of <bold>102</bold>, which also used a relatively high catalyst loading (20 mol % for 46% yield). While these authors did not report on the hydrolysis of <bold>123</bold> to prepare thailanstatin A (<bold>7</bold>), they found that the methyl ester <bold>123</bold> (IC<sub>50</sub> ≈ 0.4 μM) is nearly equipotent with <bold>7</bold> (IC<sub>50</sub> ≈ 0.65 μM).</p><fig id=\"C21\" position=\"float\"><label>Scheme 21</label><caption><p>The Ghosh synthesis of spliceostatin A (<bold>4</bold>), FR901464 (<bold>1</bold>), spliceostatin E (<bold>10</bold>), and thailanstatin methyl ester (<bold>123</bold>).</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g023\"/></fig></sec><sec><title>Fragment coupling via Wittig olefination, cross-metathesis, and Julia olefination</title><p>In their recent synthesis of a 1-phenyl analog (<bold>124</bold>) of FR901464 (<bold>1</bold>), Arisawa’s group utilized a combination of previously reported methodologies (<xref ref-type=\"fig\" rid=\"C22\">Scheme 22</xref>) [<xref rid=\"R35\" ref-type=\"bibr\">35</xref>]. Thus, the (all-<italic>cis</italic>)-2,3,5,6-tetrasubstituted aldehyde <bold>27</bold> was constructed according to Kitahara’s protocol [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>–<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]. The Wittig methenylation of <bold>27</bold>, followed by the cleavage of the Boc protecting group and the amidation with <bold>12b</bold> afforded the fragment <bold>125</bold>. The cross-metathesis of <bold>125</bold> with an excess of methacrolein in the presence of the Ru catalyst <bold>122</bold> gave the aldehyde <bold>126</bold>. This coupling is analogous to Koide’s cross-metathesis of <bold>121</bold> (cf. <xref ref-type=\"fig\" rid=\"C20\">Scheme 20</xref>). A modified Julia olefination of <bold>126</bold> with the anion generated from the sulfone <bold>78</bold>, followed by the cleavage of the 4’-TBS protecting group gave <bold>127</bold> in an acceptable yield (52%). A protecting-group adjustment finalized the synthesis of <bold>124</bold>. Notably, these authors found that switching the sulfone and aldehyde functionalities, i.e., an olefination of the aldehyde generated by the oxidation of <bold>77</bold> with Kitahara’s sulfone <bold>119</bold>, proceeded less efficiently (22% yield). In a subsequent assay for repressed cell proliferation against the human prostate cancer cell lines LNCaP, LNCaP95, and CWR22Rv, these authors reported IC<sub>50</sub> values of 63, 175, and 93 nM, respectively, for <bold>124</bold>. These can be compared to the IC<sub>50</sub> values of 1.3, 1.0, and 0.36 nM, respectively, for spliceostatin A (<bold>4</bold>) against the same cell lines.</p><fig id=\"C22\" position=\"float\"><label>Scheme 22</label><caption><p>Arisawa synthesis of a C-1-phenyl analog of FR901464 (<bold>1</bold>).</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g024\"/></fig></sec><sec><title>Fragment union via Pd-catalyzed sp<sup>2</sup>–sp<sup>2</sup> coupling</title><p>A Negishi coupling reaction [<xref rid=\"R42\" ref-type=\"bibr\">42</xref>] was used in the Jacobsen synthesis of FR901464 (<bold>1</bold>, <xref ref-type=\"fig\" rid=\"C23\">Scheme 23</xref>) [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>–<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]. The hydrozirconation of <bold>91</bold>, followed by a transmetalation provided a vinylzinc reagent that was coupled with <bold>48</bold> to afford <bold>128</bold>, for which only the <italic>E</italic>-stereoisomer was observed. Notably, the Negishi conditions were tolerant to the azide present in <bold>48</bold> and the oxirane and 1° iodoalkane present in <bold>91</bold>. The subsequent reduction of the azide and the amidation with <bold>12c</bold> afforded <bold>129</bold>. The synthesis was completed by the elimination of the 1° iodide, silyl ether cleavage, and hydration of the exocyclic enol ether. Of all the syntheses of FR901464 (<bold>1</bold>), the coupling of <bold>91</bold> with <bold>48</bold> is the most efficient sequence (6 steps, 45% yield).</p><fig id=\"C23\" position=\"float\"><label>Scheme 23</label><caption><p>Jacobsen fragment coupling by a Pd-catalyzed Negishi coupling.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g025\"/></fig><p>Nicolaou utilized the sequential application of a cross-metathesis and a Suzuki–Miyaura coupling in the syntheses of thailanstatin A and B (<bold>7</bold> and <bold>5</bold>) and spliceostatin D (<bold>9</bold>, <xref ref-type=\"fig\" rid=\"C24\">Scheme 24</xref>) [<xref rid=\"R24\" ref-type=\"bibr\">24</xref>–<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]. To this end, the methylhydrazinolysis of the phthalimide <bold>39</bold> and the amide formation with <bold>12c</bold> yielded <bold>121</bold>. The cross-metathesis of <bold>121</bold> with a five-fold excess of isopropenylboronic acid pinacol ester afforded the lynchpin vinylborane <bold>130</bold>. A Pd-catalyzed Suzuki–Miyaura coupling of <bold>130</bold> with the vinyl iodide <bold>110</bold>, <bold>111</bold>, or <bold>112</bold> gave the <italic>tert</italic>-butyl ester <bold>131</bold>, <bold>132</bold>, or <bold>133</bold> in a moderate yield (42–63%). The hydrolysis of the <italic>tert</italic>-butyl esters with formic acid gave the carboxylic acid <bold>9</bold>, <bold>7</bold>, or <bold>5</bold>. Additionally, the treatment of the thailanstatin A <italic>tert</italic>-butyl ester with lithium chloride generated the thailanstatin B <italic>tert</italic>-butyl ester.</p><fig id=\"C24\" position=\"float\"><label>Scheme 24</label><caption><p>Nicolaou syntheses of thailanstatin A and B (<bold>7</bold> and <bold>5</bold>) and spliceostatin D (<bold>9</bold>) via a Pd-catalyzed Suzuki–Miyaura coupling.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g026\"/></fig><p>Ghosh used a similar cross-metathesis/Suzuki–Miyaura coupling sequence for the preparation of spliceostatin G (<bold>11</bold>, <xref ref-type=\"fig\" rid=\"C25\">Scheme 25</xref>) [<xref rid=\"R43\" ref-type=\"bibr\">43</xref>]. The catalyst loading for the coupling of <bold>130</bold> with methyl (<italic>E</italic>)-3-iodoacrylate (20 mol %) was twice the amount used by Nicolaou (10 mol %).</p><fig id=\"C25\" position=\"float\"><label>Scheme 25</label><caption><p>The Ghosh synthesis of spliceostatin G (<bold>11</bold>) via Suzuki–Miyaura coupling.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1991-g027\"/></fig></sec></sec></sec><sec><title>Conclusion</title><p>As delineated above, a wide variety of synthetic strategies has been employed to either introduce the stereocenters present in the spliceostatins/thailanstatins or to couple the subfragments of these molecules (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). The most common route to the (2<italic>Z</italic>,4<italic>S</italic>)-4-acetoxy-2-butenoic acid fragment relies on the <italic>cis</italic>-reduction of 4-acetoxy-2-pentynoic acid; the most efficient of these routes utilizes a Noyori asymmetric reduction of an alkynyl ketone to produce the required stereocenter. Of the syntheses to date, Jacobsen’s use of an asymmetric Cr(III)-catalyzed cycloaddition stands as the most efficient route, in terms of synthetic steps and low catalyst loading, to the (all-<italic>cis</italic>)-2,3,5,6-tetrasubstituted tetrahydropyran and the C-1–C-6 tetrahydropyran fragments of FR901464 (<bold>1</bold>). The preparation of the C-1–C-5 tetrahydropyran fragment of the thailanstatins has, so far, utilized sugar-derived precursors. A variety of reactions have been utilized for union of these fragments via a diene segment. While a Ru-catalyzed cross-metathesis reaction was employed in numerous syntheses, the drawbacks of this strategy include the high catalyst loading and the excess of the olefin coupling partners. To date, the most efficient strategy for the union of the fragments relies on Pd-catalyzed sp<sup>2</sup>–sp<sup>2</sup> couplings.</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Summary of the total syntheses.</p></caption><table frame=\"hsides\" rules=\"groups\"><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">target</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">PI</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">year</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Reference</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Scheme</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">no. of steps (LLS)</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">overall yield</td></tr><tr><td align=\"center\" colspan=\"7\" valign=\"top\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">FR901464 (<bold>1</bold>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Jacobsen</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2000</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"R17\" ref-type=\"bibr\">17</xref>–<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">5, 14, 23</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">8.46%</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">FR901464 (<bold>1</bold>) 1st generation</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Kitahara</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2001</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2, 10, 19</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.63%</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">FR901464 (<bold>1</bold>) 2nd generation</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Kitahara</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2006</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2, 11, 19</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3.18%</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">FR901464 (<bold>1</bold>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Koide</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2006</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>–<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3, 15, 20</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3.77%</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">spliceostatin A (<bold>4</bold>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Ghosh</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2013</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"R15\" ref-type=\"bibr\">15</xref>–<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3, 15, 20</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">6.61%</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">FR901464 (<bold>1</bold>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Ghosh</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2013</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"R15\" ref-type=\"bibr\">15</xref>–<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">7, 13, 21</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">5.22%</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">spliceostatin E (<bold>10</bold>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Ghosh</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2014</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">7, 18, 21</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">8.24%</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">thailanstatin A (<bold>7</bold>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Nicolaou</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2016</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4, 17, 24</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2.23%</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">thailanstatin A methyl ester (<bold>123</bold>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Ghosh</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2018</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"R31\" ref-type=\"bibr\">31</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">8, 16, 21</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.54%</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">spliceostatin G (<bold>11</bold>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Ghosh</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2018</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"R43\" ref-type=\"bibr\">43</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">9, 25</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2.31%</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">spliceostatin D (<bold>9</bold>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Nicolaou</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2018</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4, 17, 24</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2.24%</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">thailanstatin B (<bold>5</bold>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Nicolaou</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2018</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4, 17, 24</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.87%</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">spliceostatin A analog</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Arisawa</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2019</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"R34\" ref-type=\"bibr\">34</xref>]</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2, 12, 22</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3.99%</td></tr></table></table-wrap></sec></body><back><ref-list><ref id=\"R1\"><label>1</label><element-citation 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"letter\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Beilstein J Org Chem</journal-id><journal-id journal-id-type=\"iso-abbrev\">Beilstein J Org Chem</journal-id><journal-title-group><journal-title>Beilstein Journal of Organic Chemistry</journal-title></journal-title-group><issn pub-type=\"epub\">1860-5397</issn><publisher><publisher-name>Beilstein-Institut</publisher-name><publisher-loc>Trakehner Str. 7-9, 60487 Frankfurt am Main, Germany</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32831955</article-id><article-id pub-id-type=\"pmc\">PMC7431758</article-id><article-id pub-id-type=\"doi\">10.3762/bjoc.16.165</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Letter</subject></subj-group><subj-group subj-group-type=\"topic\"><subject>Chemistry</subject><subj-group subj-group-type=\"topic\"><subject>Organic Chemistry</subject></subj-group></subj-group></article-categories><title-group><article-title>A complementary approach to conjugated <italic>N</italic>-acyliminium formation through photoredox-catalyzed intermolecular radical addition to allenamides and allencarbamates</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Koleoso</surname><given-names>Olusesan K</given-names></name><xref ref-type=\"aff\" rid=\"A1\">1</xref><xref ref-type=\"aff\" rid=\"A2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Turner</surname><given-names>Matthew</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0001-6508-7010</contrib-id><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Plasser</surname><given-names>Felix</given-names></name><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Kimber</surname><given-names>Marc C</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0003-2943-1974</contrib-id><email>M.C.Kimber@lboro.ac.uk</email><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Noël</surname><given-names>Timothy</given-names></name><role>Guest Editor</role></contrib></contrib-group><aff id=\"A1\"><label>1</label>School of Science, Department of Chemistry, Loughborough University, Loughborough, LE11 3TU, UK</aff><aff id=\"A2\"><label>2</label>Department of Pharmaceutical Technology, Moshood Abiola Polytechnic, Abeokuta, Nigeria</aff><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>12</day><month>8</month><year>2020</year></pub-date><volume>16</volume><fpage>1983</fpage><lpage>1990</lpage><ext-link ext-link-type=\"doi\" xlink:href=\"10.3762/bjoc.16.165\">10.3762/bjoc.16.165</ext-link><history><date date-type=\"received\"><day>19</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>5</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Koleoso et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Koleoso et al.</copyright-holder><ali:free_to_read xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\"/><license license-type=\"Beilstein\"><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\">https://creativecommons.org/licenses/by/4.0</ali:license_ref><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\">https://www.beilstein-journals.org/bjoc/terms</ali:license_ref><license-p>This is an Open Access article under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"https://creativecommons.org/licenses/by/4.0\">https://creativecommons.org/licenses/by/4.0</ext-link>). Please note that the reuse, redistribution and reproduction in particular requires that the authors and source are credited.