GRM1 expression was silenced in MDA-MB-231 cells to study changes in expression of inflammatory genes regulated by mGluR1. Results were confirmed by ELISA using GRM1-silenced and overexpressed cells and mGluR1 inhibitors. A functional role for these differentially expressed genes was determined in vitro and in vivo. 131 genes were differentially expressed in GRM1-silenced MDA-MB-231 cells, with some of these falling into four major canonical pathways associated with acute inflammation, specifically leukocyte migration/chemotaxis. Upregulation of three of these genes  and their corresponding protein was confirmed by qPCR analysis and ELISA in GRM1-manipulated TNBC cells. Upregulation of these cytokines enhanced endothelial adhesion and transmigration of neutrophils in co-culture assays and in 4T1 mouse tumors. Our results suggest mGluR1 may serve as a novel endogenous regulator of inflammation in TNBC.Breast cancer remains a major cause of death among women. 15% of these cancers are triple negative breast cancer (TNBC), an aggressive subtype of breast cancer for which no current effective targeted therapy exists. We have previously demonstrated a role for mGluR1 in mediating tumor cell growth, endothelial cell proliferation, and tumor-induced angiogenesis in TNBC. In this study, we explore a role for mGluR1 in regulating inflammation in TNBC.  The only current systemic treatment options for TNBC include cytotoxic chemotherapeutics that target rapidly replicating cells and produce significant morbidity. The identification of new molecular targets to treat TNBC thus has the potential to produce new effective therapies for TNBC while reducing toxicity associated with standard chemotherapy and addressing a critical unmet need in breast cancer therapy.Approximately 15% of all breast cancer cases in the U.S. are triple negative breast cancer (TNBC), an aggressive subtype that lacks receptors for estrogen, progesterone and human epidermal growth factor receptor 27. Determining how to stimulate the influx of these immune infiltrates could potentially reduce mortality associated with TNBC. However, the immune system can play a dual role in cancer, acting both as a suppressor or promoter of tumor growth. The system is complex and involves various factors secreted by tumor cells, surrounding stromal and invading immune cells8. In breast cancer, immune cells, including tumor-associated neutrophils (TANs) play a major role in determining tumor cell fate through expression of various inflammatory agents, including chemokines14. Thus, polarizing the TIM in favor of an anti-tumor phenotype could potentially be effective as a treatment for TNBC.Recently, a link between the tumor immune microenvironment (TIM) and TNBC has been established, in which increased immune infiltrates positively correlated with improved pathologic complete response, decreased distance recurrence, and improved progression-free survival15. mGluR1 belongs to the Group I mGluR family whose over-expression has been linked to melanoma17. We detected high levels of mGluR1 in various TNBC cells compared to normal breast epithelial cells and observed inhibiting or silencing mGluR1 inhibits breast cancer growth and angiogenesis, both in vitro and in vivo20. In addition, we have demonstrated GRM1 and mGluR1 are expressed at significantly higher levels in human breast cancer tissue compared to patient-derived normal breast tissue18. In this study, we observe mGluR1 as an inhibitor of both the production of inflammatory chemoattractants by TNBC cells as well as the induction of neutrophil (PMN) transmigration. These findings suggest mGluR1 may serve as a novel endogenous regulator of inflammation in TNBC by initiating signals in breast cancer cells that modulate PMN transmigration and function within the TIM.Previously, we identified metabotropic glutamate receptor-1 (mGluR1) as a possible therapeutic target in TNBC. Metabotropic glutamate receptors (mGluRs) are a family of G-protein coupled receptors known to mediate reflexes in the nervous systemGRM1-silenced MDA-MB-231cells . The effect of riluzole on MDA-MB-231 CXCL1 levels was not significant. Unlike riluzole, after treatment with BAY, a dose-dependent increase in CXCL1 levels did occur in both SUM159 and BT549 cells by 24 hours with a significant increase of 2-fold at the highest dose tested (10 μM). MDA-MB-231 cells were not as responsive, with a small but significant increase only at the highest dose. However, at 48 hours, these levels continued to increase with BAY significantly increasing CXCL1 levels by up to 3-fold in all 3 cell lines.mGluR1-mediated regulation of CXCL1 and IL-8 was further demonstrated using mGluR1 inhibitors BAY36-7620 (BAY) and riluzole in BT549, SUM159 and MDA-MB-231 cells, which also express mGluR1 Fig. . All 3 c24. However, after 48 hours, a dose-dependent increase in IL-8 levels were observed in SUM159 cells with a significant increase of about 1.5-fold at the highest dose tested (50 μM). BT549 cells were unresponsive to lower levels of riluzole but did induce a significant 2.5-fold increase at the highest dose tested. MDA-MB-231 cells were still unresponsive to riluzole. The response of SUM159 and MDA-MB-231 cells to BAY was much more robust with a dose-dependent increase in IL-8 levels as early as 24 hours and still evident after 48 hours. At the highest dose tested (10 μM), BAY induced a significant increase of at least 2-fold at both times points. Similar to riluzole treatment, BT549 cells were unresponsive to lower levels of BAY but a significant increase of over 2-fold was observed at higher levels (0.5–1.0 μM).Unlike CXCL1, IL-8 chemokine secretion levels varied between cell lines with SUM159 expressing high levels at 24 hours with less detection in the BT549 (just above background) and MDA-MB-231 cells Fig. . After 226. This process is triggered by binding of chemokine to its receptor on PMNs, causing integrin clustering on the cell surface of the PMN, resulting in increased binding of integrin to ICAM-1 on the endothelium. Following these events, PMNs migrate across the endothelium. mGluR1’s ability to regulate this process was examined. Endothelial monolayers were exposed for 30 minutes to conditioned medium from either GRM1-silenced or overexpressed cells (cultured for 24-hours) prior to adding labeled PMNs for 30 minutes. This incubation process allows for chemokines expressed in the medium to bind their receptors on the endothelium30. After 30 minutes, monolayers were washed to remove non-adherent PMNs and remaining PMNs were detected by fluorescence. As a positive control, monolayers were treated with TNFα for 6 hours. As expected, treatment of the endothelium with TNFα induced an almost 2-fold and 3-fold increase in PMNs adhering to the monolayers compared to NS or LACZ controls  increase in PMNs present in the medium from GRM1 silenced cells compared to NS medium  but significant decrease in the number of PMNs present compared to medium from LACZ cells.Following adhesion to the vascular endothelium, PMNs will transmigrate across the monolayer moving towards a chemokine gradient. To examine a role for mGluR1, inserts containing endothelial monolayers were placed in medium from either ium Fig. . In the GRM1-expressing 4T1 mouse tumor model to measure PMN presence in the tumors after injections with riluzole or sunitinib, an anti-angiogenic drug known to regulate PMN migration32. As our previously published growth curve demonstrates19, treatment with riluzole or sunitinib significantly decreased tumors by 50% compared to vehicle DMSO. In this same study, we measured PMN presence and found decreased tumor volume corresponded with a significant increase in PMN presence, detected by anti-Ly6G positive staining  transmigration. This was demonstrated in various TNBC cells where inhibition of mGluR1 using either the mGluR1 inhibitors  or an shRNA directed at mGluR1 increased expression of CXCL1 and IL-8 and overexpressing In vivo, we also observed that treating the syngeneic mouse 4T1 mammary cancer model with riluzole increased the presence of PMNs in the tumors, which coincided with decreased tumor growth19. This suggests that mGluR1, in addition to its direct effect on tumor cell growth and survival20 is capable of regulating inflammation within the TIM. However, recent findings suggest riluzole’s anti-tumor properties in breast cancer are likely largely mediated through mGluR1 independent mechanisms36. In that study, riluzole inhibited cell growth, invasion and migration in both GRM1 silenced and over-expressed cells suggesting lack of GRM1 involvement. In addition, a recent microarray analysis demonstrated cell cycle genes to be major pathways regulated by riluzole further suggesting alternative pathway(s) by which riluzole functions other than mGluR24. Further in vivo studies utilizing more specific mGluR1 inhibitors, such as BAY36-7620, are necessary to definitively define a role for mGluR1 in mediating inflammation within the TIM. Nonetheless, riluzole’s ability to mediate PMN migration in the 4T1 tumor model in the present study is important because riluzole is an already FDA-approved drug that can be quickly translated into the clinic.38 a link between chronic inflammation and neoplastic progression has long been recognized41. Within the TIM, there is a complex mix of cell types contributing inflammatory factors including cancer cells themselves. TANs were originally thought to promote cancer by affecting angiogenesis42 or by modulating the TIM in favor of immunosuppression. However, based on recent evidence45, it appears the TIM can be manipulated to polarize TANs to acquire anti-tumor phenotypes involving CXCL1 and TNFα48. TNBC cell’s ability to regulate PMN adhesion and migration through production of these chemoattractants strongly confirms these studies and suggests mGluR1 as a novel endogenous regulator of leukocyte phenotype within the TIM.The role of inflammation in cancer is complex, demonstrating both pro- and anti-tumor properties. Although acute inflammation is associated with anti-tumor immune responsesGRM1 silencing (95-fold increase). Recent studies demonstrate a strong association of TIL presence with increased metastasis-free survival and decreased distant recurrence in early stage TNBC5. These studies correlate with other studies involving TANs where in early-stage tumors, TANs possess anti-tumorigenic properties and actually activate TILs52. Thus, it appears targeting mGluR1 early in the treatment regime may play an important role in stimulating an adaptive immune response in TNBC. In support of this, riluzole has been shown to increase survival of CD8 T-cells in HIV-1-infected individuals and enhance proliferation of anti-CD3/CD28 stimulated T cells53.In addition to PMNs, data from the microarray analysis show lymphocyte migration and chemotaxis to be strongly upregulated by Previously, we identified mGluR1 as a promising target for breast cancer therapy based on its roles promoting angiogenesis and tumor cell growth. Our results now implicate mGluR1 and riluzole as novel endogenous inhibitors of inflammation and PMN transmigration in TNBC. Further studies into the mechanism by which mGluR1 and riluzole mediate these effects could be very useful in the development of therapeutic targets for treating TNBC cancer and would provide more insight into the role inflammation plays on the progression of TNBC.54. The human microvascular endothelial cell line HMEC-1 was obtained from Centers for Disease Control and cultured as described previously19. Cell lines were authenticated via cytogenetic analysis or used within 6 months of purchase or stored in liquid nitrogen. The specific mGluR1 inhibitor, BAY36-7260, and riluzole were purchased from Tocris Bioscience . All tumor digestion reagents were purchased from Sigma Aldrich .Cell culture reagents were purchased from Thermofisher Scientific . TNBC cell lines were purchased from ATCC except the SUM159 cell line was a kind gift from Stephen Ethier . The mouse 4T1-12B cell line was a kind gift from Fred MillerGRM1 shRNA vector (shGRM1) or non-silenced control vector (NS) were obtained from Karmanos Cancer Institute by subscription to Thermo Scientific GIPZ shRNAmir library. Lentiviral particles containing these vectors were generated by reverse transfection of these constructs with Trans-Lentiviral package mix, into HEK293T cells using Arrest-In/Express-In transfection reagent. Approximately 106 TU/ml was used to infect MDA-MB-231 cells in the presence of polybrene (10 μg/ml) and stable cultures generated by growing in puromycin (1 μg/ml) as previously described36. GRM1 silencing was confirmed by Western blot and RT-QPCR.Reagents for