</license-p><license-p>The license is subject to the Beilstein Journal of Organic Chemistry terms and conditions: (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.beilstein-journals.org/bjoc/terms\">https://www.beilstein-journals.org/bjoc/terms</ext-link>)</license-p></license></permissions><abstract><p>An intermolecular radical addition, using photoredox catalysis, to allenamides and allencarbamates is reported. This transformation synthesizes <italic>N</italic>-acyl-<italic>N’</italic>-aryl-<italic>N</italic>,<italic>N’</italic>-allylaminals, and proceeds by a conjugated <italic>N</italic>-acyliminium intermediate that previously has principally been generated by electrophilic activation methods. The radical adds to the central carbon of the allene giving a conjugated <italic>N</italic>-acyliminium that undergoes nucleophilic addition by arylamines and alcohols.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>allenamide</kwd><kwd>allene</kwd><kwd>intermolecular</kwd><kwd><italic>N</italic>-acyliminium</kwd><kwd>photoredox</kwd></kwd-group><funding-group><funding-statement>We gratefully acknowledge financial support from Loughborough University and the Tertiary Education Trust Fund (TETFund) Abuja, Nigeria (O.K.K.).</funding-statement></funding-group></article-meta><notes><p>This article is part of the thematic issue \"Advances in photoredox catalysis\".</p></notes></front><body><sec><title>Introduction</title><p>Allenamides (<xref ref-type=\"fig\" rid=\"C1\">Scheme 1</xref>, <bold>1</bold>) and their congeners have attracted considerable attention over the past 15 years due to their characteristic reactivity profiles [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]. The reactivity that an allenamide can display is distinct from a traditional allene due to the presence of an amide unit attached at the α-carbon. This substituent can donate electron density into the allene, principally onto the central β-carbon, that can be harnessed in subsequent chemical transformations leading to regiochemical confidence in the resulting products (<xref ref-type=\"fig\" rid=\"C1\">Scheme 1</xref>).</p><fig id=\"C1\" position=\"float\"><label>Scheme 1</label><caption><p>Electrophilic activation of allenamides.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1983-g002\"/></fig><p>Importantly, this unique reactivity has underpinned allenamide chemistry and led to the development of a number of innovative transformations, including cycloadditions [<xref rid=\"R5\" ref-type=\"bibr\">5</xref>–<xref rid=\"R9\" ref-type=\"bibr\">9</xref>], intramolecular cyclizations and intermolecular addition reactions [<xref rid=\"R10\" ref-type=\"bibr\">10</xref>–<xref rid=\"R18\" ref-type=\"bibr\">18</xref>], as well as the use of the allenamide building block in natural product synthesis [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>].</p><p>Addition reactions of allenamides, which can also encompass intramolecular cyclizations, are typically promoted by the electrophilic activation of the β-carbon of the allenamide. This can be achieved using various electrophilic methods, including the use of Brönsted acids [<xref rid=\"R15\" ref-type=\"bibr\">15</xref>,<xref rid=\"R19\" ref-type=\"bibr\">19</xref>–<xref rid=\"R22\" ref-type=\"bibr\">22</xref>], halogenation sources [<xref rid=\"R16\" ref-type=\"bibr\">16</xref>,<xref rid=\"R18\" ref-type=\"bibr\">18</xref>,<xref rid=\"R23\" ref-type=\"bibr\">23</xref>–<xref rid=\"R28\" ref-type=\"bibr\">28</xref>], by means of oxidation [<xref rid=\"R20\" ref-type=\"bibr\">20</xref>,<xref rid=\"R29\" ref-type=\"bibr\">29</xref>–<xref rid=\"R31\" ref-type=\"bibr\">31</xref>] or through the use of a transition metal such as Au(I) [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>,<xref rid=\"R10\" ref-type=\"bibr\">10</xref>–<xref rid=\"R16\" ref-type=\"bibr\">16</xref><xref rid=\"R32\" ref-type=\"bibr\">32</xref>–<xref rid=\"R36\" ref-type=\"bibr\">36</xref>]. The reaction of the allenamide with an electrophilic source promotes the formation of a conjugated <italic>N</italic>-acyliminium intermediate <bold>2</bold> [<xref rid=\"R37\" ref-type=\"bibr\">37</xref>–<xref rid=\"R40\" ref-type=\"bibr\">40</xref>] that subsequently undergoes an addition reaction with a suitable nucleophile (<xref ref-type=\"fig\" rid=\"C1\">Scheme 1</xref>).</p><p>Importantly, the electrophilic mediated formation of this key intermediate <bold>2</bold> have been the cornerstone for allenamide chemistry over the past 15 years. Therefore, given the importance of the allenamide building block we sought a new synthetic methodology that could generate this conjugated <italic>N</italic>-acyliminium intermediate <bold>2</bold>, that would not be dependent on conventional electrophilic activation modes (<xref ref-type=\"fig\" rid=\"C2\">Scheme 2</xref>).</p><fig id=\"C2\" position=\"float\"><label>Scheme 2</label><caption><p>The planned intramolecular radical addition to allenamides generating the conjugated <italic>N</italic>-acyliminium intermediate <bold>14</bold>.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1983-g003\"/></fig><p>In principle, a new method for its generation could greatly enhance the value of the allenamide building block, as well as potentially unlocking new chemical reactivity yet unseen in the context of electrophilic activation. We envisaged that a route to the conjugated <italic>N</italic>-acyliminium intermediate could be achieved via an intermolecular addition of an electrophilic radical to an allenamide (<bold>1</bold>) followed by an oxidation process on the subsequently formed radical (<xref ref-type=\"fig\" rid=\"C3\">Scheme 3</xref>) [<xref rid=\"R41\" ref-type=\"bibr\">41</xref>]. The thought process behind this approach is based on three observations; (i) Akita and co-workers disclosure on the photoredox-catalyzed oxytrifluoromethylation of allenes <bold>6</bold> to give 2-trifluoromethylated allyl acetates <bold>7</bold> [<xref rid=\"R42\" ref-type=\"bibr\">42</xref>–<xref rid=\"R44\" ref-type=\"bibr\">44</xref>]; (ii) that intramolecular radical cyclization of allenamides have been reported by Hsung and co-workers, where the radical adds principally to the central carbon of the allene (<bold>10</bold>) [<xref rid=\"R45\" ref-type=\"bibr\">45</xref>]; and (iii) the photoredox-catalyzed addition of radicals to enamides reported by Masson [<xref rid=\"R46\" ref-type=\"bibr\">46</xref>–<xref rid=\"R49\" ref-type=\"bibr\">49</xref>], and more recently by our own laboratory in the synthesis of <italic>N</italic>,<italic>N’</italic>-aminals [<xref rid=\"R50\" ref-type=\"bibr\">50</xref>]. Therefore, photoredox-catalysis would be employed to generate an electrophilic radical that would add to the central carbon of the allenamide <bold>1</bold> to give a transient radical <bold>13</bold>, whose oxidation, facilitated by the photoredox catalyst [<xref rid=\"R47\" ref-type=\"bibr\">47</xref>–<xref rid=\"R48\" ref-type=\"bibr\">48</xref>], would provide the conjugated <italic>N</italic>-acyliminium <bold>14</bold>. The iminium <bold>14</bold> could then undergo traditional nucleophilic addition giving the addition product. Given that the iridium complex Ir[(ppy)<sub>2</sub>(dtbbpy)]PF<sub>6</sub> had been effective [<xref rid=\"R47\" ref-type=\"bibr\">47</xref>–<xref rid=\"R48\" ref-type=\"bibr\">48</xref><xref rid=\"R50\" ref-type=\"bibr\">50</xref>] in this radical/cationic pathway with enamides we would now like to report our preliminary findings on the performance of allenamides. Specifically, the electron-withdrawing group on the allenamide and the nucleophile is examined. We provide evidence for the formation of the <italic>N</italic>-acyliminium intermediate through direct sample loop and flow injection analysis by HRESIMS, and DFT analysis of the <italic>N</italic>-acyliminium intermediate is provided to explain the addition product distribution.</p><fig id=\"C3\" position=\"float\"><label>Scheme 3</label><caption><p>Photoredox Ir-catalyzed intermolecular addition of bromide <bold>18</bold> and aniline <bold>16</bold> to allenamide <bold>15</bold>.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1983-g004\"/></fig></sec><sec><title>Results and Discussion</title><p>We began our study by using the iridium catalyst Ir[(ppy)<sub>2</sub>(dtbbpy)]PF<sub>6</sub> (<bold>17</bold>) and the reaction conditions used in the addition of amine nucleophiles to enamides [<xref rid=\"R48\" ref-type=\"bibr\">48</xref>,<xref rid=\"R50\" ref-type=\"bibr\">50</xref>]. By exploiting these reaction conditions, using diethyl bromomalonate (<bold>18</bold>), 4-anisidine (<bold>16</bold>) as the amine nucleophile, and allenamide <bold>15</bold> we were able to isolate <italic>N</italic>-acyl-<italic>N</italic>’-aryl-<italic>N</italic>,<italic>N</italic>’-allylaminal <bold>19</bold> as the sole identifiable product in 54% yield.</p><p>All of the starting allenamide <bold>15</bold> was consumed in the reaction, but the isolation of <bold>19</bold> proved very challenging and we can account for this instability by the identification of <bold>14a</bold> in the <sup>1</sup>H NMR spectrum of the crude reaction mixture. A comparable instability profile was observed in the formation of <italic>N</italic>-acyl-<italic>N</italic>’-aryl-<italic>N</italic>,<italic>N</italic>’-aminals derived from enamides [<xref rid=\"R50\" ref-type=\"bibr\">50</xref>]; however, to our knowledge, there are no reports of the isolation of analogous <italic>N</italic>-acyl-<italic>N</italic>’-aryl-<italic>N</italic>,<italic>N</italic>’-allylaminal substrates in the literature, consequently, these specific conditions were not optimized [<xref rid=\"R51\" ref-type=\"bibr\">51</xref>]. The evidence for the formation of <bold>19</bold> was the appearance of the methylene protons in <sup>1</sup>H NMR at δ 5.46 (d, <italic>J</italic> = 1.6 Hz, 1H) and δ 5.45 (d, <italic>J</italic> = 2.0 Hz, 1H) ppm, respectively. Crucially, it is apparent that using the photoredox conditions described in <xref ref-type=\"fig\" rid=\"C3\">Scheme 3</xref>, the aniline nucleophile adds primarily at the α-position of the allenamide <bold>15</bold>; this is in contrast to the archetypal electrophilic activation modes where comparable nucleophiles add to the γ-position [<xref rid=\"R37\" ref-type=\"bibr\">37</xref>,<xref rid=\"R39\" ref-type=\"bibr\">39</xref>–<xref rid=\"R40\" ref-type=\"bibr\">40</xref>].</p><p>An examination of the allenamide unit under these conditions is shown in <xref ref-type=\"fig\" rid=\"C4\">Scheme 4</xref>, and the six allenylamides/sulfonamides (<bold>15</bold>, <bold>21</bold>–<bold>25</bold>) were prepared using known conditions [<xref rid=\"R52\" ref-type=\"bibr\">52</xref>–<xref rid=\"R53\" ref-type=\"bibr\">53</xref>]. The allenamides derived from pyrrolidinone (<bold>21</bold>), piperidinone (<bold>22</bold>) and oxazolidinone (<bold>15</bold>) with 2,4-dimethylaniline all performed adequately in this reaction giving their <italic>N</italic>,<italic>N</italic>’-allylaminal products <bold>26</bold>, <bold>27</bold> and <bold>28</bold>, respectively. The isolated yields once again reflected the sensitivity of the <italic>N</italic>,<italic>N</italic>’-allylaminal functional group. The aminosulfonyl allenyl <bold>23</bold> failed to provide any discernable product, with only a complex mixture, as identified by <sup>1</sup>H NMR, being isolated. Significantly, in the <sup>1</sup>H NMR spectra of this complex mixture we observed complete fragmentation of the sulfonamide unit. Two chiral allenamides (<bold>24</bold> and <bold>25</bold>) were exposed to the photoredox conditions with 2,4-dimethylaniline, and it was observed that the predominant product in each case was <italic>Z</italic>-<bold>30</bold> and <italic>Z</italic>-<bold>31</bold>, respectively, which presumably result from γ-addition of the nucleophile. This was confirmed by <sup>1</sup>H NMR NOE analysis of <italic>Z</italic>-<bold>30</bold>, where an enhancement between the enamide proton and the methylene proton were observed, as shown in <xref ref-type=\"fig\" rid=\"C4\">Scheme 4</xref>.</p><fig id=\"C4\" position=\"float\"><label>Scheme 4</label><caption><p>Reaction scope (a) allenamide; (b) arylamine nucleophile; (c) alcohol nucleophile.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1983-g005\"/></fig><p>Given our interest in the <italic>N</italic>,<italic>N’</italic>-aminal functionality [<xref rid=\"R50\" ref-type=\"bibr\">50</xref>], we also explored variation of the arylamine nucleophile. 2,4-Dimethylaniline, <italic>o</italic>-anisole, 2-trifluoromethoxyaniline and 3,5-ditrifluoromethylaniline were all effective in this transformation, giving their labile <italic>N</italic>,<italic>N</italic>’-allylaminal products (<bold>32</bold> to <bold>35</bold>) in moderate to good isolated yields.</p><p>4-Bromo-2-fluoroaniline was also examined as a nucleophile, as we had previously shown this to be an effective aniline platform for developing linezolid analogues, and this delivered two <italic>N</italic>,<italic>N</italic>’-allylaminals <bold>36</bold> and <bold>37</bold>, respectively. Masson had previously explored oxygen nucleophiles in their photoredox-catalyzed addition to enamides [<xref rid=\"R48\" ref-type=\"bibr\">48</xref>]. Consequently, the use of excess methanol provided the addition product <bold>38</bold>, resulting from α-addition, in a modest isolated yield. The stability of product <bold>38</bold> was marginal, but an improved stability of the <italic>N</italic>,<italic>O’</italic>-allyl product was observed when ethanol was used as the nucleophile giving product <bold>39</bold> in 52% isolated yield. In contrast, isopropanol gave an inseparable mixture of the α- and γ-addition products, <bold>40a/b</bold> in 33% isolated yield. As with the case for the formation of <bold>19</bold>, all of the starting allenes were consumed in each reaction shown in <xref ref-type=\"fig\" rid=\"C4\">Scheme 4</xref>.</p><p>A tentative mechanism for this transformation is described in <xref ref-type=\"fig\" rid=\"C5\">Scheme 5a</xref>. Excitation of the Ir(III) complex <bold>17</bold> provides *Ir(III) that subsequently undergoes reductive quenching by Et<sub>3</sub>N, delivering Ir(II) [<xref rid=\"R48\" ref-type=\"bibr\">48</xref>]. Single electron transfer from Ir(II) to <bold>18</bold> then generates an electrophilic radical R<sup>•</sup> together with stoichiometric bromide ion; R<sup>•</sup> subsequently adds to the β-carbon of the allenamide <bold>15</bold>, providing an α-aminoallyl radical <bold>41</bold>. To support this hypothesis the calculated HOMO of allenamide <bold>15</bold> is shown in <xref ref-type=\"fig\" rid=\"C5\">Scheme 5b</xref>, signifying the increased nucleophilicity at the β-carbon [<xref rid=\"R54\" ref-type=\"bibr\">54</xref>]. Mechanistically, the formation of the key conjugated <italic>N</italic>-acyliminum <bold>14a</bold> from α-aminoallyl radical <bold>41</bold> is analogous to the photoredox initiated addition of radicals to enamides [<xref rid=\"R46\" ref-type=\"bibr\">46</xref>,<xref rid=\"R48\" ref-type=\"bibr\">48</xref>–<xref rid=\"R49\" ref-type=\"bibr\">49</xref>]. Consequently, the formation of <bold>14a</bold> can results from a SET event between the oxidized Ir(III) [<xref rid=\"R55\" ref-type=\"bibr\">55</xref>] and the α-aminoallyl radical <bold>41</bold>; a radical propagation pathway through the reduction of <bold>18</bold> was discounted, as the reaction required continuous irradiation.</p><fig id=\"C5\" position=\"float\"><label>Scheme 5</label><caption><p>(a) Tentative mechanism for the photoredox-catalyzed formation of the conjugated <italic>N</italic>-acyliminium intermediate; (b) the calculated HOMO [<xref rid=\"R54\" ref-type=\"bibr\">54</xref>] for allenamide <bold>15</bold>; (c) the identification of intermediate <bold>14a</bold> by direct sample loop and flow injection HRESIMS analysis.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1983-g006\"/></fig><p>To further support this mechanistic hypothesis, we used ESIMS to identify the formation of the conjugated <italic>N</italic>-acyliminium <bold>14a</bold>. The addition of <bold>18</bold> to allenamide <bold>15</bold> under the Ir-catalyzed photoredox conditions in the presence of 4-bromoaniline was monitored by direct sample loop and flow injection analysis by HRESIMS [<xref rid=\"R56\" ref-type=\"bibr\">56</xref>–<xref rid=\"R57\" ref-type=\"bibr\">57</xref>]. After 5 minutes we observed a peak at <italic>m</italic>/<italic>z</italic> 284.1129 that corresponded satisfactorily to the expected iminium complex <bold>14a</bold> (<italic>m</italic>/<italic>z</italic> 284.1129: calcd for [C<sub>13</sub>H<sub>18</sub>NO<sub>6</sub>]<sup>+</sup>); this peak persisted at 15, 30, 60 and 120 min time intervals, respectively.</p><p>Upon the oxidation of <bold>41</bold> two plausible iminium stereoisomers can be formed, <italic>Z</italic>-<bold>42</bold> and <italic>E</italic>-<bold>42</bold>, respectively, with each of these iminium stereoisomers existing in two further conformers designated <italic>E</italic>-<bold>42’</bold> and <italic>Z</italic>-<bold>42’</bold>. DFT calculations [<xref rid=\"R54\" ref-type=\"bibr\">54</xref>] were performed on all four of these proposed structures, where it was observed that <italic>Z</italic>-<bold>42</bold> was approx. 