transduction assays were purchased from ThermoFisher Scientific. GIPZ Lentiviral particles containing GRM1 vectors has been described previously36. Lentiviral particles containing the GRM1 vector or LACZ control vector were generated by reverse transfection of these constructs with Virapower packaging mix into HEK293T cells using Lipofectamine 2000 reagent. A dilution (1:1) of viral supernatant was used to infect low GRM1-expressing MDA-MB-468 cells using polybrene (10 μg/ml). Stable cultures were generated by growing in blasticidin (5 μg/ml).Construction of Lentiviral GRM1 silenced or NS MDA-MB-231 cells using puromycin (10 μg/ml) for two weeks, cells were plated in triplicate overnight and RNA extracted using RNeasy Plus Mini Kit . RNA was quality assessed using the 2100 Bioanalyzer System and hybridized to the Illumina® Human HT-12v4 array. Data was uploaded to BeadStudio, background-corrected and normalized using rank invariant algorithm. Differentially expressed genes were identified using Illumina Custom Error Model and genes differentially expressed were uploaded to Genomatix software suite to determine over-represented canonical pathways. The online DAVID tool was used to determine Gene Ontology Biological Process terms over-represented by the data.After stably selecting GRM1, CXCL1, IL6, IL8, and housekeeping gene GAPDH as listed in Table Total RNA was extracted using Qiagen’s RNeasy Plus Mini Kit and reverse transcription performed using High Capacity cDNA Reverse Transcription Kit (Thermo Scientific) according to manufacturer protocols. QPCR was performed using ABsolute QPCR Mix with ROX (Thermo Scientific) according to manufacturer using the sense/anti-sense primers for 20. Controls without RT were used to confirm lack of genomic DNA. Relative fold change in GRM1, CXCL1, IL6, or IL8 expression compared to NS or LACZ control was determined using the following equation: 2−ddCt, where −ddCt is difference between the dCt of the cytokine gene and the housekeeping gene normalized to the control values.Thermal cycling was performed as previously described30–60 μg of protein isolated from TNBC cells was separated by SDS-polyacrylamide gel electrophoresis (10%) and transferred to PVDF membranes. Immunodetection of mGluR1 was performed using anti-mGluR1 antibody  with appropriate secondary antibody and detected by chemiluminescence. Blots were further reprobed with antibody against GAPDH .36, and chemokine expression was normalized to cell counts.CXCL1 and IL-8 levels in culture supernatants from stably transduced cells or after treatment with TNFα (10 ng/ml), BAY36-7620, riluzole, or vehicle (0.01% DMSO) were measured by sandwich ELISA  according to manufacturer protocol. Since these inhibitors are known to inhibit cell growth, relative TNBC cells numbers were determined after each experiment using MTT analysis4) were injected into mammary fat pads of female BALB/c mice  and allowed to grow until tumors reached a mean volume of 62 mm3 (approximately 10 days). Mice were then divided into groups of 10 and treated daily with i.p. injections of riluzole (18 mg/kg), sunitinib (20 mg/kg), or vehicle (DMSO) for 14 days. Tumor size was measured three times a week using a Vernier caliper and tumor volume estimated using the following formula: length × width × depth/2. After 14 days of treatment, mice were euthanized and their tumors harvested, minced, and digested in enzyme solution (1 g tissue/10 ml solution) containing collagenase type IV (0.15 mg/ml), collagenase type I (0.4 mg/ml), DNase I (1.25 mg/ml) and BSA (0.5%) for 1 hour. PMNs were identified as described below. Animals were housed in a pathogen-free facility and all animal studies were performed in accordance with local IACUC at Wayne State University.4T1 cells  for 30 minutes at 37 °C. The percentage of PMNs in the tumor digestion mixture was determined by FACS analysis after washing mixture through a 70 μm nylon strainer and labeling with FITC-conjugated anti-Ly6G antibody .Heparin anti-coagulated blood was obtained from healthy volunteers after informed consent and in accordance with ethical guidelines of Wayne State University. PMNs were isolated using Ficoll-Paque followed by dextran (1%) density gradient centrifugation as described previously5/well and allowed to attach for 30 minutes. After 30 minutes, HMEC-1 monolayers were washed 3 times with PBS and remaining PMNs detected by measuring FITC fluorescence using BioTek Synergy 2 plate reader.HMEC-1 cells were grown to confluence in 96-well black plates and exposed for 30 minutes to conditioned medium from stably transduced cells (cultured for 24 hours) or to TNFα (10 ng/ml) for 6 hours as a positive control. After incubation, the medium was removed and labeled PMNs added to HMEC-1 monolayers at 2 × 106 per insert) and allowed to migrate for 1.5 hours. Media in the bottom of wells was then removed and PMNs collected by centrifugation, resuspended in PBS and detected by FITC fluorescence.HMEC-1 cells were grown to confluence on BD cell culture inserts (8 μm pore size) in 24-well plates containing EGM-2 complete medium. Upon confluence, medium in bottom of wells was replaced with 24 hour conditioned medium from stably transduced cells, after which labeled PMNs were added  for Macintosh. All numerical results are expressed as mean ± SEM and statistical analysis performed by one-way or two-way repeated-measures analysis of variance (ANOVA) followed by multiple comparison procedure with Student-Newman Keuls method. A value of p ≤ 0.05 was considered significant.Public Health Service Policy on Humane Care and Use of Laboratory Animals .All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. Animal studies were approved by the local Institutional Animal Care and Use Committee (IACUC) at Wayne State University which is structured and operated in accordance with NIH’s Office of Laboratory Animal Welfare (OLAW) All procedures performed in studies involving human participants were performed with their consent and in accordance with the Declaration of Helsinki and have been approved following Expedited Review (IRB #123016MP4E) by the Chairperson for the Wayne State University Institutional Review Board (MP4).Supplementary Information FileDataset 1


Nrf1 gene has capability to be differentially transcripted alongside with alternative mRNA-splicing and subsequent translation through different initiation signals so as to yield distinct lengths of polypeptide isoforms. Amongst them, three of the most representatives are Nrf1α, Nrf1β and Nrf1γ, but the putative specific contribution of each isoform to regulating ARE-driven target genes remains unknown. To address this, we have herein established three cell lines on the base of the Flp-In T-REx system, which are allowed for the tetracycline-inducibly stable expression of Nrf1α, Nrf1β and Nrf1γ. Consequently, the RNA-Sequencing results have demonstrated that a vast majority of differentially expressed genes (i.e. >90% DEGs detected) were dominantly up-regulated by Nrf1α and/or Nrf1β following induction by tetracycline. By contrast, the other DEGs regulated by Nrf1γ were far less than those regulated by Nrf1α/β (i.e. ~11% of Nrf1α and ~7% of Nrf1β). However, further transcriptomic analysis revealed that the tetracycline-induced expression of Nrf1γ significantly increased the percentage of down-regulated genes in total DEGs. These statistical data were further validated by quantitative real-time PCR. The experimental results indicate that distinct Nrf1 isoforms make diverse and even opposing contributions to regulating different subsets of target genes, such as those encoding 26S proteasomal subunits and others involved in various biological processes and functions. Collectively, Nrf1γ acts as a major dominant-negative inhibitor competitively against Nrf1α/β activity, such that a number of DEGs regulated by Nrf1α/β are counteracted by Nrf1γ.The single  It is important to note that the unique function of Nrf1 is finely tuned by a steady-state balance between production of the CNC-bZIP protein (i.e. translation of transcripts) and concomitantly  processing in order to give rise to distinct multiple isoforms  before being turned over. These distinct proteoforms of Nrf1 are postulated to together confer on the host robust cytoprotection against a vast variety of cellular stresses through coordinately regulating distinctive subsets of important homoeostatic and developmental genes. The transcriptional expression of such key genes are driven by antioxidant response elements (AREs) and/or other cis-regulating consensus sequences, some of which are conversed with the activating protein-1 (AP-1) binding site, within these gene promoter regions.Nuclear factor-erythroid 2-related factor 1  acts as a transcription factor belonging to the cap’n’collar (CNC) basic-region leucine zipper (bZIP) family, which is indispensable for maintaining both cellular homoeostasis and organ integrity during normal development and growth, as well as the adaptative responses to other pathophysiological processesNrf1 is allowed for differential transcriptional expression to yield multiple mRNA transcripts (between ~1.5 kb and ~5.8 kb) and subsequently alternative translation into distinct polypeptide isoforms (between ~25-kDa and ~140-kDa), which are determined to be differentially distributed in embryonic, fetal and adult tissues, including liver, brain, kidney, lung, heart, skeletal muscle, bone, testis, ovary, placenta and others7. Amongst such isoforms, the full-length Nrf1 (designated Nrf1α) is yielded by the first translation initiation signal within the main open reading frame (ORF) of alternatively spliced mRNA transcripts, in which the exon 4  is removed from its long isoform TCF11 (transcription factor 11) in the human5. Albeit Nrf1 lacks the Neh4L region, it was identified to retain a strong transactivation activity that is largely similar to the TCF11 ability8.The single gene of 9. By bioinformatic analysis, it is thus inferred that Nrf1β lacks the N-terminal domain  and its adjacent acidic domain 1 11. Later, Nrf1β is also determined to exhibit a weak transactivation activity14, but stimulation of Nrf1β activity appears to be dependent on distinct stressors that had been administrated in different cell lines15. Furthermore, a small dominant-negative isoform, called Nrf1γ13, is produced by another potential in-frame translation starting at the putative methionine of position 584, as well as by the putative endoproteolytic processing of longer Nrf1 proteins. When generation of Nrf1γ is blocked, the transactivation activity of Nrf1β is significantly increased12. On the contrary, when Nrf1γ is forcedly expressed, the consequence enables it to make a possible interference with the functional assembly of each of the active transcription factors (i.e. Nrf1α or Nrf2) with its heterodimeric partner (i.e. sMaf and other bZIP proteins), in order to down-regulate expression of AP1-like ARE-driven target genes13.By contrast with Nrf1α, the short isoform Nrf1β [which was early designated as LCR-F1 (locus control region-factor 1)] is determined to be generated through the in-frame translation that is initiated by an internal perfect Kozak’ starting signal (5′-puCCATGG-3′), which is situated within and around the four methionine codons between positions 289 and 297 in the mouseNrf1α, HEK293DNrf1β and HEK293ENrf1γ, which are allowed for stably tetracycline (Tet)-induced expression of human Nrf1α, Nrf1β, and Nrf1γ, respectively. The similarities and differences of structural domains of these three isoforms were shown diagrammatically  in the parallel experiments. The inducible expression of Nrf1α, Nrf1β and Nrf1γ was under control by Tet for distinct experimental requirements, before which the positively-selected clones of cell lines were maintained within double antibiotics 150 μg/ml hygromycin B and 15 μg/ml blasticidin. The resulting selected cell lines were named HEK293control, HEK293CNrf1α, HEK293DNrf1β and HEK293ENrf1γ, respectively. Thus they were also abbreviated to control, Nrf1α, Nrf1β, and Nrf1γ, as shown in the subsequent experimental results. Of note, the stable expression of distinct isoforms was monitored by interaction of Tet with its repressor TetR leading to release from the Tet operator and then induction of interested gene transcription.To gain an in-depth insight into distinct contributions of human Nrf1 isoforms  to the precision regulation of different subsets of its target genes, each of these isoform-expressing cell lines was herein established by Flp recombinase-mediated integration on the base of the Flp-In T-REx-293 host cells. These cell lines had been transfected with each of pcDNA5/FRT/TO-V5 expression