6 kcal/mol higher in energy, relative to the most stable isomer <italic>E</italic>-<bold>42</bold>, which was 1–2 kcal/mol lower in energy than <italic>E</italic>-<bold>42’</bold> and <italic>Z</italic>-<bold>42’</bold>, respectively. It is feasible that both <italic>E</italic>-<bold>42</bold> and <italic>Z</italic>-<bold>42’</bold> undergo nucleophilic addition at the α-position giving the observed <italic>N</italic>,<italic>N’</italic>-allylaminal product <bold>43</bold>. Conversely, addition of a nucleophile at the γ-position of <italic>E</italic>-<bold>42</bold> gives the observed <italic>Z</italic>-enamide <bold>44</bold>; and addition at the γ-position of <italic>Z</italic>-<bold>42’</bold> gives the same observed <italic>Z</italic>-enamide <bold>44</bold> after C–N bond rotation.</p></sec><sec><title>Conclusion</title><p>In conclusion, in this letter we have demonstrated the first intermolecular addition of an electrophilic radical, generated under photoredox conditions, to an allenamide building block. The addition of the radical occurs at the central carbon of the allene, giving a conjugated <italic>N</italic>-acyliminium intermediate after subsequent oxidation. We have established that the conjugated <italic>N</italic>-acyliminium intermediate can be formed from a broad range of allenamide precursors; additionally, the conjugated <italic>N</italic>-acyliminium intermediate can undergo nucleophilic addition with an arylamine or alcohol nucleophiles at the α- or γ-position, with the regioselectivity of the addition being controlled by steric factors. Significantly, the formation of the key conjugated <italic>N</italic>-acyliminium intermediate using these photoredox conditions can be seen as complementary to the well-developed electrophilic activation modes of allenamides. We are currently examining the full mechanism of this transformation, expanding the scope of substrates that can be used in the radical addition step, and alternative fates for the α-<italic>N</italic>-acyl radical <bold>13</bold> [<xref rid=\"R58\" ref-type=\"bibr\">58</xref>–<xref rid=\"R60\" ref-type=\"bibr\">60</xref>].</p></sec><sec sec-type=\"supplementary-material\"><title>Supporting Information</title><supplementary-material content-type=\"local-data\" id=\"SD1\"><label>File 1</label><caption><p>Experimental details, analytical (<sup>1</sup>H NMR, <sup>13</sup>C NMR) and ESIMS data.</p></caption><media mime-subtype=\"pdf\" mimetype=\"application\" xlink:href=\"Beilstein_J_Org_Chem-16-1983-s001.pdf\" xlink:type=\"simple\" id=\"d39e1020\" position=\"anchor\"/></supplementary-material></sec></body><back><ref-list><ref id=\"R1\"><label>1</label><element-citation publication-type=\"journal\"><person-group 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Beilstein J Nanotechnol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Beilstein J Nanotechnol</journal-id><journal-title-group><journal-title>Beilstein Journal of Nanotechnology</journal-title></journal-title-group><issn pub-type=\"epub\">2190-4286</issn><publisher><publisher-name>Beilstein-Institut</publisher-name><publisher-loc>Trakehner Str. 7-9, 60487 Frankfurt am Main, Germany</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32832315</article-id><article-id pub-id-type=\"pmc\">PMC7431759</article-id><article-id pub-id-type=\"doi\">10.3762/bjnano.11.104</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Full Research Paper</subject></subj-group><subj-group subj-group-type=\"topic\"><subject>Nanoscience</subject></subj-group><subj-group subj-group-type=\"topic\"><subject>Nanotechnology</subject></subj-group></article-categories><title-group><article-title>3D superconducting hollow nanowires with tailored diameters grown by focused He<sup>+</sup> beam direct writing</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Córdoba</surname><given-names>Rosa</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-6180-8113</contrib-id><email>rosa.cordoba.castillo@gmail.com</email><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ibarra</surname><given-names>Alfonso</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-4599-3013</contrib-id><xref ref-type=\"aff\" rid=\"A2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mailly</surname><given-names>Dominique</given-names></name><xref ref-type=\"aff\" rid=\"A3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Guillamón</surname><given-names>Isabel</given-names></name><xref ref-type=\"aff\" rid=\"A4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Suderow</surname><given-names>Hermann</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-5902-1880</contrib-id><xref ref-type=\"aff\" rid=\"A4\">4</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>De Teresa</surname><given-names>José María</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0001-9566-0738</contrib-id><email>deteresa@unizar.es</email><xref ref-type=\"aff\" rid=\"A2\">2</xref><xref ref-type=\"aff\" rid=\"A5\">5</xref><xref ref-type=\"aff\" rid=\"A6\">6</xref></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Hlawacek</surname><given-names>Gregor</given-names></name><role>Guest Editor</role></contrib><contrib contrib-type=\"editor\"><name><surname>Wolff</surname><given-names>Annalena</given-names></name><role>Guest Editor</role></contrib></contrib-group><aff id=\"A1\"><label>1</label>Instituto de Ciencia Molecular, Universitat de València, Catedrático José Beltrán 2, 46980 Paterna, Spain</aff><aff id=\"A2\"><label>2</label>Laboratorio de Microscopías Avanzadas (LMA)-Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, E-50018 Zaragoza, Spain Departamento de Física de la Materia Condensada, Universidad de Zaragoza, E-50009 Zaragoza, Spain</aff><aff id=\"A3\"><label>3</label>Centre de Nanosciences et de Nanotechnologies, CNRS, Univ Paris Sud, Université Paris Saclay, 91120 Palaiseau, France</aff><aff id=\"A4\"><label>4</label>Laboratorio de Bajas Temperaturas y Altos Campos Magnéticos, Departamento de Física de la Materia Condensada, Instituto de Ciencia de Materiales Nicolás Cabrera, Condensed Matter Physics Center (IFIMAC), Universidad Autónoma de Madrid, 28049, Madrid, Spain</aff><aff id=\"A5\"><label>5</label>Instituto de Nanociencia y de Materiales de Aragón (INMA), Universidad de Zaragoza-CSIC, E-50009 Zaragoza, Spain</aff><aff id=\"A6\"><label>6</label>Departamento de Física de la Materia Condensada, Universidad de Zaragoza, E-50009 Zaragoza, Spain</aff><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><volume>11</volume><fpage>1198</fpage><lpage>1206</lpage><ext-link ext-link-type=\"doi\" xlink:href=\"10.3762/bjnano.11.104\">10.3762/bjnano.11.104</ext-link><history><date date-type=\"received\"><day>6</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>14</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Córdoba et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Córdoba et al.</copyright-holder><ali:free_to_read xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\"/><license license-type=\"Beilstein\"><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\">https://creativecommons.org/licenses/by/4.0</ali:license_ref><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\">https://www.beilstein-journals.org/bjnano/terms</ali:license_ref><license-p>This is an Open Access article under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"https://creativecommons.org/licenses/by/4.0\">https://creativecommons.org/licenses/by/4.0</ext-link>). Please note that the reuse, redistribution and reproduction in particular requires that the authors and source are credited.</license-p><license-p>The license is subject to the Beilstein Journal of Nanotechnology terms and conditions: (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.beilstein-journals.org/bjnano/terms\">https://www.beilstein-journals.org/bjnano/terms</ext-link>)</license-p></license></permissions><abstract><p>Currently, the patterning of innovative three-dimensional (3D) nano-objects is required for the development of future advanced electronic components. Helium ion microscopy in combination with a precursor gas can be used for direct writing of three-dimensional nanostructures with a precise control of their geometry, and a significantly higher aspect ratio than other additive manufacturing technologies. We report here on the deposition of 3D hollow tungsten carbide nanowires with tailored diameters by tuning two key growth parameters, namely current and dose of the ion beam. Our results show the control of geometry in 3D hollow nanowires, with outer and inner diameters ranging from 36 to 142 nm and from 5 to 28 nm, respectively; and lengths from 0.5 to 8.9 µm. Transmission electron microscopy experiments indicate that the nanowires have a microstructure of large grains with a crystalline structure compatible with the face-centered cubic WC<sub>1−</sub><italic><sub>x</sub></italic> phase. In addition, 3D electron tomographic reconstructions show that the hollow center of the nanowires is present along the whole nanowire length. Moreover, these nanowires become superconducting at 6.8 K and show high values of critical magnetic field and critical current density. Consequently, these 3D nano-objects could be implemented as components in the next generation of electronics, such as nano-antennas and sensors, based on 3D superconducting architectures.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>electron tomography</kwd><kwd>focused ion beam induced deposition (FIBID)</kwd><kwd>helium ion microscope</kwd><kwd>magneto-transport measurements</kwd><kwd>nano-superconductors</kwd><kwd>tungsten carbide (WC)</kwd></kwd-group><funding-group><funding-statement>This work was supported by the financial support from Spanish Ministry of Economy and Competitiveness through the projects MAT2017-82970-C2-2-R, PIE201760E027, including FEDER funding, FIS2017-84330-R, CEX2018-000805-M, EU ERC (Grant Agreement No. 679080), from the EU-H2020 research and innovation programme under grant agreement No 654360 NFFA-Europe, and Nanocohybri COST CA-16218, from regional Gobierno de Aragón (grants E13_20R and E28-20R) with European Social Fund (Construyendo Europa desde Aragón) and Comunidad de Madrid through project NANOMAGCOST-CM (Grant No. (S2018/NMT-4321)). The project that gave rise to these results received the support of a fellowship from ”la Caixa” Foundation (ID 100010434). The fellowship code is LCF/BQ/PR19/11700008. The French RENATECH network (French national nanofabrication platform).</funding-statement></funding-group></article-meta><notes><p>This article is part of the thematic issue \"Ten years of the helium ion microscope\".</p></notes></front><body><sec><title>Introduction</title><p>Superconductors are dissipationless carriers of electric current and provide macroscopic, and thus robust, quantum coherence. This allows for a wide range of applications, particularly at the nanometer-scale, where they can be easily integrated in circuits and used as ultrasensitive sensors of magnetic fields, temperature and as key elements for quantum computation. The behavior of nanosized superconductors as one-dimensional quantum oscillators [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>], Josephson junction arrays [<xref rid=\"R2\" ref-type=\"bibr\">2</xref>], electronic transport devices [<xref rid=\"R3\" ref-type=\"bibr\">3</xref>–<xref rid=\"R7\" ref-type=\"bibr\">7</xref>], very small-scale devices [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>–<xref rid=\"R9\" ref-type=\"bibr\">9</xref>], micrometer-scale coolers [<xref rid=\"R10\" ref-type=\"bibr\">10</xref>], or thermal and spin sensors [<xref rid=\"R11\" ref-type=\"bibr\">11</xref>–<xref rid=\"R12\" ref-type=\"bibr\">12</xref>] has been studied in detail.</p><p>Nowadays, research on manufacturing highly energy-efficient three-dimensional (3D) structures [<xref rid=\"R13\" ref-type=\"bibr\">13</xref>] is critical for the development of future electronics. However, when approaching the nanometer-scale, the number of works on real 3D nano-superconductors [<xref rid=\"R14\" ref-type=\"bibr\">14</xref>–<xref rid=\"R19\" ref-type=\"bibr\">19</xref>] decreases dramatically, mostly due to the complex fabrication and characterization. A technique successfully utilized for fabricating 3D nano-objects is direct writing by a focused beam of positively charged particles, the so-called focused-ion-beam induced deposition (FIBID) [<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]. A very promising development of FIBID is based on Ga<sup>+</sup> ions. Functional 3D nanomaterials have been grown by Ga<sup>+</sup> FIBID in the last decade [<xref rid=\"R21\" ref-type=\"bibr\">21</xref>–<xref rid=\"R26\" ref-type=\"bibr\">26</xref>]. In particular, Ga<sup>+</sup> FIBID in combination with W(CO)<sub>6</sub> as precursor material yielded 3D superconducting W-based wires with a critical temperature (<italic>T</italic><sub>c</sub>) below 5 K and a critical magnetic field (µ<sub>0</sub><italic>H</italic><sub>c2</sub>(0)) up to 9.5 T [<xref rid=\"R14\" ref-type=\"bibr\">14</xref>–<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]. Alternatively, in combination with Nb(NMe<sub>2</sub>)<sub>3</sub>(N-<italic>t</italic>-Bu), Ga<sup>+</sup> FIBID yielded NbC wires with a broadened <italic>T</italic><sub>c</sub> range from 4 to 11 K [<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]. One significant limitation is that 3D elements below 100 nm in diameter cannot be obtained with Ga<sup>+</sup> FIBID, mainly due to the relatively large Ga<sup>+</sup> beam diameter (approx. 5 nm) and a high proximity effect generated by Ga<sup>+</sup> ion scattering.</p><p>Regarding a higher spatial resolution, the helium ion microscope (HIM) [<xref rid=\"R27\" ref-type=\"bibr\">27</xref>], based on a gas field-ionization source, has emerged as a tool for direct writing of complex 3D nano-objects taking advantage of its small beam diameter (approx. 0.3 nm) and low proximity effect [<xref rid=\"R28\" ref-type=\"bibr\">28</xref>]. When precursor molecules from the gas phase are adsorbed on a substrate surface, He<sup>+</sup> FIB dissociates them into non-volatile and volatile products. The non-volatile products attach to the surface, resulting in a deposit, whereas the volatile products ones are pumped out of the process chamber. Normally, the final deposit is a mixture of carbon, metallic elements and oxygen. As clearly described using analytical modelling [<xref rid=\"R29\" ref-type=\"bibr\">29</xref>] and Monte-Carlo simulations [<xref rid=\"R30\" ref-type=\"bibr\">30</xref>], the vertical growth of 3D nano-objects by He<sup>+</sup> FIBID is mainly caused by secondary electrons of the first order produced from the primary ion beam, whereas the lateral growth is induced by secondary electrons of the second order generated from scattered ions. Thus, the direct contribution of the primary He<sup>+</sup> ion beam and the scattered He<sup>+</sup> ions is almost negligible for the growth of these 3D nano-objects. Nevertheless, it is worth mentioning that its resolution, volume per dose and throughput are very sensitive to the selected growth conditions such as ion beam energy, ion beam current, precursor flux, surface interactions with the beam, and precursor molecules [<xref rid=\"R29\" ref-type=\"bibr\">29</xref>–<xref rid=\"R30\" ref-type=\"bibr\">30</xref>]. Hence, the He<sup>+</sup> FIBID technique is highly recommended for direct writing of 3D nano-objects with high resolution and aspect ratio [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>,<xref rid=\"R19\" ref-type=\"bibr\">19</xref>,<xref rid=\"R31\" ref-type=\"bibr\">31</xref>–<xref rid=\"R35\" ref-type=\"bibr\">35</xref>]. A successful example of tailored 3D nano-objects grown by He<sup>+</sup> FIBID has been reported by Kohama and co-workers [<xref rid=\"R35\" ref-type=\"bibr\">35</xref>]. The authors deposited W-based pillars with diameters down to approx. 40 nm and aspect ratios of approx. 50. The microstructure of the grown material consisted of fcc WC<sub>1−</sub><italic><sub>x</sub></italic> and W<sub>2</sub>(C,O) grains. Moreover, when the He<sup>+</sup> beam was well focused the authors observed columnar voids created at the center of the pillars with a diameter ranging from 1 to 15 nm, showing the path to build complex 3D nano-objects beyond standard nanowires (NWs). Recent breakthroughs in the growth of 3D WC superconducting nano-objects with extremely large aspect ratios using He<sup>+</sup> FIBID have been reported by some of the authors, such as hollow NW-like nanotubes as small as 32 nm in diameter [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>] and nanohelices with controllable geometries, including the smallest and most densely packed nanohelix to date with a diameter of 100 nm [<xref rid=\"R19\" ref-type=\"bibr\">19</xref>].