constructs for distinct cDNA sequences encoding Nrf1α, Nrf1β and Nrf1γ  ≤0.001 (ref.17), along with an absolute value of Log2 (fold change) ≥1, and identified by calculating each gene expression in sample groups versus controls as indicated in the Stat Chart  were up-regulated  but the other 44 genes (i.e. 4.4%) were down-regulated (green bar). And, transcriptional expression of as many as 1675 genes was also significantly regulated upon stable expression of Nrf1β in the Tet-inducible HEK293DNrf1β cells, 94.8% of which (i.e. 1588 genes) were up-regulated and the other 5.2% (i.e. 87 genes) were down-regulated  were down-regulated, the other 86 genes (i.e. 76.1%) were still up-regulated . An overview of the primary sequencing data was then depicted in Table art Fig. . By compart Fig.  in theirted Fig. , red barted Fig. . By strited Fig. . This imAfter normalization by the control, each isoform-specific or their common DEGs amongst three Nrf1 isoforms were statistically shown with the Venn diagram Fig. . Their sdatabase for annotation, visualization and integrated discovery (DAVID) of bioinformatic resources consists of an integrated biological knowledgebase with analytic tools, which is used for systematic extraction of biological features and/or meanings associated with large lists of genes18. Therefore, in order to investigate the relationship and difference in gene regulation by amongst these three Nrf1 isoforms, the enriched functional annotation of GO terms . This is helpful to further understand biological functions of distinct subsets of genes regulated by each Nrf1 isoform.Further data analysis of thousands of DEGs involved in distinct biological processes is an important downstream task following RNA-Seq to understand relevant meanings of those ‘interested’ genes regulated by each isoform of Nrf1. Of note, the gene ontology (GO) is an internationally-standardized functional classification system of genes, which offers a dynamically-updated controllable vocabulary with a strictly defined concept to describe comprehensively properties of distinct genes and their products in any organisms, and also covers three major domains, including cellular component, molecular function and biological process. The enrichment analysis of GO provides all relevant terms that are significantly enriched in the DEGs, by comparison to the genomic background, and then filters the DEGs that correspond to potential biological functions. The s Tables –S7 was ms Tables –S10 was The top 20 of significantly enriched functional annotation terms Fig. . Almost Furtherly, the results obtained from relevant pathway enrichment analysis Tables –S10 reve20 ) being down-regulated by Nrf1β. By contrast, 6 of the top 10 RPKM Nrf1γ-specific genes were up-regulated, whereas the other 4 genes were down-regulated by this isoform ) was down-regulated. Another 9 of the top 10 RPKM genes were commonly up-regulated by three Nrf1 isoforms, with only an exception of LCP1 (lymphocyte cytosolic protein 1) that was significantly down-regulated by Nrf1β but up-regulated by the other two isoforms 22 and STRING (lower three panels)23, respectively. Distinct expression profiles of these putative genes involved in the networks were extracted from distinct RNA-Seq data sets, which were reflected with different gradient colors in accordance with fold changes , BRD9 (bromodomain containing 9) and FBXW7 (F-box and WD repeat domain containing 7) were differentially regulated by Nrf1γ from other two isoforms, another three genes RUSC2 (RUN and SH3 domain containing 2), CAPN1  and HCFC1 (host cell factor C1) were differentially regulated by Nrf1β, the remaining one gene C8ORF33 (chromosome 8 open reading frame 33) was differentially regulated by Nrf1α. By comparison of the network identified with the STRING database, 5 of 11 genes showed an uniform regulation trend, whilst the other 6 genes were differently regulated: i) three genes MAFG, NRF1 (nuclear respiratory factor 1) and FBXW7 were differentially regulated by Nrf1γ from other two isoforms; ii) two genes C3ORF35 (chromosome 3 open reading frame 35) and MAFK (v-maf musculoaponeurotic fibrosarcoma oncogene homolog K) were differentially regulated by Nrf1β; and iii) only one gene SP7 (Sp7 transcription factor) was differentially regulated by Nrf1α. Collectively, the interaction network analysis indicates that three isoforms of Nrf1 could diversely regulate its target genes. Such changes in the expression abundance of one isoform may also influence its overall transcriptional regulation of Nrf1-target genes. In fact, transcriptional expression of different subsets of target genes (driven by AP1-like AREs) was principally attributable to the precision regulation by distinct functional heterodimers of CNC-bZIP family members  with small Maf or other bZIP factors24. Such transcription of these factors was also further compared to determine distinct Nrf1-specific effects . This notion also appeared to be further supported by the observation that both the mRNA levels , siRNA (i.e. siNrf2) or constructive activation of Nrf2 mutant (i.e. caNrf2ΔN). In turn, these results also unravel that significant changes in the expression of Keap1 are accompanied by altered expression of Nrf2, but not Nrf1α. This implies a feedback circuit existing between Nrf2 (rather than Nrf1) and its negative regulator Keap1.Intriguingly, the N-terminal Neh2 (Nrf2–ECH homology 2) domain of Nrf2, which contains a redox-sensitive Keap1 (Kelch-like ECH-associated protein 1)-binding degron targeting the homoeostatic CNC-bZIP protein to ubiquitin ligase cullin-3 and Rbx1-dependent proteasomal degradation pathwayels Fig.  and protels Fig.  of Keap129. Importantly, coordinated regulation of proteasomes by Nrf1, but not Nrf2, occurs through proteasome-limited proteolytic processing of the former CNC-bZIP protein into a mature active factor to mediate Nrf1-target proteasomal gene expression in the ‘bounce-back’ response to relative lower doses of proteasomal inhibitors33. Nrf1-mediated induction of proteasomes subunits results in significant increases in mRNA expression levels of all proteasomal subunits only upon exposure to lower concentrations of proteasomal inhibitors, but this feedback compensatory response is prevented by high concentrations of proteasomal inhibitors34. Therefore, there exists a bidirectional regulatory feedback circuit between Nrf1 and the proteasome35. However, our data revealed that almost none of proteasomal subunits were differentially expressed at basal levels, because they appeared to be unaffected by stably-induced expression of any one of Nrf1 isoforms  is crucial for eukaryotic cells to adjust its capacity of protein degradation to changing proteolytic requirements, because these proteins are marked with polyubiquitin chains targeting for their degradation by the 26S proteasome, an ATP-dependent complex consisting of the 20S proteolytic particle capped by one or two of the 19S regulatory particlesrms Fig. .Figure 5PSMA1, PSMA4, PSMB7, PSMC2 and PSMD12 by real-time qPCR  of BTZ enabled for significant stimulation of Nrf1γ to increase mRNA expression levels of all the examined proteasomal subunits, albeit the detailed mechanism(s) remains to be further explored.To address this, we validated Nrf1-stimulated induction of proteasomal subunits by its inhibitors. Experimental cells that had pretreated with 1 μg/ml of Tet alone or plus a low (0.01 μmol/L) or a high (10 μmol/L) concentration of bortezomib (BTZ) for 16 h were subjected to further determination of mRNA expression levels of some proteasomal subunits, including PCR Fig. . As expe38, GCLM , GCLC 42, MT1E 43, PGC-1β 44 and LPIN1 (lipin1)45. As expected, the experimental results revealed that these Nrf1-target genes exhibited different trends to be expressed specifically in distinct isoform-expressing stable cells , and then determined by real-time qPCR. As shown in , KPNB1 (karyopherin subunit beta 1), FOXC1 (forkhead box C1) and ELOVL5 (ELOVL fatty acid elongase 5) were differentially up-regulated by Nrf1β, at less increased levels than equivalents regulated by Nrf1α and Nrf1γ  and KRT19 (keratin 19) were differentially down-regulated by Nrf1β  was down-regulated by Nrf1α and Nrf1γ, but up-regulated by Nrf1β 24. The transcriptional expression of AREs-driven genes is thus determined by differential recruitment of Nrf1, Nrf2 and Nrf3, in different combinations with each of their heterodimeric partners , to target gene promoters. Of note, Nrf1 and Nrf2 are two important CNC-bZIP transcription factors expressed ubiquitously in various vertebrate tissues and thus elicit their putative combinational or competitive functions. Relative to the well-known water-soluble Nrf2, less attention has hitherto been drawn to the membrane-bound Nrf124. However, major discoveries that had been made in the past twenty-five years have convincingly revealed that Nrf1, but not Nrf2, has been shown to be indispensable for maintaining cellular homoeostasis and organ integrity during normal development and healthy growth, as well as a vast variety of other patho-physiological processes. Importantly, several significant pathological phenotypes were developed in different transgenic mice (expressing distinct mutants of loss-of-function of Nrf1), including embryonic lethality, fetal anemia, lipid metabolic disorder, obesity, fatty liver, NASH, liver cancer, neurodegenerative diseases, hyperinsulinemia, diabetes, Warbug effect with high glycolysis. In addition, other mice expressing gain-of-function mutants of Nrf1 displayed glucose metabolic disorder, insulin resistance, diabetes and reduced body-weight. Thus it is inferable that the functional activity of Nrf1 is finely tuned, to a robust homeostatic extent, by a steady-state balance between its production and the concomitant processing into distinct isoforms before being turned over, which are together coordinated to confer on the host cytoprotection against a variety of cellular stresses.Differential expression of different subsets of cognate genes is dependent on their enhancers and promoter regions containing distinct Nfe2l1/Nrf1 gene, though differentially expressed, in different mammalian species. These isoforms are synthesized by translation through distinct initiation signals  embedded in different lengths of open reading frames, portions of which can be alternatively spliced from intact or longer transcripts. The resulting variations in the abundance of each isoform may not only influence the whole transcriptional functions of Nrf1 to regulate distinct subsets of cognate target genes and also contribute to the nuance in between distinct pathological phenotypes24. Therefore, it is of crucial important to determine differences in transcriptional regulation of cognate genes mediated by each Nrf1 isoform. Although this is hard, our present study has identified differential expression profiles of distinct target genes regulated by Nrf1α, Nrf1β and Nrf1γ alone or in their cooperation respectively. Here, we have further determined differences in the transcriptional regulation of Nrf1-target genes by between each Nrf1 isoforms. Notably, Nrf1α and Nrf1β are two major isoforms contributing to the main Nrf1-mediated transcription of downstream genes at RNA levels, such that a vast majority of differentially expressed genes are up-regulated by Tet-inducible expression of these two isoforms. On the contrary, stably Tet-inducible expression of Nrf1γ as a putative dominant-negative inhibitor is likely to interfere with the putative functional assembly of active transcription factors , leading to down-regulation of several key genes, some of which are up-regulated by Nrf1α and Nrf1β. These findings are consistent with our previous reports48. Collectively, these findings are very helpful to elucidate which isoforms of Nrf1 contribute to different transcription of distinct subsets of target genes that are involved in those significant pathological phenotypes. Thus, this study has provided three cell models to facilitate the future development of Nrf1 isoform-specific targets for chemoprevention against relevant diseases 49.Accumulating evidence has also unraveled that over eleven of distinct Nrf1 isoforms are produced from the single 48, which revealed that this low molecular weight isoform acts as a dominant-negative inhibitor of Nrf1 competitively against the functional heterodimeric assembly of either an active transcription factor  or another homologous trans-repressor (i.e. Bach1 and Bach2) with one of their cognate partner sMaf or other bZIP proteins . Therefore, it is plausible that either transactivation or transrepression of distinct subsets of similar and/or different target genes