</p><p>In this work, we present the direct writing of 3D WC crystalline superconducting hollow NWs with tailored diameters grown using a HIM. The hollow NW geometry is successfully controlled by tuning the ion beam current and dose from 0.65 to 7 pA and from 0.1 to 0.4 nC, respectively, resulting in NWs with outer diameters from 36 to 142 nm and with inner diameters from 5 to 28 nm, and total length from 0.5 to 8.9 µm (aspect ratio ≈ 196). These values are significantly better than those reported in previous works [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>,<xref rid=\"R35\" ref-type=\"bibr\">35</xref>]. The NWs microstructure consists of large grains of fcc WC<sub>1−</sub><italic><sub>x</sub></italic>, in good agreement with [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>,<xref rid=\"R35\" ref-type=\"bibr\">35</xref>]. In addition, the NWs are hollow along the whole NW length, which could make them nonconventional nanopipettes, as demonstrated in 3D reconstructions of electron tomography experiments. Finally, these 3D hollow NWs exhibit superconductivity below 6.8 K (<italic>T</italic><sub>c</sub>), as well as high upper critical magnetic fields µ<sub>0</sub><italic>H</italic><sub>c2</sub> ≈ 14.7 T, and large critical current densities <italic>J</italic><sub>c</sub> ≈ 0.15 MA/cm<sup>2</sup>.</p></sec><sec><title>Results and Discussion</title><sec><title>Growth of 3D hollow nanowires by He<sup>+</sup> FIBID</title><p>We use a HIM in combination with a W(CO)<sub>6</sub> precursor to grow individual, out-of-plane WC NWs in a single step, controlling inner and outer diameter and total length. The precursor gas is delivered to the process chamber and adsorbed onto the substrate surface, while the He<sup>+</sup> FIB spot remains fixed during the deposition favoring continuous vertical growth along [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>].</p><sec><title>Dimensional control for nanowires</title><p>We investigated the dimensional control for these NWs by optimizing in the deposition the following parameters: the ion beam current and ion dose. SEM images of typical NWs grown with ion beam currents ranging from 0.54 to 6.47 pA and doses from 0.1 to 1.4 nC are depicted in <xref ref-type=\"fig\" rid=\"F1\">Figure 1a</xref>–d.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>(a–d) SEM images of hollow NWs grown by He<sup>+</sup> FIBID; (a) ion beam current: 0.54 pA, dose: 0.1–0.3 nC from right to left; (b) ion beam current: 1.17 pA, dose: 0.1–0.6 nC from left to right and upwards; (c) ion beam current: 2.91 pA, dose: 0.1–0.6 nC from left to right and upwards; (d) ion beam current: 6.47 pA, dose: 0.229, 0.456, 0.696, 0.935, 1.158, and 1.402 nC from left to right and upwards. (e) NW volume in (a) as a function of the ion beam dose.</p></caption><graphic xlink:href=\"Beilstein_J_Nanotechnol-11-1198-g002\"/></fig><p>Varying these parameters enables us to fabricate 3D NWs with diameters ranging from 45 to 125 nm, lengths ranging from 0.5 to 8.9 µm, and with aspect ratios up to 198. Further details regarding the growth conditions are described in the Experimental section. We found a linear dependence of the NW volume (determined as π × (outer diameter/2 – inner diameter/2)<sup>2</sup> × height) as a function of the ion dose for the mentioned ion beam currents (<xref ref-type=\"fig\" rid=\"F1\">Figure 1e</xref>). Moreover, we noted that the NW volume rapidly decreases as a function of the ion beam current for the same dose (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). When using high currents several effects can play a role in this dependence such as precursor depletion, local heating, which decreases the precursor molecule sticking coefficient, and low precursor diffusion from the substrate to the top of the pillar [<xref rid=\"R36\" ref-type=\"bibr\">36</xref>–<xref rid=\"R37\" ref-type=\"bibr\">37</xref>]. This shows the need for future systematic experiments varying the dwell time in pulsed growth or varying the flux of precursor gas.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>NW volume for a specific dose, as a function of the ion beam current.</p></caption><graphic xlink:href=\"Beilstein_J_Nanotechnol-11-1198-g003\"/></fig></sec></sec><sec><title>(High-resolution) scanning transmission electron microscopy</title><sec><title>Dependence of NW inner diameter on the ion beam current</title><p>To investigate the dependence of the NW inner diameter on the ion beam current, scanning transmission electron microscopy (STEM) experiments were performed. We found that inner diameter of the hollow NWs changes from 5 to 28 nm, whereas the outer diameter changes from 36 to 143 nm upon increasing the ion beam current from 1.3 to 7 pA. STEM images of these hollow NWs are shown in <xref ref-type=\"fig\" rid=\"F3\">Figure 3a</xref>. The observed non-uniform shape of the cavity in the central nanowire could be explained by several reasons, such as He<sup>+</sup> FIB instability or irregular substrate surface. We find a linear dependence of the inner diameter on the ion beam current (<xref ref-type=\"fig\" rid=\"F3\">Figure 3b</xref>), which indicates that the ion beam forms the cavity due to a milling effect. Thus via tuning the ion beam current and dose we have full control to tailor the diameters of the hollow 3D NWs. The specific deposition parameters and NW diameters are listed in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>.</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>(a) STEM images of hollow NWs grown at 1.3, 2.3, and 7 pA, from left to right. (b) NW diameter in (a) as a function of the ion beam current.</p></caption><graphic xlink:href=\"Beilstein_J_Nanotechnol-11-1198-g004\"/></fig><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Growth parameters and diameters of hollow WC NWs.</p></caption><table frame=\"hsides\" rules=\"groups\"><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">hollow nanowire type</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td align=\"left\" colspan=\"4\" valign=\"top\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ion beam current (pA)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.3</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2.3</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">7.0</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">outer diameter (nm)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">71</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">142</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">inner diameter (nm)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">28</td></tr></table></table-wrap></sec><sec><title>Electron tomography</title><p>In order to further examine the NW diameters along their length, electron tomography experiments on two specific NWs were carried out. <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref> shows the tomographic reconstruction of hollow WC NWs grown at (a) 7 pA and (b) 2 pA. One can see from the images that the cavities are present up to the tip of the NW. On the left panel of <xref ref-type=\"fig\" rid=\"F4\">Figure 4a</xref>, a STEM image of the NW with outer and inner diameter of 142 and 28 nm, respectively, is shown. On the right panel, a snapshot of the colored 3D tomographic reconstruction is depicted. <xref ref-type=\"fig\" rid=\"F4\">Figure 4b</xref> shows a STEM image of the NW with outer and inner diameter of 77 and 8 nm, respectively, on the left panel, and a snapshot of the colored 3D tomographic reconstruction is displayed on the right panel. Three movies of the tomographic reconstruction for each hollow NW are added in <xref ref-type=\"supplementary-material\" rid=\"SD1\">Supporting Information File 1</xref>–7, including a transversal (<italic>x</italic>–<italic>y</italic>) and a longitudinal (<italic>y</italic>–<italic>z</italic>) section, and a colored three-dimensional reconstruction.</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>(a) Tomography of a hollow WC NW grown at 7 pA, with an outer diameter of 142 nm and an inner diameter of 28 nm; left panel: STEM image, right panel: snapshot of the 3D tomographic reconstruction. (b) Tomography of a hollow WC NW grown at 2 pA, with an outer diameter of 77 nm and inner diameter of 8 nm; left panel: STEM image, right panel: snapshot of the 3D tomographic reconstruction.</p></caption><graphic xlink:href=\"Beilstein_J_Nanotechnol-11-1198-g005\"/></fig></sec><sec><title>Microstructure</title><p>Concerning the microstructure of the NWs, high-resolution scanning transmission electron microscopy (HRSTEM) images have been acquired sequentially and processed to extract the crystallographic structure (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>). We indexed the spots indicated in the fast Fourier transform (<xref ref-type=\"fig\" rid=\"F5\">Figure 5b</xref>) of the image in <xref ref-type=\"fig\" rid=\"F5\">Figure 5a</xref> with the planes (−11−1), (−200) and (−1−11), and the [011] zone axis of the WC<sub>1−</sub><italic><sub>x</sub></italic> fcc structure, with a lattice parameter of <italic>a</italic> = 0.4272 nm. A lower magnification STEM image of the NW grown at 1.3 pA is depicted in <xref ref-type=\"fig\" rid=\"F5\">Figure 5c</xref>. These results are in good agreement with the previous work reported by some of the authors [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>].</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>(a) HRSTEM image of a typical hollow WC NW grown at 1.3 pA. (b) Fast Fourier transform of the image in (a), showing the crystalline nature of the material and indexed as the [011] zone axis of the fcc WC<sub>1−</sub><italic><sub>x</sub></italic> structure. (c) Lower magnification STEM image of the WC NW in (a).</p></caption><graphic xlink:href=\"Beilstein_J_Nanotechnol-11-1198-g006\"/></fig></sec><sec><title>Magneto-electrical-transport study</title><p>To determine the critical superconducting parameters in NWs grown at 0.65, 1.3, and 2.18 pA (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref> and <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>), a magneto-electrical transport study using the typical four-point-probe configuration has been performed. Following the procedure described in [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>], first 3D NWs were placed flat on the SiO<sub>2</sub> layer of a Si/SiO<sub>2</sub> substrate by means of a nano-manipulator. Then, four Pt FIBID contacts were grown to connect the NWs to pre-patterned Ti pads. Finally, we made four-point-probe electrical measurements at low temperature (down to 0.5 K) and under a magnetic field perpendicular to the substrate plane (up to 9 T).</p><fig id=\"F6\" position=\"float\"><label>Figure 6</label><caption><p>(a) Normalized resistance as a function of the temperature of hollow NWs grown using ion beam currents indicated in the legend. <italic>R</italic><sub>N</sub> is the resistance for the normal state at <italic>T</italic> = 10 K. The inset shows the resistance as function of the temperature (full range) for a NW grown at 0.65 pA, <italic>I</italic><sub>bias</sub> = 100 nA. (b) Upper critical magnetic field (µ<sub>0</sub><italic>H</italic><sub>c2</sub>) as a function of the temperature of NWs grown using ion beam currents indicated in the legend. Data is fitted to a power-law equation. The inset shows the resistance as a function of the temperature for a NW grown at 0.65 pA under perpendicular magnetic fields from 0 to 9 T.</p></caption><graphic xlink:href=\"Beilstein_J_Nanotechnol-11-1198-g007\"/></fig><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Superconducting parameters of NWs estimated from experimental magneto-transport measurements.</p></caption><table frame=\"hsides\" rules=\"groups\"><tr><td align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">hollow nanowire type</td><td align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">3</td></tr><tr><td align=\"left\" colspan=\"4\" valign=\"middle\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ion beam current (pA)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.65</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1.30</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2.18</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">outer diameter (nm)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">41</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">72</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><italic>R</italic><sub>N</sub> (Ω)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1959</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2101</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">430</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><italic>T</italic><sub>c</sub> (K)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">6.78</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">6.16</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">5.45</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">µ<sub>0</sub><italic>H</italic><sub>c2</sub> (0 K) (<italic>T</italic>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">14.66</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">14.48</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><italic>J</italic><sub>c</sub> (0 T) (MA/cm<sup>2</sup>)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.083 (0.6 K)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.151 (0.6 K)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.026 (1 K)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">ξ (0 K) (nm)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4.74</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4.77</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">λ (0 K) (nm)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">839</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">720</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">874</td></tr></table></table-wrap><p>The NWs change from the normal to the superconducting state at <italic>T</italic><sub>c</sub> (0.5<italic>R</italic><sub>N</sub>) values between 5.45 and 6.78 K (<xref ref-type=\"fig\" rid=\"F6\">Figure 6a</xref> and <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). No clear trend was visible in <italic>T</italic><sub>c</sub> values for NWs grown using different currents, although the identified <italic>T</italic><sub>c</sub> range is in good agreement with the previously reported results [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>]. Also, it is up to 1.5 times higher than that of Ga<sup>+</sup> FIBID nanostructures of similar dimensions [<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]. The inset of <xref ref-type=\"fig\" rid=\"F6\">Figure 6a</xref> shows the measured resistance as a function of the temperature in the full temperature range investigated for a NW grown at 0.65 pA. The value of µ<sub>0</sub><italic>H</italic><sub>c2</sub> as a function of the temperature for NWs grown at 0.65 and 1.3 pA is depicted in <xref ref-type=\"fig\" rid=\"F6\">Figure 6b</xref>. The values of µ<sub>0</sub><italic>H</italic><sub>c2</sub> (0.9<italic>R</italic><sub>N</sub>) are extracted from the resistance-vs-temperature curves under perpendicular magnetic field (inset of <xref ref-type=\"fig\" rid=\"F6\">Figure 6b</xref>). By fitting µ<sub>0</sub><italic>H</italic><sub>c2</sub>(<italic>T</italic>) to a power-law equation µ<sub>0</sub><italic>H</italic><italic><sub>c</sub></italic><sub>2</sub>(<italic>T</italic>) ∝ (1 –<italic>T</italic>/<italic>T</italic><sub>c</sub>)<italic><sup>n</sup></italic>, µ<sub>0</sub><italic>H</italic><sub>c2</sub>(0 K) is estimated to be approx. 14.5 T for the different NWs. The coherence length, ξ, at 0 K is around 4.77 nm and the estimated magnetic field penetration depth, λ, [<xref rid=\"R38\" ref-type=\"bibr\">38</xref>–<xref rid=\"R39\" ref-type=\"bibr\">39</xref>] ranges from 720 to 874 nm. Additionally, <italic>J</italic><sub>c</sub> (0.6 K, 0 T) is approx. 0.15 MA/cm<sup>2</sup>.</p><p>Summarizing, the estimated superconducting parameters (<italic>T</italic><sub>c</sub>, µ<sub>0</sub><italic>H</italic><sub>c2</sub>, <italic>J</italic><sub>c</sub>, ξ, λ) for these NWs (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>) are mostly compatible with those reported for He<sup>+</sup> FIBID out-of-plane WC nanotubes [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>], nanohelices [<xref rid=\"R19\" ref-type=\"bibr\">19</xref>], and in-plane NWs used in hybrid microwave resonators [<xref rid=\"R40\" ref-type=\"bibr\">40</xref>]. They are potential building blocks for highly packed 3D nano-resonators, superconducting logic gates [<xref rid=\"R41\" ref-type=\"bibr\">41</xref>], quantum switches [<xref rid=\"R42\" ref-type=\"bibr\">42</xref>], and single-photon detectors [<xref rid=\"R43\" ref-type=\"bibr\">43</xref>–<xref rid=\"R45\" ref-type=\"bibr\">45</xref>].</p></sec></sec></sec><sec><title>Conclusion</title><p>We report a direct writing methodology to create 3D superconducting hollow NWs with tailored diameters using W(CO)<sub>6</sub> precursor with a highly focused He<sup>+</sup> beam. The resulting 3D hollow NWs have inner and outer diameters from 5 to 28 nm and from 36 to 142 nm, respectively, and aspect ratios above 196, which is unachievable by other additive manufacturing methods. The electron tomography study proved that the center hole is present along the whole length of the NWs.</p><p>As expected, the microstructure corresponds to the fcc WC<sub>1−</sub><italic><sub>x</sub></italic> phase. By studying their magnetotransport properties, we found <italic>T</italic><sub>c</sub> ≈ 6.8 K, as well as µ<sub>0</sub><italic>H</italic><sub>c2</sub> ≈ 14.7 T and <italic>J</italic><sub>c</sub> ≈ 0.15 MA/cm<sup>2</sup>. The presented methodology yields an advanced bottom-up approach for the fabrication of innovative 3D nano-architectures, in which nano-superconductivity may provide an advantage, for future electronic components, particularly for sensors, energy-storage components, and quantum computing.</p></sec><sec><title>Experimental</title><sec><title>Growth of 3D hollow WC nanowires</title><p>He<sup>+</sup> FIBID hollow WC NWs have been fabricated in a ZEISS ORION NanoFab instrument equipped with a helium ion beam column and a single-needle gas injection system (GIS) through which W(CO)<sub>6</sub> gas is delivered to the process chamber.</p><p>The NWs were deposited on top of the pre-patterned Ti pads (150 nm in thickness) to prevent charge effects on the insulator layer (250 nm thick of SiO<sub>2</sub>) thermally grown on a silicon wafer [<xref rid=\"R23\" ref-type=\"bibr\">23</xref>]. These chips were fabricated following a routine recipe for UV optical lithography using a lift-off method. For the electron tomography and (HR)STEM experiments, NWs were directly grown on Cu TEM grids. Typical deposition conditions used for the He<sup>+</sup> FIBID process were as follows; precursor material: tungsten hexacarbonyl, W(CO)<sub>6</sub>; <italic>T</italic><sub>precursor</sub> = 55 °C; GIS<sub>needle diameter</sub> ≈ 500 µm; GIS<italic><sub>z</sub></italic> ≈ 500 µm; GIS<italic><sub>x,y</sub></italic> ≈ 60 µm; <italic>P</italic><sub>base</sub> ≈ 3 × 10<sup>−7</sup> mbar; <italic>P</italic><sub>process</sub> ≈ 4 × 10<sup>−6</sup> mbar; acceleration voltage = 30 kV; pattern shape: spot mode; ion beam current range of 0.54 to 6.47 pA and dose range of 0.1 to 1.4 nC.</p></sec><sec><title>Microstructure and tomography at the nanometer-scale</title><p>Scanning transmission electron microscopy (STEM) imaging and EDS were carried out in a probe-corrected FEI Titan 60–300 operated at 300 kV and equipped with a high-brightness X-FEG and a CETCOR <italic>C</italic><sub>s</sub> corrector for the condenser system to provide sub-angstrom probe size.</p><p>STEM high-angle annular dark-field (HAADF) tomography was performed using a Thermo Fisher Tecnai field-emission gun operated at 300 kV. The angular range for the tilt series was ±70° with pictures taken every 1°. Image alignment and 3D reconstruction was carried out with FEI tomography acquisition software Inspect 3D after the acquisition of 140 images. The movies of the tomographic reconstruction for each hollow NW were performed using Amira 3D software.</p></sec><sec><title>Magneto-electrical transport study</title><p>The magneto-electrical transport measurements on the NWs were carried out using a ''Physical Property Measurement System'' (PPMS), from Quantum Design equipped with a helium-3 refrigerator insert.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supporting Information</title><p>Movies of 3D tomographic reconstruction.</p><supplementary-material content-type=\"local-data\" id=\"SD1\"><label>File 1</label><caption><p>Electron tomography_3D reconstruction_hollow NW grown at 2 pA and 0.6 nC.</p></caption><media mime-subtype=\"x-ms-wmv\" mimetype=\"video\" xlink:href=\"Beilstein_J_Nanotechnol-11-1198-s001.wmv\" xlink:type=\"simple\" id=\"d39e1151\" position=\"anchor\"/></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SD2\"><label>File 2</label><caption><p>Electron tomography_3D longitudinal_hollow NW grown at 2 pA and 0.6 nC.</p></caption><media mime-subtype=\"x-ms-wmv\" mimetype=\"video\" xlink:href=\"Beilstein_J_Nanotechnol-11-1198-s002.wmv\" xlink:type=\"simple\" id=\"d39e1158\" position=\"anchor\"/></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SD3\"><label>File 3</label><caption><p>Electron tomography_longitudinal section_hollow NW grown at 2 pA and 0.6 nC.</p></caption><media mime-subtype=\"x-ms-wmv\" mimetype=\"video\" xlink:href=\"Beilstein_J_Nanotechnol-11-1198-s003.wmv\" xlink:type=\"simple\" id=\"d39e1165\" position=\"anchor\"/></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SD4\"><label>File 4</label><caption><p>Electron tomography_transversal section_hollow NW grown at 2 pA and 0.6 nC.</p></caption><media mime-subtype=\"x-ms-wmv\" mimetype=\"video\" xlink:href=\"Beilstein_J_Nanotechnol-11-1198-s004.wmv\" xlink:type=\"simple\" id=\"d39e1172\" position=\"anchor\"/></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SD5\"><label>File 5</label><caption><p>Electron tomography_3D reconstruction_hollow NW grown at 7 pA and 1.009 nC.</p></caption><media mime-subtype=\"x-ms-wmv\" mimetype=\"video\" xlink:href=\"Beilstein_J_Nanotechnol-11-1198-s005.wmv\" xlink:type=\"simple\" id=\"d39e1179\" position=\"anchor\"/></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SD6\"><label>File 6</label><caption><p>Electron tomography_3D longitudinal_hollow NW grown at 7 pA and 1.009 nC.</p></caption><media mime-subtype=\"x-ms-wmv\" mimetype=\"video\" xlink:href=\"Beilstein_J_Nanotechnol-11-1198-s006.wmv\" xlink:type=\"simple\" id=\"d39e1186\" position=\"anchor\"/></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SD7\"><label>File 7</label><caption><p>Electron tomography_transversal section_hollow NW grown at 7 pA and 1.009 nC.</p></caption><media mime-subtype=\"x-ms-wmv\" mimetype=\"video\" xlink:href=\"Beilstein_J_Nanotechnol-11-1198-s007.wmv\" xlink:type=\"simple\" id=\"d39e1193\" position=\"anchor\"/></supplementary-material></sec></body><back><ack><p>The authors highly acknowledge Rubén Valero for the UV lithography process. The microscopy works have been conducted in the “Laboratorio de Microscopías Avanzadas” at “Instituto de Nanociencia de Aragón - Universidad de Zaragoza”. The authors acknowledge the LMA-INA for offering access to their instruments and expertise. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"letter\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Beilstein J Org Chem</journal-id><journal-id journal-id-type=\"iso-abbrev\">Beilstein J Org Chem</journal-id><journal-title-group><journal-title>Beilstein Journal of Organic Chemistry</journal-title></journal-title-group><issn pub-type=\"epub\">1860-5397</issn><publisher><publisher-name>Beilstein-Institut</publisher-name><publisher-loc>Trakehner Str. 7-9, 60487 Frankfurt am Main, Germany</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32831954</article-id><article-id pub-id-type=\"pmc\">PMC7431760</article-id><article-id pub-id-type=\"doi\">10.3762/bjoc.16.164</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Letter</subject></subj-group><subj-group subj-group-type=\"topic\"><subject>Chemistry</subject><subj-group subj-group-type=\"topic\"><subject>Organic Chemistry</subject></subj-group></subj-group></article-categories><title-group><article-title>Metal-free synthesis of phosphinoylchroman-4-ones via a radical phosphinoylation–cyclization cascade mediated by K<sub>2</sub>S<sub>2</sub>O<sub>8</sub></article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Liu</surname><given-names>Qiang</given-names></name><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0001-8342-712X</contrib-id><xref ref-type=\"aff\" rid=\"A1\">1</xref><xref ref-type=\"aff\" rid=\"A2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lu</surname><given-names>Weibang</given-names></name><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Xie</surname><given-names>Guanqun</given-names></name><email>2018008@dgut.edu.cn</email><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Xiaoxia</given-names></name><xref ref-type=\"aff\" rid=\"A1\">1</xref></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Nay</surname><given-names>Bastien</given-names></name><role>Associate Editor</role></contrib></contrib-group><aff id=\"A1\"><label>1</label>Dongguan University of Technology, Dongguan 523808, P. R. China</aff><aff id=\"A2\"><label>2</label>Department of Applied Chemistry, School of Science, Xi’an Jiaotong University, Xi’an 710049, P. R. China</aff><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>12</day><month>8</month><year>2020</year></pub-date><volume>16</volume><fpage>1974</fpage><lpage>1982</lpage><ext-link ext-link-type=\"doi\" xlink:href=\"10.3762/bjoc.16.164\">10.3762/bjoc.16.164</ext-link><history><date date-type=\"received\"><day>9</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>31</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Liu et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Liu et al.</copyright-holder><ali:free_to_read xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\"/><license license-type=\"Beilstein\"><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\">https://creativecommons.org/licenses/by/4.0</ali:license_ref><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\">https://www.beilstein-journals.org/bjoc/terms</ali:license_ref><license-p>This is an Open Access article under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"https://creativecommons.org/licenses/by/4.0\">https://creativecommons.org/licenses/by/4.0</ext-link>). Please note that the reuse, redistribution and reproduction in particular requires that the authors and source are credited.</license-p><license-p>The license is subject to the Beilstein Journal of Organic Chemistry terms and conditions: (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.beilstein-journals.org/bjoc/terms\">https://www.beilstein-journals.org/bjoc/terms</ext-link>)</license-p></license></permissions><abstract><p>A variety of chroman-4-ones bearing phosphine oxide motifs were conveniently synthesized from readily available diphenylphosphine oxides and alkenyl aldehydes via a metal-free tandem phosphinoylation/cyclization protocol. The reaction utilizes K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> as oxidant and proceeds in DMSO/H<sub>2</sub>O at environmentally benign conditions with a broad substrate scope and afforded the title compounds in moderate yields.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>chroman-4-ones</kwd><kwd>diphenylphosphine oxide</kwd><kwd>metal-free</kwd><kwd>potassium persulfate</kwd><kwd>radical cyclization</kwd></kwd-group><funding-group><funding-statement>We are grateful for the financial supports from the Science Foundation for Distinguished Scholars of Dongguan University of Technology (No. GC200906-10) and the Postdoctoral Foundation of Dongguan University of Technology (No. 196100040006).</funding-statement></funding-group></article-meta></front><body><sec><title>Introduction</title><p>The chroman-4-one framework is a privileged structural motif found in numerous natural products and pharmaceuticals with extraordinary biological and pharmaceutical activities such as anticancer activities and anti-HIV activity among others (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>) [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]. Therefore, the preparation of functionalized chroman-4-one derivatives has attracted great attention of experts and scientists in the field of organic synthesis and pharmaceutical sciences over the last few years [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R4\" ref-type=\"bibr\">4</xref>–<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]. In general, chroman-4-one derivatives could be obtained via a polarity reversal strategy enabled by the N-heterocyclic carbene (NHC)-catalyzed intramolecular Stetter reaction [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>–<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]. Besides, chroman-4-one derivatives were also constructed via intramolecular oxa-Michael additions of 2’-hydroxychalcones [<xref rid=\"R9\" ref-type=\"bibr\">9</xref>–<xref rid=\"R10\" ref-type=\"bibr\">10</xref>], or through condensation cyclization reactions of <italic>o</italic>-hydroxyacetophenones with ketones/aldehydes [<xref rid=\"R11\" ref-type=\"bibr\">11</xref>–<xref rid=\"R12\" ref-type=\"bibr\">12</xref>], in addition to other alternative transformations [<xref rid=\"R13\" ref-type=\"bibr\">13</xref>–<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]. Moreover, radical cascade cyclizations of <italic>o</italic>-allyloxybenzaldehydes by employing appropriate radical precursors through visible-light promoted systems [<xref rid=\"R15\" ref-type=\"bibr\">15</xref>–<xref rid=\"R16\" ref-type=\"bibr\">16</xref>], transition-metal-catalyzed systems [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>–<xref rid=\"R18\" ref-type=\"bibr\">18</xref>], or transition-metal-free systems [<xref rid=\"R19\" ref-type=\"bibr\">19</xref>–<xref rid=\"R20\" ref-type=\"bibr\">20</xref>], have emerged as a powerful strategy for the synthesis of diversely functionalized chroman-4-one derivatives.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Biologically active compounds featuring the chroman-4-one framework.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1974-g002\"/></fig><p>Organophosphorus compounds are well-known for their medicinal, biological, or specific material-related properties and have found wide applications in pharmaceutical chemistry, biochemistry, and materials science [<xref rid=\"R21\" ref-type=\"bibr\">21</xref>–<xref rid=\"R26\" ref-type=\"bibr\">26</xref>]. They represent also excellent ligands for many metals and have been used in catalytic systems for a huge number of organic reactions [<xref rid=\"R21\" ref-type=\"bibr\">21</xref>–<xref rid=\"R26\" ref-type=\"bibr\">26</xref>]. Due to the importance of the chroman-4-one scaffold on one hand and that of organophosphorus compounds on the other, the development of concise and efficient approaches for the synthesis of chroman-4-one derivatives decorated with phosphorus functionalities [<xref rid=\"R27\" ref-type=\"bibr\">27</xref>–<xref rid=\"R28\" ref-type=\"bibr\">28</xref>], thus combining the characteristics of both components in one molecule may find useful applications. However, there are only few ways to prepare such compounds. For example, in 2008 Rovis et al. [<xref rid=\"R27\" ref-type=\"bibr\">27</xref>] reported an intramolecular Stetter reaction of alkenyl aldehydes to synthesize a series of phosphine oxide and phosphonate-functionalized chroman-4-ones. Unfortunately, the preparation of the substrates involved a Rh-catalyzed hydrophosphinylation of a protected functional alkyne, and the subsequent deprotection with Hg(O<sub>2</sub>CCF<sub>3</sub>)<sub>2</sub>, which is not environmentally benign (<xref ref-type=\"fig\" rid=\"C1\">Scheme 1a</xref>). Besides, in 2016 Li’s group [<xref rid=\"R28\" ref-type=\"bibr\">28</xref>] reported a silver-catalyzed straightforward approach for the synthesis of phosphonate-functionalized chroman-4-ones via a phosphoryl radical-initiated cascade cyclization of 2-(allyloxy)arylaldehydes using K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> as an oxidant, however, diphenylphosphine oxide (DPPO) was not suitable for the transformation (<xref ref-type=\"fig\" rid=\"C1\">Scheme 1b</xref>). So the development of metal-free and greener methods to approach chroman-4-ones bearing a phosphine oxide moiety is still highly desirable. Herein, we present a transition-metal-free radical cascade cyclization to access the desired chroman-4-one derivatives in one pot under environmentally benign conditions (<xref ref-type=\"fig\" rid=\"C1\">Scheme 1c</xref>).</p><fig id=\"C1\" position=\"float\"><label>Scheme 1</label><caption><p>Methods to produce phosphonate-substituted chroman-4-ones.