driven by AP1-like ARE/EpRE batteries depends possibly on a nuance between different temperospatially-assembled heterodimeric complexes of these CNC-bZIP factors with their partners. This is to say, understandably, that the dominant-negative form of Nrf1γ is much likely to counteract (or interfere with) the putative activity of its prototypic factors Nrf1α/β to transactivate or transrepress their downstream genes. For this reason, it is thus deduced that if some genes are down-regulated by Nrf1α/β, this down-regulation is abolished or even reversed to allow for their activation by Nrf1γ. In addition, there also exists an exception that a few number of Nrf1-target genes , whereas antibody against KEAP1 was purchased from Sangon Biotech . Mouse monoclonal antibody against the V5 epitope was from Invitrogen Ltd, whilst anti-β-actin and secondary antibodies were obtained from Zhongshan Jinqiao Co .All chemicals were of the highest quality commercially available. Hygromycin-B and blasticidin were purchased from Invitrogen Ltd, which served as double screening drugs to select the positive clones by final concentrations of 150 μg/ml and 15 μg/ml, respectively. The inducible reagent tetracycline hydrochloride was from Sangon Biotech Co  and used at a final concentration of 1 μg/ml. The proteasome inhibitor bortezomib was purchased from ApwxBio (USA). The antibody against endogenous Nrf1 proteins was acquired from our lab  containing 2 μg/ml protease inhibitor cocktail . The protein concentration of lysates was quantified by using BCA Protein Assay Reagent . Equal amounts of protein prepared from cell lysates were loaded into each electrophoretic well so as to be separated by SDS-PAGE, followed by visualization by immunoblotting with each of indicated antibodies as described previously53, and β-Actin was served as an internal control to verify amounts of proteins that were loaded in each well.Experimental cells were harvested in a denatured lysis buffer . Then, 1.5 μg of total RNAs were used as a template for the subsequent synthesis of cDNA by using a RevertAid first strand cDNA synthesis kit . The resulting cDNA products (15 ng) served as the templates of quantitative real-time PCR within 5 μl of the MixGoTaq qPCR Master Mix . Each of RT-qPCR with distinct pairs of primers , along with the negative control cells, were allowed for growth in 6-well plates, before being induced by tetracycline (1 μg/ml) for 12 h. Such similar three independent experiments were conducted to prepare the samples on the same experimental conditions. Total RNAs were extracted by using an RNAsimple total RNA kit  and the integrity of RNAs was checked by an Agilent Bioanalyzer 2100 system . For each sample, equal amounts of total RNAs from three independent experiments were pooled for RNA-Seq. Subsequently, RNA-Seq was carried out by Beijing Genomics Institute  on an Illumina HiSeq 2000 sequencing system  after the sample library products are ready for sequencing.54. Then, the clean reads were mapped to the reference human genome (GRCh37/hg19 from UCSC database) by using SOAP2 (ref.55), and gene expression levels were calculated by using the RPKM (Reads Per Kilobase of feature per Million mapped reads) method16. The expression of genes regulated by each Nrf1 isoforms, relative to the control sample, was calculated as Log2 (fold change), with a P-value calculated corresponding to the differential gene expression test and FDR , and the differential expressed genes (DEGs) were further identified by the Poisson distribution model method (PossionDis)56, which was developed referring to “The significance of digital gene expression profiles”57 by BGI. Similar methods have also been applied in recent publications60. Both FDR ≤0.001 and the absolute value of Log2 (fold change) ≥1 were herein taken as the threshold to identify differentially expressed genes. To give a better understanding of potential functions of the DEGs, both GO and KEGG pathway analysis were performed by using the online tools DAVID (https://david.ncifcrf.gov/) and KEGG (http://www.kegg.jp/) databases, respectively. In addition, the putative interaction networks of Nrf1-related genes were searched from the databases of BioGRID (https://thebiogrid.org/) and STRING (https://string-db.org/), before being annotated with sequencing results by the Cytoscape software61. The sequencing data have been submitted to NCBI SRA (PRJNA501789). For detailed descriptions, please see relevant supplementary contents at http://www.genomics.cn/en/index.Before sequencing analysis, all mRNAs were fragmented into short fragments (about 200 bp) and the low-quality reads in which its percentage  is greater than 50% were removed during data filtering. After examining the sequence quality and removing the “dirty” raw reads, which contain low quality reads and/or adaptor sequences, clean reads  were generated and stored as the FASTQ formatt-test or Fisher’s exact test, as appropriate. The resulting value of p < 0.05 was considered as a significant difference. In addition, statistical determination of the ‘dry’ sequencing analysis was aforementioned.The ‘wet’ experimental data provided in this study were represented as the mean ± SD and were analyzed using the Student’s Supplementary Info FileDataset 1


When focused, this source can ionize gas targets, which we demonstrate here through the ionization of atomic xenon at wavelengths ranging from 5 μm to 9 μm. This opens up new opportunities for fundamental atomic and molecular physics, enabling experimental tests of strong-field ionization theories in the extreme long-wavelength, few-cycle limit and the direct excitation of vibrational transitions in organic molecules.For the last several decades, the wavelength range accessible for strong-field, few-cycle studies has remained limited to the visible, near infrared and mid-wave infrared regimes. In particular, sources in the long-wave infrared have been lacking. We report the development of a 1 kHz, few-cycle laser source with up to a 9  These applications would strongly benefit from longer wavelength driving sources as the pondermotive energy of an electron accelerated by a laser field scales with the square of the wavelength. The extension of strong-field, ultrafast science into the MWIR regime9 has allowed for scientific advances such as the first demonstration of a table-top coherent X-ray source11, the generation of isolated attosecond pulses with photon energies up to 300 eV2, as well as the first femtosecond-resolved measurement of chemical bond dynamics with angstrom resolution5.The interaction of strong laser fields with atoms and molecules has been of considerable interest for decades. Technical developments in this area have led to, among other breakthroughs, the generation of attosecond pulsesμm) regime. The quadratic wavelength scaling of the energy of the photoelectrons presents an enticing reason for pushing to longer wavelengths. Additionally, broadband pulses in this regime allow for the simultaneous excitation of many molecular ro-vibrational energy levels13. Unlike LWIR sources emphasizing high average power, intense few-cycle fields in the LWIR promise to deliver new strong-field studies in molecular phenomena14. Other interesting possibilities with intense, ultrafast sources in this wavelength regime include the observation of dynamics due to the breakdown of the dipole approximation at modest intensities16 and extreme modification of optical waveforms via shock formation17. However, the development of high energy, ultrafast laser sources in the LWIR has proven to be a technologically challenging endeavor.Such studies have motivated the development of intense ultrafast sources at even longer wavelengths in the long-wave infrared  durations18 and have progressed slowly since their early use in strong-field ionization experiments19. A standard method for generating femtosecond pulses in this region employs difference frequency generation (DFG), where two high frequency fields are mixed in a suitable nonlinear medium to produce a new field which can possess a much longer wavelength24. Recently, a new source based on optical parametric chirped-pulse amplification has demonstrated 200 μJ, ~8-cycle pulses with a MWIR wavelength of 7 μm25, while another based on DFG has achieved sub-cycle fields at 30 μJ, spanning 2–9 μm, but with a central wavelength of 4.2 μm26. To date, no strong-field ionization studies have been reported with these sources due to the difficulty of achieving the required peak intensities.The most intense sources currently available in the LWIR, based on COμm. Using this apparatus we demonstrate, to the best of our knowledge, the first strong-field ionization in the LWIR with femtosecond pulses. These results utilize a collinear DFG scheme, which has several advantages. First and foremost, the technique requires only one nonlinear crystal after an optical parametric amplifier (OPA) while simultaneously delivering fields without spatial chirp (ensuring feasibility for achieving high peak intensities). Secondly, the short path length and minimal number of transmissive optics produces near transform-limited, few-cycle pulses. Finally, with a front-end based on Ti:Sapphire and an OPA, technology present in most strong-field science laboratories, this technique can be readily implemented at other facilities.In this work we demonstrate an intense, tunable light source in the LWIR possessing only 2.8 cycles under the FWHM at a central wavelength of 8.9 et al.27. DFG between the signal and idler of the OPA is performed in an interferometer-based setup. Collinear overlap is ensured by observing the spatial chirp of the LWIR mode in the far field. This allows the beam to be focused to the smallest possible area and, thus, produce the highest peak intensities.As shown in Fig. 2(I) crystal . With this crystal, up to 4.2 mJ  of the OPA can be used in the DFG process. Incident energies greater than 4.2 mJ accelerate degradation of the crystal and show signs of nonlinear back-conversion. By tuning the central wavelength of the signal from 1300 nm to 1450 nm, with the corresponding idler changing from 2000 nm to 1730 nm, the central wavelength of the DFG output is tuned from approximately 3.5 μm to 9 μm.For DFG, we use a 1 × 1 × 0.1 cm, anti-reflective (AR) coated AgGaSμm region that is highly reflective (≈90%) of the OPA signal. This increases the usable peak power in the LWIR by as much as a factor of 2.2 at wavelengths greater than 8 μm without hindering other characteristics of the LWIR field, such as pulse duration (see section 2 of Supplementary Information). As shown in Fig. μJ at 5.3 μm and up to 80 μJ at 8.9 μm center wavelengths.As we require a collinear arrangement for high peak intensities, the LWIR field must be separated from the higher frequency beams using a 1 mm thick, AR coated, low-pass, Germanium (Ge) filter. However, the OPA’s large fluence on the Ge surface produces a high density of free carriers that can attenuate the LWIR pulse by as much as 55%. We reduce the free carrier density by incorporating a coated, 1 mm thick, zinc selenide window before the Ge filter, which has an AR coating for the 3–12 28. As shown in Fig. μm. Figure μm central wavelength, Fig. μm yield pulses with fewer than three optical field cycles under the FWHM.A characterization of the electric field is performed using a cross-correlation frequency resolved optical gating (XFROG) deviceμm on the top row and down to 5.3 μm on the final row.In addition to the retrieved fields shown in 2(b) and 2(c), XFROG results over a broad range of wavelengths from our LWIR apparatus are shown in Fig. 27. This, combined with the OPA’s passively CEP stable idler29, provides a straightforward route to phase stable LWIR fields.We plot the electric field for a carrier envelope phase (CEP) of zero for Fig. μm, 8 μm, and 5.3 μm (not shown) using an ion time-of-flight (iTOF) apparatus. Figure χ(3) processes in the AGS crystal was investigated and, to our best knowledge, these processes are not present in this system. The iTOF was designed to support the 2.5 cm back-focus required for the experiment, otherwise it is a common arrangement with an extractor, repeller, and drift tube. Figure + ionized using a λ = 8.9 μm pulse which clearly shows all the abundant Xe isotopes. No higher charge states of Xe were observed. Further details of the detection system are found in the Methods section. Figure + as a function of input pulse energy (top axis) in log-log scale for λ = 8 μm. These results represent, to the best of our knowledge, the first strong-field ionization results, with femtosecond pulses, in the LWIR. The wavelength dependence of ionization versus