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1974-g004\"/></fig></sec><sec><title>Results and Discussion</title><p>Motivated by the desire to develop a metal-free and environmentally benign protocol for the construction of phosphine oxide-functionalized chroman-4-ones, we focused on the cascade cyclization employing 2-(allyloxy)benzaldehyde (<bold>1a</bold>) and diphenylphosphine oxide (DPPO, <bold>2a</bold>) as the model substrates with K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> as the oxidant, which is a cheap, readily available, and versatile oxidant. On the basis of literature reports [<xref rid=\"R29\" ref-type=\"bibr\">29</xref>–<xref rid=\"R30\" ref-type=\"bibr\">30</xref>] and our continuing interest in green chemistry [<xref rid=\"R31\" ref-type=\"bibr\">31</xref>–<xref rid=\"R32\" ref-type=\"bibr\">32</xref>], we set the temperature at 70 °C based on the fact that K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> thermally decomposes forming sulfate radicals (SO<sub>4</sub><sup>·−</sup>) [<xref rid=\"R29\" ref-type=\"bibr\">29</xref>–<xref rid=\"R30\" ref-type=\"bibr\">30</xref>], which may react with the substrates to furnish such a cascade cyclization. To our delight, the anticipated product <bold>3aa</bold> was obtained in 42% yield in DMSO/H<sub>2</sub>O (4:1) as reaction medium in one pot (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, entry 1). The structure of <bold>3aa</bold> was unambiguously confirmed by X-ray diffraction analysis of a single crystal (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>) and by NMR spectroscopy (see <xref ref-type=\"supplementary-material\" rid=\"SD1\">Supporting Information File 1</xref>) [<xref rid=\"R33\" ref-type=\"bibr\">33</xref>]. The increase of the amount of K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> to 3 equiv resulted in the improvement of the yield of <bold>3aa</bold> to 52% (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, entry 2). However, adjusting the amount of the oxidant K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> to 4 equiv (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, entry 3) did not further improve the yield. By further screening of a few solvents, such as MeCN/H<sub>2</sub>O, DMF/H<sub>2</sub>O, DMA/H<sub>2</sub>O, dioxane/H<sub>2</sub>O, THF/H<sub>2</sub>O, EtOH/H<sub>2</sub>O, DCE/H<sub>2</sub>O, and NMP/H<sub>2</sub>O, it turned out that the highest yield was achieved in the DMSO/H<sub>2</sub>O (4:1) system (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, entries 4–13). It is notable that product <bold>3aa</bold> was not observed at room temperature or in the absence of K<sub>2</sub>S<sub>2</sub>O<sub>8</sub>, indicating that the reaction was mainly mediated by K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, entries 14 and 15). Increasing the reaction temperature to 80 °C afforded better product yields as compared with the reactions performed at either 70 °C or 90 °C (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, entries 2, 16, and 17). Then, various oxidants such as (NH<sub>4</sub>)<sub>2</sub>S<sub>2</sub>O<sub>8</sub>, Na<sub>2</sub>S<sub>2</sub>O<sub>8</sub>, TBHP (<italic>tert</italic>-butyl hydroperoxide), DTBP (di-<italic>tert</italic>-butyl peroxide), and dioxygen were tested and the results showed that K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> exhibited the best efficiency (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, entries 18–22).</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Optimization of the reaction conditions.<sup>a</sup></p></caption><table frame=\"hsides\" rules=\"groups\"><tr><td align=\"center\" colspan=\"5\" valign=\"middle\" rowspan=\"1\"><inline-graphic xlink:href=\"Beilstein_J_Org_Chem-16-1974-i001.jpg\"/></td></tr><tr><td align=\"center\" colspan=\"5\" valign=\"middle\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Entry</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Oxidant</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Solvent</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Temp. (°C)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Yield (%)<sup>b</sup></td></tr><tr><td align=\"left\" colspan=\"5\" valign=\"top\" rowspan=\"1\"><hr/></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (2.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMSO/H<sub>2</sub>O (4:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">42</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMSO/H<sub>2</sub>O (4:1v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">52</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (4.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMSO/H<sub>2</sub>O (4:1v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">50</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">MeCN/H<sub>2</sub>O (4:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">32</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMF/H<sub>2</sub>O (4:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">24</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMA/H<sub>2</sub>O (4:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">dioxane/H<sub>2</sub>O (4:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">trace</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">THF/H<sub>2</sub>O (4:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">trace</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">EtOH/H<sub>2</sub>O (4:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">trace</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DCE/H<sub>2</sub>O (4:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">trace</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">NMP/H<sub>2</sub>O (4:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">18</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMSO/H<sub>2</sub>O (1:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">32</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMSO/H<sub>2</sub>O (8:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">44</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">–</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMSO/H<sub>2</sub>O (4:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMSO/H<sub>2</sub>O (4:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">rt<sup>c</sup></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMSO/H<sub>2</sub>O (4:1v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">80</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">58</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMSO/H<sub>2</sub>O (4:1v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">90</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">54</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">(NH<sub>4</sub>)<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMSO/H<sub>2</sub>O (4:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">80</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">40</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">19</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Na<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMSO/H<sub>2</sub>O (4:1v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">80</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">50</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DTBP (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMSO/H<sub>2</sub>O (4:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">80</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">TBHP (3.0 equiv)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMSO/H<sub>2</sub>O (4:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">80</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">O<sub>2</sub></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">DMSO/H<sub>2</sub>O (4:1 v/v)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">80</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0</td></tr></table><table-wrap-foot><fn id=\"TFN1\"><p><sup>a</sup>Reaction conditions: <bold>1a</bold> (0.3 mmol, 1 equiv), <bold>2a</bold> (1.5 equiv), solvent (v/v, 5 mL), N<sub>2</sub> atmosphere, 18 h. <sup>b</sup>Isolated yields. <sup>c</sup>Room temperature.</p></fn></table-wrap-foot></table-wrap><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>X-ray structure of compound <bold>3aa</bold> (CCDC 2002878).</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1974-g003\"/></fig><p>With the optimal reaction conditions in hand (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, entry 16), we next explored the scope and generality of this protocol using various 2-(allyloxy)arylaldehydes <bold>1</bold> for the reaction with <bold>2a</bold>. As shown in <xref ref-type=\"fig\" rid=\"C2\">Scheme 2</xref>, substrates <bold>1</bold> with a range of functional groups, such as electron-donating groups Me- (<bold>1b</bold> and <bold>1c</bold>), <italic>t</italic>-Bu- (<bold>1d</bold> and <bold>1e</bold>), and electron-withdrawing groups Cl- (<bold>1f</bold>), Br- (<bold>1g</bold>), and F- (<bold>1h</bold>) were well tolerated in this transformation, providing the desired products <bold>3aa</bold>–<bold>ha</bold> in 48–62% yields. Furthermore, the transformation also proceeded with naphthyl substrate <bold>1i</bold> giving the desired product <bold>3ia</bold> in 45% yield. Notably, when the substrate was 2-allylbenzaldehyde (<bold>1j</bold>), the protocol was also compatible affording the indanone derivative <bold>3ja</bold> with comparable yield. This outcome is of special interest, because indanone derivatives are also privileged structural motifs found in numerous natural products and pharmaceuticals with extraordinary biological and pharmaceutical activities [<xref rid=\"R34\" ref-type=\"bibr\">34</xref>–<xref rid=\"R35\" ref-type=\"bibr\">35</xref>]. However, no desired product (<bold>3ka</bold>) was obtained when there was a nitro group in the substrate (<bold>1k</bold>). Then, <italic>N</italic>-allyl-<italic>N</italic>-(2-formylphenyl)-4-methylbenzenesulfonamide (<bold>1l</bold>) was examined in the cascade cyclization and the desired products <bold>3la</bold>, <bold>3lf</bold> were obtained in 44% and 40% yield.</p><fig id=\"C2\" position=\"float\"><label>Scheme 2</label><caption><p>Scope of 2-(allyloxy)arylaldehydes. Reaction conditions: <bold>1</bold> (0.3 mmol, 1 equiv), <bold>2a</bold> (1.5 equiv) [<bold>2f</bold> for product <bold>3lf</bold>], DMSO/H<sub>2</sub>O (4:1 v/v, 5 mL), K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv), N<sub>2</sub> atmosphere, 18 h. Yields are isolated yields.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1974-g005\"/></fig><p>Next, to further demonstrate the generality of this strategy, the scope of different diphenylphosphine oxide <bold>2</bold> was examined as shown in <xref ref-type=\"fig\" rid=\"C3\">Scheme 3</xref>. Simple diphenylphosphine oxides, such as 2-Me-DPPO (<bold>2b</bold>), 4-Me-DPPO (<bold>2c</bold>), and 4-<italic>t</italic>-Bu-DPPO (<bold>2d</bold>) furnished the corresponding products in good yields. Also multisubstituted diarylphosphine oxides <bold>2e</bold> and <bold>2f</bold> were well tolerated under these reaction conditions. Gratifyingly, diphenylphosphine oxides bearing fluoro-substituents (<bold>2g</bold>) reacted smoothly furnishing the anticipated product <bold>3ag</bold> in 58% yield. Furthermore, 1-naphthyl-DPPO (<bold>2h</bold>) was also suitable for this transformation, and afforded the expected product <bold>3ah</bold> in 50% yield. The reaction between diethyl phosphonate (<bold>2j</bold>) and <bold>1a</bold> proceeded less efficiently under the conditions and a low yield of <bold>3aj</bold> was obtained. Dimethylphosphine oxide (<bold>2k</bold>) did not participate in the reaction, likely due to its high oxidation potential and poor ability to undergo tautomerization [<xref rid=\"R36\" ref-type=\"bibr\">36</xref>].</p><fig id=\"C3\" position=\"float\"><label>Scheme 3</label><caption><p>Scope of diphenylphosphine oxides. Reaction conditions: <bold>1a</bold> (0.3 mmol, 1 equiv), <bold>2</bold> (1.5 equiv), DMSO/H<sub>2</sub>O (4:1 v/v, 5 mL), K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> (3.0 equiv), N<sub>2</sub> atmosphere, 18 h. Yields are isolated yields.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1974-g006\"/></fig><p>To demonstrate the practicability of this methodology, a gram-scale experiment was next performed, employing <bold>1b</bold> and <bold>2a</bold> as substrates under the optimized conditions (<xref ref-type=\"fig\" rid=\"C4\">Scheme 4</xref>). The reaction afforded the desired product <bold>3ba</bold> in a good yield of 56%, and the structure was also confirmed by X-ray diffraction (see <xref ref-type=\"supplementary-material\" rid=\"SD1\">Supporting Information File 1</xref>) [<xref rid=\"R33\" ref-type=\"bibr\">33</xref>].</p><fig id=\"C4\" position=\"float\"><label>Scheme 4</label><caption><p>Gram-scale reaction.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1974-g007\"/></fig><p>To gain an insight into the reaction mechanism, we carried out some control experiments (<xref ref-type=\"fig\" rid=\"C5\">Scheme 5</xref>). When the reaction was conducted in the presence of radical scavengers such as 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO) and butylated hydroxytoluene (BHT), the reactions were completely shut down, which indicated that the reaction proceeds through a radical pathway [<xref rid=\"R37\" ref-type=\"bibr\">37</xref>–<xref rid=\"R41\" ref-type=\"bibr\">41</xref>]. Also, we successfully separated a small amount of byproduct <bold>4</bold> which was identified by NMR spectroscopy. These experiments clearly support a phosphorus-centered radical reaction pathway. It has been reported that phosphorus-centered radicals could be generated from phosphine oxides in the presence of potassium persulfate [<xref rid=\"R42\" ref-type=\"bibr\">42</xref>–<xref rid=\"R44\" ref-type=\"bibr\">44</xref>]. Based on literature precedent [<xref rid=\"R29\" ref-type=\"bibr\">29</xref>–<xref rid=\"R30\" ref-type=\"bibr\">30</xref><xref rid=\"R42\" ref-type=\"bibr\">42</xref>–<xref rid=\"R46\" ref-type=\"bibr\">46</xref>] and preliminary mechanistic experiments, a plausible mechanism was proposed in <xref ref-type=\"fig\" rid=\"C5\">Scheme 5</xref> which was different from the predominant mechanism observed in the Ag-catalyzed radical cascade for the preparation of phosphonate-functionalized chroman-4-ones [<xref rid=\"R28\" ref-type=\"bibr\">28</xref>]. Initially, K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> thermally decomposes to form sulfate radical anions (SO<sub>4</sub><sup>•−</sup>) [<xref rid=\"R29\" ref-type=\"bibr\">29</xref>–<xref rid=\"R30\" ref-type=\"bibr\">30</xref>], which react with diphenylphosphine oxide (DPPO, <bold>2</bold>) to give the phosphorus-centered radical <bold>I</bold> [<xref rid=\"R42\" ref-type=\"bibr\">42</xref>–<xref rid=\"R44\" ref-type=\"bibr\">44</xref>]. Then, the phosphorus centered radical <bold>I</bold> added to the C–C double bond of <bold>1</bold> to generate a new carbon-centered radical <bold>II</bold>, with sequential attack on the aldehyde group. The oxygen radical <bold>III</bold> thus formed undergoes a formal 1,2-H shift to generate the benzyl radical <bold>IV</bold> [<xref rid=\"R45\" ref-type=\"bibr\">45</xref>–<xref rid=\"R46\" ref-type=\"bibr\">46</xref>]. Finally, hydrogen abstraction by the sulfate radical anion (SO<sub>4</sub><sup>•−</sup>) from the benzyl radical <bold>IV</bold> affords the final products <bold>3</bold> [<xref rid=\"R45\" ref-type=\"bibr\">45</xref>–<xref rid=\"R46\" ref-type=\"bibr\">46</xref>].</p><fig id=\"C5\" position=\"float\"><label>Scheme 5</label><caption><p>Control experiments and proposed mechanism.</p></caption><graphic xlink:href=\"Beilstein_J_Org_Chem-16-1974-g008\"/></fig></sec><sec><title>Conclusion</title><p>In summary, an environmentally benign and practical radical cascade cyclization was developed to synthesize a series of phosphonate-functionalized chroman-4-ones from 2-(allyloxy)benzaldehydes and diphenylphosphine oxides. This protocol proceeds under metal-free conditions and uses cheap K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> as oxidant with easy handling and a broad substrate scope. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"systematic-review\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Oncol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Oncol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Oncol.</journal-id><journal-title-group><journal-title>Frontiers in Oncology</journal-title></journal-title-group><issn pub-type=\"epub\">2234-943X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850319</article-id><article-id pub-id-type=\"pmc\">PMC7431761</article-id><article-id pub-id-type=\"doi\">10.3389/fonc.2020.01112</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Oncology</subject><subj-group><subject>Systematic Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Gemcitabine-Based Neoadjuvant Treatment in Borderline Resectable Pancreatic Ductal Adenocarcinoma: A Meta-Analysis of Individual Patient Data</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Giovinazzo</surname><given-names>Francesco</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/544991/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Soggiu</surname><given-names>Fiammetta</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/552840/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Jang</surname><given-names>Jin-Young</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Versteijne</surname><given-names>Eva</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>van Tienhoven</surname><given-names>Geertjan</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/719331/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>van Eijck</surname><given-names>Casper H.</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Han</surname><given-names>Youngmin</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/685352/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Choi</surname><given-names>Seong Ho</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Kang</surname><given-names>Chang Moo</given-names></name><xref ref-type=\"aff\" rid=\"aff7\"><sup>7</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zalupski</surname><given-names>Mark</given-names></name><xref ref-type=\"aff\" rid=\"aff8\"><sup>8</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/786376/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ahmad</surname><given-names>Hasham</given-names></name><xref ref-type=\"aff\" rid=\"aff9\"><sup>9</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Yentz</surname><given-names>Sarah</given-names></name><xref ref-type=\"aff\" rid=\"aff8\"><sup>8</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Helton</surname><given-names>Scott</given-names></name><xref ref-type=\"aff\" rid=\"aff10\"><sup>10</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Rose</surname><given-names>J. Bart</given-names></name><xref ref-type=\"aff\" rid=\"aff11\"><sup>11</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/937534/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Takishita</surname><given-names>Chie</given-names></name><xref ref-type=\"aff\" rid=\"aff12\"><sup>12</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/936228/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Nagakawa</surname><given-names>Yuichi</given-names></name><xref ref-type=\"aff\" rid=\"aff12\"><sup>12</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Abu Hilal</surname><given-names>Mohammad</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff13\"><sup>13</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/639464/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Surgery, University Hospital of Southampton NHS Foundation Trust</institution>, <addr-line>Southampton</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Hepato-Pancreato-Biliary and Liver Transplant Unit, Royal Free Hospital</institution>, <addr-line>London</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Surgery, Seoul National University Hospital</institution>, <addr-line>Seoul</addr-line>, <country>South Korea</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Radiation Oncology, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Department of Surgery, Erasmus MC Cancer Institute</institution>, <addr-line>Rotterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff6\"><sup>6</sup><institution>Department of Surgery, Sungkyunkwan University School of Medicine</institution>, <addr-line>Seoul</addr-line>, <country>South Korea</country></aff><aff id=\"aff7\"><sup>7</sup><institution>Division of HBP Surgery, Department of Surgery, Yonsei University College of Medicine</institution>, <addr-line>Seoul</addr-line>, <country>South Korea</country></aff><aff id=\"aff8\"><sup>8</sup><institution>Department of Medicine, University of Michigan</institution>, <addr-line>Ann Arbor, MI</addr-line>, <country>United States</country></aff><aff id=\"aff9\"><sup>9</sup><institution>Department of Surgery, University Hospital of Leicester NHS Trust</institution>, <addr-line>Leicester</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff10\"><sup>10</sup><institution>Section of General, Thoracic and Vascular Surgery, Department of Surgery, Virginia Mason Medical Center</institution>, <addr-line>Seattle, WA</addr-line>, <country>United States</country></aff><aff id=\"aff11\"><sup>11</sup><institution>Section of Surgical Oncology, University of Alabama</institution>, <addr-line>Birmingham, AL</addr-line>, <country>United States</country></aff><aff id=\"aff12\"><sup>12</sup><institution>Department of Gastrointestinal and Pediatric Surgery, Tokyo Medical University</institution>, <addr-line>Tokyo</addr-line>, <country>Japan</country></aff><aff id=\"aff13\"><sup>13</sup><institution>Depatment of Surgery, Fondazione Poliambulanza Istituto Ospedaliero Multispecialistico</institution>, <addr-line>Brescia</addr-line>, <country>Italy</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Savio George Barreto, Medanta the Medicity, India</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Christoph W. Michalski, Heidelberg University Hospital, Germany; Alex Nicolas Gordon-Weeks, University of Oxford, United Kingdom</p></fn><corresp id=\"c001\">*Correspondence: Francesco Giovinazzo <email>giovinazzo_francesco@live.com</email></corresp><corresp id=\"c002\">Mohammad Abu Hilal <email>abuhilal9@gmail.com</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Surgical Oncology, a section of the journal Frontiers in Oncology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>1112</elocation-id><history><date date-type=\"received\"><day>14</day><month>10</month><year>2019</year></date><date date-type=\"accepted\"><day>03</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Giovinazzo, Soggiu, Jang, Versteijne, van Tienhoven, van Eijck, Han, Choi, Kang, Zalupski, Ahmad, Yentz, Helton, Rose, Takishita, Nagakawa and Abu Hilal.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Giovinazzo, Soggiu, Jang, Versteijne, van Tienhoven, van Eijck, Han, Choi, Kang, Zalupski, Ahmad, Yentz, Helton, Rose, Takishita, Nagakawa and Abu Hilal</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p><bold>Background:</bold> Non-randomized studies have investigated multi-agent gemcitabine-based neo-adjuvant therapies (GEM-NAT) in borderline resectable pancreatic ductal adenocarcinoma (BR-PDAC). Treatment sequencing and specific elements of neoadjuvant treatment are still under investigation. The present meta-analysis aims to assess the effectiveness of GEM-NAT on overall survival (OS) in BR-PDAC.</p><p><bold>Patients and Methods:</bold> A meta-analysis of individual participant data (IPD) on GEM-NAT for BR-PDAC were performed. The primary outcome was OS after treatment with GEM-based chemotherapy. In the Individual Patient Data analysis data were reappraised and confirmed as BR-PDAC on provided radiological data.</p><p><bold>Results:</bold> Six studies investigating GEM-NAT were included in the IPD metanalysis. The IPD metanalysis was conducted on 271 patients who received GEM-NAT. Pooled median patient-level OS was 22.2 months (95%CI 19.1–25.2). R0 rates ranged between 81 and 95% (<italic>I</italic><sup>2</sup> = 0%, <italic>p</italic> = 0.64), respectively. Median OS was 27.8 months (95%CI 23.9–31.6) in the patients who received NAT-GEM followed by resection compared to 15.4 months (95%CI 12.3–18.4) for NAT-GEM without resection and 13.0 months (95%CI 7.4–18.5) in the group of patients who received upfront surgery (<italic>p</italic> < 0.0001). R0 rates ranged between 81 and 95% (<italic>I</italic><sup>2</sup> = 0%, <italic>p</italic> = 0.64), respectively. Overall survival in the R0 group was 29.3 months (95% CI 24.3–34.2) vs. 16.2 months (95% CI 7·9–24.5) in the R1 group (<italic>p</italic> = 0·001).</p><p><bold>Conclusions:</bold> The present study is the first meta-analysis combining IPD from a number of international centers with BR-PDAC in a cohort that underwent multi-agent gemcitabine neoadjuvant therapy (GEM-NAT) before surgery. GEM-NAT followed by surgical resection improve survival and R0 resection in BR-PDAC. Also, GEM-NAT may result in a good palliative option in non-resected patients because of progressive disease after neoadjuvant treatment. Results from randomized controlled trials (RCTs) are awaited to validate these findings.</p></abstract><kwd-group><kwd>gemcitabine</kwd><kwd>gemcitabine-based neoadjuvant</kwd><kwd>neoadjuvant treatment of pancreatic cancer</kwd><kwd>Pancreatic ductal adenocancinoma</kwd><kwd>Borderline resectable pancreaic adenocarcinoma</kwd></kwd-group><counts><fig-count count=\"4\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"41\"/><page-count count=\"9\"/><word-count count=\"4698\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Pancreatic ductal adenocarcinoma (PDAC) is a not uncommon lethal malignancy, with a 5-year survival of 8% for all the stages. Patients undergoing resection have 20% survival rate at 5-years which may be as high as 32% in case of complete resection and 40% in the subgroup with node-negative disease. Only a minority of patients, however, are eligible for surgery at the time of diagnosis, due to metastatic or locally advanced disease (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B2\" ref-type=\"bibr\">2</xref>).</p><p>A borderline resectable (BR)-PDAC is a tumor with a variable degree of vascular contact or involvement that may not permit a complete resection without vascular resection and/or reconstruction, making resection challenging, although technically possible. Various definitions of BR-PDAC have been proposed but the literature supports that this group of patients does benefit from surgical resection if a complete resection (R0) can be reached (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B4\" ref-type=\"bibr\">4</xref>). In addition, recent studies have suggested that the use of a multimodal neoadjuvant treatment approach may better select the patient that will benefit from surgery by increasing the R0 resection rate and improving overall survival (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>–<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Previous studies have focused on the benefit of neoadjuvant treatment compared to upfront surgery in PDAC, but there is paucity of evidence on long term outcomes in BR-PDAC (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>).</p><p>To date no standard neoadjuvant protocol has been agreed in BR-PDAC (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>–<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). Palliative gemcitabine has been a standard of care in locally advanced or metastatic PDAC for many years and is used alone or in multi-agent combinations, and in association with radiotherapy (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). The type of treatment choice is influenced by individual patient features and status and based on data available from metastatic PDAC.</p><p>The present meta-analysis of individual participant data (IPD) aimed to assess the impact of the use of neoadjuvant therapies with Gemcitabine based protocols in patients with borderline resectable PDAC. The primary outcome of the meta-analysis was overall survival (OS) in patients who received GEM-NAT followed by resection.</p></sec><sec sec-type=\"methods\" id=\"s2\"><title>Methods</title><sec><title>Search Strategy</title><p>The systematic review and meta-analysis were conducted in accordance to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.</p><p>A computerized search of PubMed, Embase, Ovid Medline, and Cochrane Library was carried out. Articles published from time of inception to February 2020 were included. An advanced search was performed with the following search mesh terms: “pancreatic neoplasm and neoadjuvant therapy.” Reference lists of all obtained and relevant articles were screened manually and cross-referenced to identify any additional studies. The site clinicaltrials.gov was interrogated for any ongoing or concluded trial on the topic with available results.</p></sec><sec><title>Outcomes of Interest</title><p>The primary outcome was median overall survival (OS) in patients with BR-PDAC treated with Gemcitabine-based neo-adjuvant treatments (GEM-NAT) with or without surgical resection. Secondary outcomes were: complete resection (R0) and resection rate. In the IPD analysis all stages were re-assessed and confirmed as BR-PDAC according to NCCN guidelines based on provided radiological data.</p></sec><sec><title>Inclusion Criteria</title><p>Studies that reported results on patients diagnosed with BR-PDAC with the outcome of interest were included in the review. Studies including both patients with BR-PDAC and with locally advanced PDAC were only included when the primary outcome was available for the separate cohort of BR-PDAC. Only data from the centers who provided anonymized individual patient data with results on GEM-NAT were included in the IPD meta-analysis. Articles focused on pancreatic neuroendocrine neoplasia or other histology were excluded. When two or more articles were reported from the same institution and/or author, the one of higher quality or the most recent publication was included in the analysis.</p><p>Abstracts, letters, comments, editorials and expert opinions, unpublished articles and abstracts, reviews without original data, case reports were excluded from the meta-analysis.</p><p>Two reviewers (FS and HA) independently screened the titles and abstracts of all retrieved articles. The full texts of articles with the potential to fulfill the inclusion criteria were obtained and checked for eligibility. The following information was extracted from each article: first author, year of publication, study design, study population characteristics, number of subjects treated, type of neo-adjuvant treatment, dropout rate, procedure-related mortality and morbidity, median and disease-free survival complete oncological resection (R0), and resection rate.</p><p>After selecting eligible articles for meta-analysis, contact details of authors were gathered from recent articles or the internet and authors were asked to collaborate with us. A second request was sent to non-responders 4 weeks later. Authors who agreed to collaborate were requested to provide anonymous IPD for clinico-pathological and radiological characteristics, treatment, postoperative, and long term outcomes.</p></sec><sec><title>Data Analysis</title><p>The meta-analysis was performed using R software suite (v3.4.0, <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.R-project.org\">https://www.R-project.org</ext-link>), and Kaplan Meier curves were calculated with SPSS (v24, Chicago, IL, USA). Pooled effect was calculated using either the fixed effects or the random effects model. Time-to-event methods were used for the median survival. Hazard Ratio (HR) was derived from ln(HR) and Standard Error (SE). Studies not reporting <italic>p</italic>-value for survival analysis were excluded (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Statistical heterogeneity between trials was evaluated by χ<sup>2</sup> and <italic>I</italic><sup>2</sup>, with significance being set at <italic>p</italic> ≤ 0.10 (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). In the absence of statistically significant heterogeneity, the fixed-effect method was used to combine the results. When heterogeneity was confirmed (<italic>p</italic> ≤ 0.10), the random-effect method was used. Potential publication bias was investigated by funnel plot, Egger's test, was used to assess funnel plot asymmetry (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>) and Makaskill's test was used to quantify the bias (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>). <italic>P</italic> < 0.050 (two-tailed) was considered to indicate statistical significance. The methodological quality of the individual studies was assessed with the Critical Appraisal Skill Program (CASP) tool (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>).</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>Literature Search</title><p>The number of studies screened, assessed, and excluded is reported in the PRISMA flow diagram (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Eighteen full text articles were assessed for eligibility and six studies provided data at an IPD (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>) (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>–<xref rid=\"B20\" ref-type=\"bibr\">20</xref>).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>PRISMA 2009 flow diagram.</p></caption><graphic xlink:href=\"fonc-10-01112-g0001\"/></fig><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Studies included in the individual patient data (IPD) meta-analysis.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Author</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Year</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Study design</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Neoadjuvant treatment</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Median F/U (months)</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Treatment</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>N</italic></bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Resection rate (%)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>R0 resection (%)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Median OS (months)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Median DFS (months)</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rose JB.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2014</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Retrospective</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gem + Docetaxel +/– RT 50.4 Gy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21.6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NAT/resection</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">48.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">87.