intensity will be presented in an upcoming publication.As proof of this source’s ability to deliver intense fields across a large wavelength range, we measure the strong-field ionization of xenon (Xe) atoms at 8.9 μm, we undertook two measures for determining the intensity range. First, we determined the beam waist at the gas target in the iTOF using a scanning slit and a thermopile detector. The measurement, described in section 3 of the supplement, determined that the setup could achieve a 1/e2 radius waist of 30 μm for the back-focusing system in Fig. 2. Further improvement in the beam quality, such as using a spatially filtered OPA30, can dramatically improve the focusing characteristics without sacrificing the peak power. Our second route to determining the peak intensity implemented a fit of our data to the Perelomov-Popov-Terent’ev (PPT)31 formulation of strong-field ionization theory. PPT was recently shown to accurately describe experimental results for multi-cycle pulses from 400 nm to 3.9 μm32. Fitting our data, shown as the dashed blue line in Fig. 2 (bottom axis), which is a factor of 2.5 lower than the estimate from our beam waist measurement. As the source delivers few-cycle fields, the effect of a varying CEP was also considered and found to have an insignificant change in the overall ionization rate. If we consider the intensities predicted from our fit to PPT, we observe Xe+ ionization at a Keldysh parameter33 value of 0.23 and in a region where the HHG cutoff19 is nearly 400 eV. With more attention to the focusing quality of the source, it should be possible to reach the 100 TW/cm2 mark.To better understand the ionization versus intensity results at 8 μm to 8.9 μm. Our collinear DFG scheme maintains broadband transform-limited pulses in this wavelength range. We experimentally measured the pulse durations to be between 70 to 90 fs, depending on the wavelength, delivering a 5.8 cycle field at 5.3 μm and 2.8 cycles at 8.9 μm. Over this entire wavelength range, the source can be focused on target to peak intensities high enough to induce strong-field ionization in Xe. To the best of our knowledge, these are the longest wavelengths that a femtosecond pulse has been used to ionize neutral atomic targets, paving the way for many new and exciting studies in the intense long-wave infrared regime.We have demonstrated the generation of intense, few-cycle pulses in the LWIR tunable from 5.3 2 Type (I) crystal (AGS). This allows the weak sum frequency beam to be spatially separated from the much stronger gating field using an iris. After collecting the generated sum frequency signal in a spectrometer, a spectrogram  pulse, as compared to using only the Ge window. A series of additional tests were performed to determine the nature of this observation. The analysis determined that the transmission of LWIR light through Ge increases as the fluence of the OPA signal decreases on the Ge surface. This can be explained by the OPA signal (0.85–0.9 eV) performing a one photon transition to Ge’s direct band gap (0.8 eV), which subsequently allows LWIR photons to be lost to free carrier absorption (FCA). This is further supported by two facts. First FCA rates increase wavelength squared, making 8 μm more susceptible than 5 μm, which is what we see. Secondly, the AR coating on the ZS window reflects more than 90% of the OPA signal, which removes the first step of the proposed mechanism.When a 1 mm thick ZS window is inserted before the 1 mm thick Ge window in the path leading out of the AGS crystal, we find that the available energy for the LWIR pulse increases approximately 20% (220%) for a 5 −7 torr, consisting mainly of common atmospheric gases. The Xe gas sample was introduced by backfilling the vacuum chamber to a pressure of approximately 2 × 10−5 torr for the studies at 8.0 μm. For each intensity point, a time-of-flight spectrum is recorded for 1.2 million laser shots, which requires 20 minutes. Ion yields shown in Fig. All ionization experiments were performed using an ion time-of-flight (iTOF) mass spectrometer. The chamber background pressure was 1⋅10μJ) amount of LWIR leakage on the PyD. This allows for single-shot monitoring of the LWIR pulse energy by recording the peak of the electronic signal with an analogue to digital converter synchronously with the iTOF signal. The single-shot PyD was calibrated for each wavelength, by measuring the average power with a thermopile sensor and also taking into account the in-coupling window of the vacuum chamber. Finally, a periscope steers the main LWIR into the iTOF apparatus and is back focused inside the chamber with a spherical concave mirror of 25 mm focal length.Simultaneously, a pyroelectric detector (PyD) was used to the measure a small fraction of the LWIR beam to allow for single-shot pulse energy tagging. For this tagging, we used the Fresnel reflection from a Ge window See Fig.  filteredSupplementary information


Protocatechuic acid exerts multiple health‐promoting effects such as anticancer, anti‐atherosclerosis, and neuroprotection in animal models. While protocatechuic acid produced in the lower gastrointestinal tract by microbial catabolism of several flavonoids is bioavailable, the pharmacokinetics of protocatechuic acid has not been evaluated so far in humans following its oral consumption. In this open‐label and single‐dose pharmacokinetic trial, 16 healthy adults followed a low‐phytochemical diet for three days. Next, after overnight fasting, participants consumed 150 g of chicory containing 248 μmol of protocatechuic acid. Blood, urine, and fecal samples were collected before and up to 24 hr after chicory consumption. Protocatechuic acid in the free and glucuronide/sulfate‐conjugated forms was almost undetectable in serum, urine, and fecal samples before chicory consumption. Chicory consumption increased the levels of protocatechuic acid and its glucuronide/sulfate conjugates in biological samples. The maximum serum concentrations of protocatechuic acid in the free‐, glucuronide‐, and sulfate‐conjugated forms were 3,273, 519, and 340 nmol/L, respectively. The recovery of total protocatechuic acid in blood circulation, urine, and feces was 23.79%, 12.17%, and 12.79% of the ingested dose, respectively. Moreover, glucuronide and sulfate conjugates of protocatechuic acid made up 34.79%, 60.15%, and 72.70% of its total recovery in blood circulation, urine, and feces, respectively. Collectively, protocatechuic acid from chicory is bioavailable and undergoes partial glucuronidation and sulfation in human adults, and its regular consumption may exert health‐promoting effects. Chicory was from Hebei Vilof Agritech Co., Ltd, which were transported in a light‐proof foam box with a cold supply chain and consumed by the subjects in 24–36 hr after harvest.Protocatechuic, gallic, caffeic, 5‐caffeoylquinic, and chicoric acid were purchased from Chengdu Must Bio‐Technology Co., Ltd. Caftaric acid was from Extrasynthese . Solutions of standard phenolic acids were prepared in 50% methanol–water (volume/volume). ‐glucuronidase  and women (n = 8) participants by word of mouth among the physical examinees at the Physical Examination Center of Guangdong Second People's Hospital. The participants were nonsmokers and not taking any medication or nutritional supplements in the last three months. All participants had regular daily bowel movements. None of the women participants were pregnant. They were healthy as judged by a medical questionnaire, with normal blood values for blood pressure, aspartate aminotransferase, and alanine aminotransferase. All participants were informed about the purpose of the study and gave written informed consent before their inclusion in the trial. This trial was approved by the Ethics Committee of the School of Public Health at Sun Yat‐sen University [2017 No. 009] and registered at ClinicalTrials.gov as ChiCTR1800014393. This trial was conducted in accordance with the Declaration of Helsinki of 1975 as revised in 2008.We recruited healthy adult men  bioavailability of protocatechuic acid from chicory juice. The flowchart of participants in this pharmacokinetic trial is summarized in Figure 2.4Phenolic acids in chicory and biological samples were extracted and determined by a high‐performance liquid chromatography assay with electrochemical detection (HPLC‐ECD) as previously described with minor modifications ; time to achieve maximum serum concentrations (Tmax); the area under the concentration–time curve to 24 hr (AUC0‐24); and terminal elimination half‐life (T1/2z). The apparent bioavailability of protocatechuic acid together was quantified using AUC0‐24 divided by its ingested amount. Urinary and fecal recoveries were calculated using the total content of urinary and fecal protocatechuic acid divided by its ingested amount.The pharmacokinetic parameters of protocatechuic acid in the free, glucuronide, and sulfate conjugates were calculated using DAS 2.1 (BioGuider Co.), with a noncompartmental model: maximum serum concentrations (C2.7SD. The significance of differences between the baseline (0 hr) and the indicated time points after chicory consumption was assessed by ANOVA for repeated measures and Dunnett's 2‐tailed t test, assuming the baseline values as the reference category. p < .05 was considered significant.Data are presented as means ± 33.1Phenolic acids consist of derivatives of hydroxybenzoic acids and hydroxycinnamic acids. Representative HPLC chromatograms of phenolic acid extracts from chicory are shown in Figure 3.2All participants, which included eight men and eight women, were generally healthy with normal blood biochemistry and body mass index Table .3.3max of 3,273 ± 729 nmol/L at 1 hr postconsumption  was incubated in fresh human fecal suspensions with gut microbiota, and the content of protocatechuic acid was then quantified. The initial substrate dose was 1 µmol, from which 95.8 ± 3.3%, 86.0 ± 3.0%, 79.3 ± 5.7%, 91.5% ± 4.7%, and 72.8 ± 4.0% for gallic, caffeic, 5‐caffeoylquinic, caftaric, and chicoric acid, respectively, were recovered at the initial time point (0 hr) from the fecal suspensions  is highly bioavailable (Czank et al., ‐/‐ mice through similar mechanisms by promoting endothelium‐dependent vasodilation (Liu et al., Because in vitro studies have shown that protocatechuic acid and its glucuronide/sulfate conjugates at the dosage of 1,000 nmol/L exert anti‐atherosclerosis‐related effects in macrophage‐derived foam cells and vascular endothelial cells (Amin et al., Previous studies in humans and animal models have shown that gut microbiota metabolism of certain polyphenols is able to generate protocatechuic acid (Czank et al., Glucuronidation and sulfation are the most important phase II metabolic pathways for polyphenols (Manach, Scalbert, Morand, Remesy, & Jimenez, 1/2z of sulfated conjugate, recoveries of urinary glucuronide conjugate, and fecal‐free form of protocatechuic acid were significantly different between men and women. These data highlighted that much work is needed to be accomplished in the issue of gender‐dependent pharmacokinetics of polyphenols in humans, which might help to explain the variable health‐promoting efficacy between men and women (Campesi, Marino, Cipolletti, Romani, & Franconi, Although the pharmacokinetics of polyphenols in humans has been well studied, little is known about their differences between men and women (Lu & Anderson, Two limitations in this study should be born in mind. Firstly, although our findings from the in vitro fermentation assays showed that gallic, caffeic, 5‐caffeoylquinic, or chicoric acid could not be converted to protocatechuic acid by fresh human feces with gut microbiota, we did not exclude the possibility that the conversion of those phenolic acids to protocatechuic acid happens in humans owing to the fact that only a small proportion of gut microbiota can be cultivated in vitro (Gilbert et al., 5In conclusion, we have demonstrated for the first time that protocatechuic acid is bioavailable and undergoes partial glucuronidation and sulfation in healthy human adults following chicory consumption. Because protocatechuic acid possesses multiple biological effects in animal models, future clinical trials are worthy to confirm whether chicory recapitulates its effects in humans.None.D.W. conceived and designed the experiments; J.Z., H.X., L.H., H.W., and Q.L. performed experiments; J.Z, H.X., L.H., H.W., and Q.L. analyzed data; W.L. revised the manuscript; and D.W. and J.Z. wrote the paper. All authors read and commented the manuscript.This study was approved by the Ethics Committee of the School of Public Health at Sun Yat‐sen University. Click here for additional data file.