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>NR</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.2</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NAT/no resection</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.4</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Kim EJ.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2013</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Prospective</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gem + Ox + RT 30 Gy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NAT/resection</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">93.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NAT/no resection</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.9</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lee JH</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2015</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Prospective</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gem +/– Cisplatinum +/– Cap +/– 45 or 50.4 Gy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NAT/resection</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">93.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NAT/no resection</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19.5</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nagakawa Y.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2017</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Prospective</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gem + S1+ RT 50.4Gy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20.0</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NAT/resection</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">76.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">94.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NAT/no resection</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.3</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Jang JY</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2018</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RCT</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gem + RT 45 + 9 Gy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NAT/resection</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">51.8%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">82.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NAT/no resection</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Versteijne E</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2020</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RCT</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gem + RT 36 Gy (total)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.0</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NAT/resection</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">61.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">71.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.1</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NAT/no resection</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><p><italic>NAT, Neoadjuvant Treatment; Cap, Capecitabine; Gem, Gemcitabine; Ox, Oxaliplatin; 5-FU, 5-Fluorouracil; RT, Radiotherapy; FDR, Fixed-Dose Rate; NR, Not Reached; RCT, Randomized controlled trial</italic>.</p></table-wrap-foot></table-wrap></sec><sec><title>Individual Patient Data Meta-Analysis</title><p>The IPD metanalysis was conducted on the data provided on 271 patients who received GEM-NAT. The included studies showed evidence of an asymmetrical distribution in overall survival (Test for funnel plot asymmetry: <italic>t</italic> = 1.4439, df = 4, <italic>p</italic> = 0.2223) (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>), resection rate (Test for funnel plot asymmetry: <italic>t</italic> = 3.2400, df = 4, <italic>p</italic> = 0.0317) (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>) and R0 rate (Test for funnel plot asymmetry: <italic>t</italic> = 4.3507, df = 4, <italic>p</italic> = 0.0122) (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>). There was no heterogeneity differences with regards to resection rate, which ranged from 47 to 76% (random effect model 0.60 (95% CI 0.50–0.69) <italic>I</italic><sup>2</sup> = 54%, <italic>p</italic> = 0.06) (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>), R0 rate, which ranged from 81 to 95% (random effect model 0.86, 95% CI 0.79–0.91, <italic>I</italic><sup>2</sup> = 0%, <italic>p</italic> = 0.64) (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>).</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p><bold>(A)</bold>. Funnel plot for survival outcomes. Test for funnel plot asymmetry: <italic>t</italic> = 1.4439, df = 4, <italic>p</italic> = 0.2223. <bold>(B)</bold>. Funnel plot for resection outcomes. Test for funnel plot asymmetry: <italic>t</italic> = 3.2400, df = 4, <italic>p</italic> = 0.0317. <bold>(C)</bold>. Funnel plot for RO outcomes. Test for funnel plot asymmetry: <italic>t</italic> = 4.3507, df = 4, <italic>p</italic> = 0.0122.</p></caption><graphic xlink:href=\"fonc-10-01112-g0002\"/></fig><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Forest plots showing HR of survival <bold>(A)</bold>, rates of Resection Rate <bold>(B)</bold> and RO <bold>(C)</bold>.</p></caption><graphic xlink:href=\"fonc-10-01112-g0003\"/></fig><p>Depending on the different centers' protocols, Gemcitabine was used as single agent in 110 (40.6%) patients or in combination with Docetaxel (67 patients, 24.7%) oxaliplatin (53 patients, 19.5%) S-I (25 patients, 9.2%) or other agents such and Cisplatin, Capecitabine and 5FU. Median OS was 27.8 months (95%CI 23.9–31.6) in the patients who received NAT-GEM followed by resection compared to 15.4 months (95%CI 12.3–18.4) for NAT-GEM without resection and 13.0 months (95%CI 7.4–18.5) in the group of patients who received upfront surgery from the study published by Jang et al. (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>) (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Patients receiving a multimodality treatment with NAT-GEM followed by surgery had a significantly better OS compared to both patients who did not complete the treatment with surgery and the patients who had upfront surgery (<italic>p</italic> = 0.000)?. Median OS for NAT-GEM +/– resection in the single studies included in the IPD analysis was similar (<italic>p</italic> = 0.813): 26.9 months (95%CI 21.1–32.6) for Nagakawa et al. (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>), 25.3 months (95%CI 18.3–32.2) for Rose et al. (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>), 21.2 months (95%CI 8.6–33.7) for Kim et al. (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>), 22.2 (95%CI 16.5–27.8) for Lee et al. (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>), 17.5 months (95%CI 10.9–24.0) for Versteijne et al. (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>) and 22.9 months (18.8–26.7) for Jang et al. (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>) (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>).</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Kaplan-Meier curves for pooled overall survival in patients receiving NAT-GEM following by resection and patients who received GEM-NAT but did not undergo resection or underwent upfront surgery <bold>(A)</bold> and amongst Treatment Protocols <bold>(B)</bold>.</p></caption><graphic xlink:href=\"fonc-10-01112-g0004\"/></fig><p>Data on radiological response following NAT-GEM was available for 200 patients, of which 76 (38%) achieved downstaging. Median OS was significantly improved in patients who achieved downstaging (28.7 months 95%CI 22.0–35.3 vs. 22.0 months; 95%CI 18.6–25.3; <italic>p</italic> = 0.026) (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure 1</xref>).</p><p>Median disease-free survival (DFS) in the entire cohort was 11.8 (95%CI 9.3–14.3). Data on toxicity was available for 244 patients: 27 (11.2% did not experience toxicity from GEM-NAT, 139 patients (56.9%) had toxicity grade I-II, whilst toxicity III and IV was seen in 92 (37.7%) and 20 (8.2%) of the patients.</p><p>Information on oncological complete resection was available for 158 patients who underwent resection following GEM-NAT. R0 resection was achieved in 130 patients (82.3%) with a median OS of 29.3 months (95% CI 24.3–34.2) vs. 16.2 months (95% CI 7.9–24.5) in the R1 group (<italic>p</italic> = 0.001).</p></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Different strategies combining surgery and chemoradiotherapy are being investigated for BR- PDAC (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). Although neoadjuvant therapy had shown beneficial effects in BR-PDAC by improving overall survival and the rate of R0 resection, treatment sequencing and specific elements of neoadjuvant treatment are still under investigation (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B22\" ref-type=\"bibr\">22</xref>–<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). The benefit of gemcitabine-based combination therapies is hotly debated and reported results have shown conflicting conclusions (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>–<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). A previous meta-analysis of 20 phase III RCT (<italic>n</italic> = 6,296) has shown no differences in overall survival between single agent gemcitabine and combination gemcitabine therapy in inoperable pancreatic cancer (RR 0.93, 95% CI 0.84–1.03, <italic>p</italic> = 0.17) (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Therefore, the evidence to recommend a specific neoadjuvant regimen is limited and practices vary with regard to the use of combination chemotherapy and/or radiotherapy (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>–<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). This meta-analysis of different gemcitabine-based protocols has shown similar results to other reported neoadjuvant regimens (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>), with a pooled median patient-level OS of 27.2 (95% CI 23.0–31.3) months in resected patients.</p><p>Up front surgery for BR-PDAC is still considered an option in some centers (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>, <xref rid=\"B29\" ref-type=\"bibr\">29</xref>). The number of RCTs comparing neoadjuvant therapy in BR-PDAC vs. up front surgery are very limited and the outcomes of different regimens still unknown (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>, <xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Recently, OS in a RCT was significantly better in the gemcitabine-based neoadjuvant chemoradiation treatment than in the upfront surgery group (21 vs. 12 months <italic>p</italic> = 0.028) and the results has been confirmed by the present meta-analysis (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). The Dutch Pancreatic Cancer Group reported in a randomized multicenter phase III trial a better OS in the preoperative gemcitabine arm than in the immediate surgery group (median 13.5 vs. 17.1 months; HR 0.71; <italic>p</italic> = 0.074) (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). In the present meta-analysis, the group of patients that did not proceed to surgery had similar median OS [20.4 (95% CI 12.7–28.0)] to the upfront surgery group in these two RCTs. Therefore, we speculate that this result confirms the findings and supports the use of neoadjuvant therapy to select patients with a favorable biological disease and to provide a palliative option in the non-responder or unresected groups.</p><p>Studies have demonstrated that neoadjuvant treatment does not decrease the rate of surgical resection in BR-PDAC and may lower surgical complication rates (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>–<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). This is likely due to a selection bias for healthier patients that are able to complete neoadjuvant treatment. Chemotherapy combinations are likely more efficacious in the neoadjuvant setting but are associated with increased toxicities. In advanced disease, gemcitabine has less Grade 3–4 toxicity as compared to FOLFIRINOX. As NAT, in one phase II study, a significant number of patients (23%) did not proceed to surgery due to toxicities or poor performance status following neoadjuvant gemcitabine and oxaliplatin (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). The present results seem to confirm that Gem-based chemotherapy is better tolerated than multidrug regimens with a minimal drop-out rate not related to progression disease.</p><p>Neoadjuvant therapy has permitted tumor down-staging and resection with similar survival rates after surgery and a decrease in the rate of margin-positive resections (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>–<xref rid=\"B38\" ref-type=\"bibr\">38</xref>). The resection rates of patients with BR-PDAC that ultimately undergo pancreatectomy after neoadjuvant therapy range from 47 to 76% with an associated 81 to 95% R0 resections (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>–<xref rid=\"B20\" ref-type=\"bibr\">20</xref>, <xref rid=\"B39\" ref-type=\"bibr\">39</xref>). In this meta-analysis, despite the variability amongst the protocols, no difference was observed within the IPD cohort in the R0 rates among drug regimens with the majority of resected patients having negative margins.</p><p>The strength of the present study is that all the data were reappraised and reclassified as BR-PDAC at an IPD level according to the radiological criteria. However, the main limitation of the present study is the heterogeneity of the included studies, although only one study is a retrospective study. In particular, some studies failed to report eligibility criteria for neo-adjuvant treatment expect that for the BR-PDAC stage (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B20\" ref-type=\"bibr\">20</xref>). A second limitation is related to the evaluation of the safety and tolerability of neoadjuvant therapy. Characteristically, retrospective studies did not include information about dose reduction or discontinuation of study drugs due to adverse events. Finally, there was paucity of data on adjuvant or second line chemotherapy. Therefore, we were not able to assess their impact on the survival outcomes.</p><p>In conclusion, to the best of our knowledge, the present meta-analysis is the first evidence-based data aggregation of gemcitabine-based Neoadjuvant Treatment in BR-PDAC. The results support the use of GEM-NAT for BR-PDAC in routine practice to select patients where surgery may contribute the most benefit. Moreover, the general tolerability of GEM-NAT may improve the rate of patients undergoing surgery following NAT and receiving adjuvant treatment related to lower post-operative complications and/or decline in the functional status (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>, <xref rid=\"B41\" ref-type=\"bibr\">41</xref>).</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The datasets presented in this article are not readily available due to maintaining patient confidentiality. Requests to access the datasets should be directed to <email>giovinazzo_francesco@live.com</email>.</p></sec><sec id=\"s6\"><title>Author Contributions</title><p>FG and FS contributed to study design, data collection, data analyses, data interpretation, writing, and reviewing. HA and FS did the literature search and figures. J-YJ, EV, GT, CE, YH, SC, CK, MZ, SY, SH, JR, CT, YN, and MA contributed to data collection and reviewing of the report. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s7\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><sec sec-type=\"supplementary-material\" id=\"s8\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fonc.2020.01112/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fonc.2020.01112/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Image_1.JPEG\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yeo</surname><given-names>CJ</given-names></name><name><surname>Abrams</surname><given-names>RA</given-names></name><name><surname>Grochow</surname><given-names>LB</given-names></name><name><surname>Sohn</surname><given-names>TA</given-names></name><name><surname>Ord</surname><given-names>SE</given-names></name><name><surname>Hruban</surname><given-names>RH</given-names></name><etal/></person-group>. <article-title>Pancreaticoduodenectomy for pancreatic adenocarcinoma: postoperative adjuvant chemoradiation improves survival</article-title>. <source>Ann Surg.</source> (<year>1997</year>) <volume>225</volume>:<fpage>621</fpage>–<lpage>36</lpage>. <pub-id pub-id-type=\"doi\">10.1097/00000658-199705000-00018</pub-id><pub-id pub-id-type=\"pmid\">9193189</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Siegel</surname><given-names>R</given-names></name><name><surname>Naishadham</surname><given-names>D</given-names></name><name><surname>Jemal</surname><given-names>A</given-names></name></person-group>. <article-title>Cancer statistics, 2013</article-title>. <source>A Cancer J Clin</source>. 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