Digital, art‐ and story‐based resources can be viable and engaging knowledge translation strategies in health care. Understanding the usability of these approaches can help maximize their impact. The aim of this work is to understand what aspects of ‘My Asthma Diary’, an art‐based digital knowledge translation tool for parents of children with asthma, has an impact on usability.Sequential explanatory mixed methods pilot study.Eighteen parents of children with asthma reviewed ‘My Asthma Diary’ in a paediatric emergency department and completed a usability questionnaire. Follow‐up interviews were conducted with five parents and analysed with qualitative description.We identified four themes which complemented the quantitative results: (a) the eBooks are relatable and mirror personal experience; (b) the digital format is convenient and easy to navigate; (c) the narrative structure aids learning; and (d) the narrative and illustrations are synergistic. We summarize core usability considerations for subsequent research and creative knowledge translation tool development in other contexts. How can creative methods of representing research be optimized to improve research engagement and optimize illness self‐management? Creative knowledge translation (KT) strategies are increasingly regarded as viable means of supporting this engagement by providing research evidence in palatable, meaningful and comprehensible formats  and two mediating factors   as visible in Figures 2.2hosted  arts‐based KT resources usable, in part because of the emergent status of such approaches   were approached on a convenience basis to discuss the study. Eligible parents were English speaking and identified as a primary caregiver for a child with asthma or asthma‐related concern. Parents were invited to review one of four eBook prototypes (i.e. prototype three), complete a paper‐based 27‐question usability questionnaire and consent to a follow‐up interview. Parents were fully informed about the study prior to providing verbal consent to participate.).The questionnaire included four pre‐ and post‐test visual analog scale (VAS) measures  anchored at 0  and 10 (‘very much’), 15 descriptive VAS measures specific to three dominant usability constructs  and four open‐ended questions. The VAS was developed based on recognized usability constructs and additional measures adapted for arts‐based KT  and descriptive statistics. Statistical significance was set at .05. Open‐ended questions were content analysed. Qualitative data were analysed inductively, guided by qualitative description. Audio recordings were listened to repeatedly, detailed notes and analytic memos were made, and data were loosely grouped thematically. Themes were revised and categories regrouped as analysis progressed. Rigour was enhanced through detailed memos and through team meetings where emerging findings were discussed and rigorously questioned. Problems encountered while using the eBooks were classified as critical or general incidences (i.e. usability issues that do or do not impair use), and participants were asked for suggestions to improve the eBooks function in relation to occurring incidences. We then used a sequential mixed analysis approach to analysing and integrating qualitative and quantitative data , parametric , which they regarded as an appropriate length . Descriptive statistics illustrate positive ratings on constructs related to effectiveness, efficiency and satisfaction. Outliers were encountered; for instance, participants occasionally provided very low ratings on the enjoyment, contribution of drawings to information and quality of information subscales. Open‐ended responses and follow‐up qualitative interviews suggest that participants may not have accessed the information icons  embedded throughout the tool, relying exclusively on the storyline to receive information. One participant who provided a low enjoyment rating found the story was ‘scary and intimidating’ for first time parents dealing with asthma , confidence in managing asthma day‐to‐day (p = .055) and confidence in managing asthma exacerbations (p = .021) using the related‐samples Wilcoxon signed‐ranks test . We ran paired t tests to obtain data on mean differences and standard deviations. Paired t tests revealed significant improvements in knowledge (t(17) = 3.09, p = .007) between pre  and post  measures; and significant improvements in confidence in managing asthma exacerbations (t(17) = 2.33, p = .032) between pre  and post  measures.Given the small quantitative sample and lack of random sampling, we conducted pre–post evaluations of knowledge gain (6.2n = 1), technical school (n = 3) and university (n = 1) levels. Interviews lasted an average of 36 min (range: 24–41.83 min). Iterative data collection and analysis confirmed that participants were identifying few usability issues; it appeared unlikely that further incidents would be encountered through additional data collection.Of eight parents who consented for follow‐up, five  completed a telephone interview. All self‐identified as the child's primary caregiver; had been managing asthma for a period ranging from 1–8 years; and had educational attainments at the high school ( = 8.29), including its format, content, method of delivery and length. Four themes were identified from the qualitative analysis and are presented here as narrative statements: (a) the eBooks are relatable and mirror personal experience; (b) the digital format is convenient and easy to navigate; (c) the narrative structure aids learning; and (d) the narrative and illustrations are synergistic. These themes are discussed in reference to affiliated quantitative measures and open‐ended questionnaire responses.Parents had an overwhelmingly positive response to the eBooks. They consistently reported that the information was clear  and relevant, the digital format was appealing and easy to use , and the storyline (mean = 7.46) and characters (mean = 7.03) were relatable and engaging. Parents unanimously reported satisfaction with the eBook (mean6.2.1(participant 3)It talks to you…and everyone can find a way to relate to it. Participants unanimously reported that the eBooks were highly relatable (mean = 7.46), regardless of whether the storyline reflected precisely their experiences. The emotional connection made possible through the telling and reliving of the fictional mother's story was a source of relatability for many participants and the uncertainty surrounding the asthma trajectory often mirrored participants' experiences. Delivering information through a first‐person narrative enhanced participants' emotional connection and provided emotional validation —the eBook was regarded as more personal than other methods of delivering information (e.g. pamphlets) and enabled participants to ‘put yourself in their shoes… it's a real person… it's real information… it was very comforting’ (participant 3).All participants emphasized how the eBook was appropriate for use with their child with asthma. The engaging illustrations and story made the eBook useful for reading with their child . Participant 3 expressed, ‘you can't put a medical document in front of a four‐year old’ and ‘here's a story and… she relates to this!’. Participants one and four credited the eBook to opening a dialogue about asthma with their child. Parents recognized the need for children to learn about his/her asthma and the eBook was seen as a useful tool to facilitate this learning.Participants expressed that the storyline reflected the reality of living with childhood asthma, thereby increasing its relatability. Specifically, participants noted that the ending of the story, wherein the central character (a 6‐year‐old boy) achieves asthma control but does not outgrow asthma, was particularly salient, believable (participants 1–5) and hopeful (participant 3). As participant 1 emphasized, the ‘growing out of asthma’ discourse was prevalent among her family and friends; the concluding sentiments in the eBook provided a needed reflection of the reality of living with asthma. For others, the turning point signified by the asthma clinic referral (participant 4), or the seasonal variations in asthma  present in the narrative were particularly reflective of parents' experiences.One participant identified that the use of multicultural names and characters effectively ‘reflects the communities we are living in’ and signified inclusivity (participant 5). While other participants did not explicitly identify this as a factor having an impact on relatability, it may have influenced the high quantitative (mean = 7.46) and unanimously positive qualitative relatability ratings among the diverse participant sample. Namely, all participants, including the fathers, were able to relate to the story, despite the narrator being a mother. As participant 2 explained, he was still able to relate to the narrative because stories need not be interpreted literally and because having a mother as the central character may indeed reflect a reality of the caregiving experience.6.2.2(participant 3)The digital format was convenient. It gives instant access anytime I need it.  = 8.29). Participants identified that convenience, portability and accessibility were augmented through the digital delivery. Digital delivery was also linked with ease of use for participants, a finding supported by the quantitative ‘ease of finding information’ measure, (mean = 7.77). Although each participant used the digital format in different ways, all participants used the navigational arrows to advance through the eBook. However, if and how participants accessed the ‘important’ information icons and external resource links varied, with few participants making use of the external resources .All participants were satisfied with the digital delivery format, consistent with the quantitative results .Most participants preferred the digital delivery to a paper‐based format . One participant (participant 3) expressed nostalgia for paper‐books but concurrently stated that the eBook was a highly useful and convenient modality for asthma education. Similarly, in an open‐ended response, one participant requested paper copies so that the information could be re‐visited. Overwhelmingly, participants recognized the digital format as appropriate and reflective of the current digital era: a belief that ‘everyone has a computer’ (participant 4) or would use their hand‐held device to access information (participant 2) rendered the eBook a contemporary educational modality. For one participant (participant 5), the digital approach enhanced the eBooks' credibility: ‘you see some of the brochures and you just know that they've been there for 20 years and are stale and out of date, not that reliable… because the format was digital you think, okay this is new, or newer, than a dusty old brochure or pamphlet and likely easier to update’.6.2.3(participant 4)The storyline helped understand the parent's perspective… and triggers your memory. Participants found the story to be an effective communication approach because it helped organize information in a manner that ‘flowed’. The narrative gave structure to information that otherwise would be a series of facts. Parents expressed satisfaction with the quality of information provided; embedding information within a sequential narrative allowed participants to follow and make sense of the information (participants 1–5). In this way, the narrative ‘made the information manageable’ (participant 3), improved information recall  and enhanced the eBooks' ease of use, thereby supporting the quantitative findings. = 7.05, enjoyment in reading eBook measure). As participant 3 expressed, ‘Putting the important information into a story, makes me feel better… it's not just some medical information booklet that you get and you read it and your bored and you don't understand half of what they're saying…’. An important component of this appeal was that the storyline situated the information in the context of family life: ‘this wasn't just a generic asthma brochure but was a story of a family. That reinforced the seriousness of the condition’ (participant 5).Participants identified that the flow of information was appealing , which supports the quantitative findings . In addition to the seasons, participants often identified the discussion of triggers , specifically pertaining to the family pet, as reflective of their own experiences (participant 5). When the storyline did not reflect the specifics of the participant's experience, the eBook was regarded as useful in ‘getting the conversation going’ (participant 1).6.2.4(participant 4)The narrative and the visuals work together.  = 6.97, contribution of drawings to information; mean = 8.1 contribution of story to information; mean = 9.04, ease of understanding), which suggests that the narrative and visual components of the eBook work together synergistically. Indeed, participants reinforced this notion through statements such as ‘the storyline triggers your memory… and the pictures help us think’ (participant 4).The illustrations enhanced the eBooks' aesthetic and aided in its functionality. Participants expressed that the illustrations contributed to comprehension, engagement and overall appeal (participants 1–5). They referred to the interplay between the story and illustrations, a finding supported by participants' quantitative ratings (meanThe illustrations reinforced and complimented the eBook content. Participants emphasized how the illustrations improved information clarity and made the research‐based information more comprehensible (participants 1–5); the amount of detail in the illustrations reinforced the key learning points  and at times, helped re‐orientate participants to what their children were experiencing. A particularly effective illustration in this respect was a visual rendering of constricted bronchial tubes Figures , 3, 4, tThe participants enjoyed the illustrations and style, which helped relate to and engage with the information provided. As one participant expressed, ‘if there were no drawings, or illustrations, [the eBook] would be very boring’ (participant 2). While four out of five participants related to the illustrations and one felt the depictions were ‘dead‐on’ (participant 1), participant 4 found the mother looked exceedingly worried; she expressed concern that this could worry her child if they read the eBook together. Interestingly, the same participant felt that the eBook could emphasize the experience of uncertainty more thoroughly. Overall, the qualitative results support and expand on the quantitative findings: the story contributed more to the information than the illustrations, but both were integral to the effectiveness, efficiency and satisfaction of engaging with the eBook.6.3(participant 2)Color is more attractive and captures our imaginations. Participants unanimously preferred the coloured versions of the eBooks  to the black and white versions . The black and white versions were seen as ‘unfinished’ (participant 5), less relatable and the illustration details  and ‘familiar’ (participant 4) graphic novel style of prototype four, while the remaining two preferred the illustration style of prototype three. Participants were divided into whether the comic‐like depiction was engaging, or whether it appeared juvenile.Participants had mixed perspectives about font. Participant 1 preferred the boldness and ease of reading of the comic font , where participant 2 found the comic font difficult to read, stating, ‘there is no sign of where words start or stop’. Two participants commented on how the text font ‘matched’ the illustration styles for all prototypes; the sans‐serif font was regarded as a ‘softer, more conversational’ match for the diary (participant 5).6.4We encountered two general incidents. First, 80% of participants did not notice the Table of Contents icon in the lower corner of each page. We classified this as a general incident since it did not impede participants' ability to navigate sequentially through the eBook. Participants suggested various strategies to improve icon visibility . Second, two participants identified that the ‘important’ icons  could be more noticeable. Three additional open‐ended responses suggested that the ‘important’ icons were not accessed. Participants suggested describing these icons and their importance in the eBooks' introduction, or adding colour or animation to the icons.6.5There were limitations to our study. We did not conduct real‐time, in‐person usability testing in a naturalistic environment, which could have revealed different usability issues , fit with purpose (e.g. does it communicate research in a meaningful and comprehensible way?), efficiency  and satisfactory display (e.g. is using the tool enjoyable?). Regardless of the creative modality employed, consideration to user‐centred design principles is necessary to ensure that KT tools are functional and appealing Bastien, . This isThe authors have no conflicts of interest to report.The Research Ethics Board at the University of Alberta granted approval for this study. Informed consent was obtained prior to parent's involvement in the quantitative study arm. Parents consented to follow‐up qualitative interviews following completion of the questionnaire. Verbal consent was obtained prior to completing the telephone interviews.


PM2.5 samples from three rural  and two urban (more populated) sites (Bayamón and Ponce) from Puerto Rico were analyzed for various inorganic constituents. A total of 59 trace elements were analyzed, of which eight were considered with the greatest toxic potential. The highest annual average concentration of PM2.5 was reported at the urban site of Ponce (5.82 ± 1.40 μg m−3), while Bayamón’s average concentration was not as high (4.69 ± 1.30 μg m−3) compared to concentrations at the rural sites Humacao, Guayama, and Guayanilla  compared to that of all the rural sites (~ 6.40 μg m−3). The lowest summer PM2.5 values were obtained at the Humacao site with an average of 5.76 μg m−3. Average Cu and Zn concentrations were 3- and 2-fold higher at the urban sites (0.68 ng m−3 and 6.74 ng m−3 respectively) compared to the rural sites (0.17 ng m−3 and 4.11 ng m−3). Relative toxicity of inorganic PM extract indicates Bayamón (urban) and Guayama with similar low LC50 followed by Humacao, Guayanilla, and finally Ponce (urban) with the highest LC50. Of the eight potential toxic metals considered, only Fe was found to be higher at the rural sites. To our understanding, there are different sources of emission for these metals which potentially indicate main anthropogenic sources, together with the trade winds adding periodically volcanic and African Dust Storm particulates that affect Puerto Rico. These results are the first of their kind to be reported in Puerto Rico.The exposure to airborne particulate matter (PM) and its constituents is an important factor to be considered when evaluating their potential health risk. Transition metals found in PM are known to contribute significantly to the exacerbation of respiratory ailments. Exposure to these constituents results in the induction of oxidative stress in the bronchial epithelium, thus promoting the secretion of inflammatory mediators. Therefore, it is important to know the contributions of PM Furthermore, inorganic materials like toxic metals, minerals , salts (ammonium sulfates and nitrates), and soil dust particles are also part of airborne PM complex mixture . This type of study and information has neither been previously reported nor has been a comparison done between rural and urban sites on the island. With the current understanding of the importance of PM metal toxicity, we must further explore the presence of these constituents, and fill the gap in the understanding of the etiology and source of exposure against the Puerto Rican population.In this study, we evaluate and compare particle pollution in two main urban cities of Puerto Rico, one on the north coast (Bayamón) and one on the southern (Ponce). We also evaluate PM2.5 have been measured and reported in a systematic way around the island of Puerto Rico  on PTFE 2.0-μm Teflon filters by the Puerto Rico Environmental Quality Board (PR-EQB), which maintains a PM monitoring stations network at various locations in the Island as part of their monitoring program , May to August (S2), and September to December (S3) for Bayamon and Humacao. For the other stations  two period were included, Jan-May and August to December. S2 was of major interest given that it is associated with the passing of African dust events which are seasonal and enriched particulate matter with both organic and inorganic compounds  and Bayamón (n = 44 filters); and the rural stations: Humacao (n = 43 filters), Guayama (n = 94 filters), and Guayanilla (n = 94 filters). These sites can be observed in Fig. PM2.5 filters obtained from the PR-EQB were soaked in 50 mL of hexane/acetone 1:1 ratio and extracted using a microwave extraction apparatus (MAE) . Extraction glassware was washed using a modified cleanup procedure from a previous study  and heated at 90 °C for 2 h to minimize polyatomic ion interferences. The final volume of extracts was completed to 2.50 mL with Nanopure water . The element concentration was determined for all samples after two readings of 5 replicates each (10 readings per sample of which the instrument discards 3 and the average obtained from the remaining). Analyses included the following elements: silver (Ag), aluminum (Al), arsenic (As), gold (Au), barium (Ba), beryllium (Be), bismuth (Bi), bromide (Br), calcium (Ca), cerium (Ce), cesium (Cs), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), dysprosium (Dy), europium (Eu), erbium (Er), iron (Fe), gallium (Ga), gadolinium (Gd), germanium (Ge), mercury (Hg), holmium (Ho), lanthanum (La), lithium (Li), lutetium (Lu), potassium (K), magnesium (Mg), manganese (Mn), molybdenum (Mo), sodium (Na), nickel (Ni), neodymium (Nd), niobium (Nb), praseodymium (Pr), lead (Pb), rubidium (Rb), rhenium (Re), sulfur (S), antimony (Sb), scandium (Sc), selenium (Se), silicon (Si), samarium (Sm), tin (Sn), strontium (Sr), tantalum (Ta), terbium (Tb),thorium (Th), titanium (Ti), thallium (Tl), thulium (Tm), uranium (U), vanadium (V), tungsten (W), yttrium (Y), ytterbium (Yb), zinc (Zn), and zirconium (Zr). Analytical curves were prepared using PE5 and PE29 standard multi-element stock solutions , in a 5% v/v HNO3 solution. An Rh solution (40 μg L−1 in 1% v/v HNO3) was used as an internal standard, injected online with every solution.Dried samples of 0.10 to 1.00 mg were sent out for elemental analyses using an ICP-MS . Each sample was diluted in 100 μL of bi-distilled nitric acid  following the manufacturer’s procedure on human lung cells (BEAS-2B). Briefly, an average of 104 cells was seeded per well in a 96-well plate and left adhering for 24 h, then treated with PM2.5 extract for another 24 h at a concentration range of 25 to 200 μg/mL in cell media. After incubation, each well was carefully aspirated to remove treatment media and the MTT -2,5-diphenyl tetrazolium bromide) reagent added at 50 μL of media + 50 μL/MTT Reagent per well. The plates were incubated for 3 h at 37 °C, aspirated carefully to remove the MTT reagent containing media, and then 150 μL of MTT solubilizing solution added to each well. The plate was placed on a shaker gently and left for 15 min to facilitate the solubilization of the formed formazan salts. The absorbance was measured at near 570 nm using a microplate reader.The cytotoxicity of PM2.5 concentrations was carried out using Student’s t test for paired comparisons between municipalities ). The toxic metal distribution and sampling site comparison graphs were generated and plotted using GraphPad Prism 8.0.0 for Windows . The determination of the LC50 for the airborne PM2.5 graphs was generated using the ATT Bioquest LC50 Calculator . The statistical analysis to assess the relationship between sampling site PM2.5 concentrations are shown in Figs. 2.5 concentration was registered in Ponce (7.58 μg m−3) followed by Guayama (6.92 μg m−3), Bayamón (6.57 μg m−3), Guayanilla (6.5 μg m−3), and Humacao (5.76 μg m−3). All of the summer PM2.5 averages were significantly higher (p < 0.0001) when compared to the rest of the year at each municipality , while the relative humidity ranged from 72.00 to 80.30%. The prevailing wind directions were E/SE (4.2 and 1.4%) and NE (7.1 and 1.4%). The overall wind speed was an average of 5.7 mph. Bayamón (north) and Ponce (south) were among the municipalities covering more than 50% of the manufacturing operations according to the manufacturing industry census of Puerto Rico (2015). The annual average PM2.5 concentration at the Ponce urban site was significantly higher than those of any of the other rural and urban (Bayamón) sites, with an approximate mean of 4.69 ± 0.41 μg m−3, while Ponce had an average of 5.82 ± 0.22 μg m−3. Guayama and Guayanilla followed with an annual mean concentration of 4.93 ± 1.20 μg m−3 and 4.88 ± 1.50 μg m−3 respectively. Humacao’s annual PM2.5 concentration mean was the lowest of all the concentrations (4.33 ± 0.22 μg m−3) recorded for the 12 months.The annual PM2.5 extracts at all sites  was rich in As and Ni, followed by Guayanilla which is located downwind west from Ponce. The Guayanilla extract was the richest in Hg followed by Bayamón. The relative abundance of Hg in the extract was encountered during the period from January to May. The annual level of Hg in the Guayanilla PM2.5 extract was significantly higher than any of the other sites studied. Nevertheless, Hg was found to be present at all sites in airborne PM2.5 extracts . Bayamón and Humacao are similar in the sense that they both have relatively high concentrations of Fe, Al, Zn, Cu Fig. –c and e,ama Fig. . In summ−3respectively), while not measured in Ponce, and the lowest concentration in Guayama (0.34 ng m−3). These concentrations need to be adjusted considering the extraction efficiency of the method employed (see the “−3 respectively) followed by Guayanilla (1.90 ng m−3) and Ponce; respectively, the lowest concentrations found were in Guayama (0.04 ng m−3). The highest levels of Ni in the air were characteristic of Ponce with an annual average of 2.50 ng m−3 followed by Guayanilla and Guayama (0.17 and 0.07 ng m−3 respectively). This presents supporting evidence that Ni is a toxic metal characteristic of the southern region. Similarly, the highest concentrations of ambient Hg were detected in Guayanilla (0.4 ng m−3) while the concentration of Hg in the rest of the sites was in the picogram per cubic meter range (Bayamón and Ponce followed). Therefore, Hg is also characteristic of the southern ambient air particles, particularly in hot spots. Another characteristic toxic metal in the south coast was As. The highest levels of As found in the sites studied in Puerto Rico ambient air were found in Ponce (0.30 ng m−3), while Guayanilla and Guayama exhibited values ≈ 0.06 ng m−3 and Bayamón and Humacao values of ≈ 0.02 ng m−3. Metals characteristic of the northern coast were Cu in Bayamón and Humacao sites with concentrations in the orders of 0.70 and 0.20 ng m−3 respectively, while the other southern stations ranged from 0.09 to 0.02 ng m−3 . Concentrations of chromium also found in Bayamón and Humacao were very similar in air ≈ 0.18 ng m−3, followed by Ponce (0.13 ng m−3) and Guayanilla and Guayama (0.04 and 0.02 ng m−3 respectively). Vanadium also had a similar concentration profile compared to Cr, with the highest concentrations in the air found in Bayamón and Humacao (0.12 and 0.15 ng m−3 respectively), followed by Ponce (0.1 ng m−3), and thereafter by Guayanilla and Guayama (0.05 and 0.02 ng m−3 respectively). Concentrations of Cd and Pb were very similar between all stations. Therefore, we describe Cr as to be at higher concentrations and thus associated with urban activities.Atmospheric toxic metal concentrations throughout the year are higher in urban sites compared to those of rural sites. The highest annual average concentrations were observed in Bayamón and Humacao sites  and a positive control (Triton X-100) , a similar concentration of 25 μg/mL did not show any relative toxicity, while the cell viability was comparable to the control. A higher concentration of this extract (50 μg/mL) killed almost 31.79 ± 0.52% of the cultured cells. The LC50 for the inorganic PM2.5 extract from Humacao was 68.89 μg/mL. However, when we obtain the relative toxicity of the other airborne PM extracts, we determined that Guayama (LC50 43.5 μg/mL) and Bayamón (44.71 μg/mL) have comparable LC50 exhibiting similar toxic responses on human lung epithelial cells. These two sites comprise airborne PM2.5 that generates the most toxic response among all the sites studied. Guayanilla and Humacao follow with also close LC50s of 52.7 μg/mL and 68.89 μg/mL respectively. Finally, Ponce (urban site) in the south coast includes airborne PM2.5 which generates the least toxic effect with the highest LC50 (78.5 μg/mL). There were large differences in the toxicity between (Ponce and Humacao) when compared to Bayamón, Guayama, and Guayanilla  was not as toxic as our northern counterpart Bayamón, as an exposure of 100 μg/mL, saw a decrease of 36.49 ± 0.01% in cultured cells. Instead, the rural site of Guayama had a higher cytotoxic effect from the inorganic particles extract, with a 100-μg/mL exposure decreasing their survivability by 38.21 ± 0.08%. Guayanilla  shows more toxicity than its closest urban site of Ponce.BEAS-2B cell cultures were exposed to the annual PM00) Fig. . The res2.5 extracts from five different sites in Puerto Rico, selected by their location  and urban (north and south), during the 12 months in 2013. The samples were selected at random from a previously sampled pool granted by the Environmental Quality Board of Puerto Rico. This assessment provides a first-of-its-kind insight into the inorganic constituents of PM2.5 in the northeastern and southern regions of Puerto Rico, classifying the sample sites as rural or urban locations. Besides, it provides the basis for an initial comparison of PM2.5 in the Caribbean region.Here, we report the measurement of 59 trace elements found in airborne PM2.5 from the municipalities of Guayama and Guayanilla  of which 4 days of ash fallings were reported for Puerto Rico.Bayamón and Ponce (both urban sites) are considered among the most developed areas with industrial growth in Puerto Rico. Hence, the enrichment of particle pollution increases due to elevated anthropogenic activity. Humacao , located in the east of the Island, is considered to have low industrial activity, most of which is related to the pharmaceutical industry. Humacao receives most of its airborne particulate from the reposition and deposition from the North Atlantic trade winds and the Caribbean Sea including local natural and anthropogenic sources. This study also includes PM50 43.5 μg/mL). Some of these toxic metals, such as Ni and V, are products originating from fossil fuel combustion, reported in the air samples monitored during the year  compared to the other sites throughout the island of Puerto Rico, particularly more rural areas like Humacao, Guayama, and Guayanilla. As much as 1728 pounds of several toxic metals  were released into the ambient air in Guayama by just one company in 2013 (EPA Toxic Release Inventory (TRI)). Incidentally, the ambient air from Guayama represents one of the most toxic extracts evaluated , we choose to focus on the most commonly associated with industrial activity. The most abundant toxic metals at the different sites are shown and summarized in Fig. Considering the differences among the distribution of toxic metals based on location, we found that V, Al, Pb, and Fe were higher at the rural sites compared to the urban sites; meanwhile, Cu and Zn, which are known to be vastly deposited in more urbanized regions due to fossil fuel combustion and biomass burning, were found to be elevated at the urban sites. It appears that the main reason for the high concentration levels of the toxic metals studied are the various sources of emission previously discussed in our study areas. Likewise, trade winds transporting African dust also contribute to the overall load of higher metal concentrations during the periods of May–September Fig. .2.5 extract’s inorganic constituents, both rural and urban sites in the E/NE region of Puerto Rico had the highest toxic potential  compared to the southern region. This is could very well be associated with an earlier deposition of particles (E/NE) transported by the trade winds. In terms of the relative toxicity of PM2.5 inorganic extracts, the urban site of Bayamón and rural site of Humacao and Guayama had the most significant cytotoxic effect, with an LC50 difference of 35.10% for the average toxic response. Considering the urban and rural sites in the south region, there is a distinguishable trend towards a higher geological toxic potential increasing to the northeast. Guayama’s PM2.5 toxicity closely resembles that of Bayamón’s, compared to the other southern sites, followed by Ponce and the least toxic being Guayanilla (Fig. 2.5 concentrations to which humans are exposed to, these extracts do not consider the coarse particulate fraction found in PM2.5. The concentrations to which we are exposed could be pari passu to the ones used as treatments in this study. PM2.5 constituents are known to be inducers of oxidative stress, shown by a significant reduction in the intracellular GSH concentrations and an increase in the expression of antioxidizing enzymes (Palleschi et al. Evaluating the toxic potential of the PMlla Fig. . The dat2.5 as previously stated. Furthermore, this study did not consider organic pollutants, which constitute another source of exposure to humans. There were some limitations to this study given the lack of available air filter samples provided by the PR-EQB due to distribution with other proyect. However these samples are valuable since after  Hurricane Maria September of 2017 all filter samples at the agency’s storage bank were lost. This adds to the importance and significance of our work since similar information will not be available regarding trace metal concentrations on the island of Puerto Rico before those years. We believe these results will be the beginning of major studies regarding the change of particle pollution in the Puerto Rico environment before the events of Hurricane Maria, The Great Earthquake of January 7, 2020, and how anthropogenic pollution changed during the COVID-19 crisis.The results of this study show that the toxic metals associated with industrial activity, particularly biopharmaceuticals, contribute to the enrichment of the local particle pollution in the Puerto Rico environment. Although this seems to be the case, addressing our previous study and contrasting the trace metal content, there has been a considerable decrease in airborne toxic metal content in Puerto Rico (Molinelli et al.