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Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2-induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway and/or gastrointestinal barrier damage and mitigate virus spread.

Retinoic acid (RA) has been shown to improve epithelial and endothelial barrier function and development and even suppress damage inflicted by inflammation on these barriers through regulating immune cell activity. This paper thus sought to determine whether RA could improve baseline barrier function and attenuate TNF-α-induced barrier leak in the human bronchial epithelial cell culture model, 16HBE14o- (16HBE). We show for the first time that RA increases baseline barrier function of these cell layers indicated by an 89% increase in transepithelial electrical resistance (TER) and 22% decrease in 14 C-mannitol flux. A simultaneous, RA-induced 70% increase in claudin-4 attests to RA affecting the tight junctional (TJ) complex itself. RA was also effective in alleviating TNF-α-induced 16HBE barrier leak, attenuating 60% of the TNF-α-induced leak to 14 C-mannitol and 80% of the leak to 14 C-inulin. Interleukin-6-induced barrier leak was also reduced by RA. Treatment of 16HBE cell layers with TNF-α resulted in dramatic decrease in immunostaining for occludin and claudin-4, as well as a downward “band-shift” in occludin Western immunoblots. The presence of RA partially reversed TNF-α’s effects on these select TJ proteins. Lastly, RA completely abrogated the TNF-α-induced increase in ERK-1,2 phosphorylation without significantly decreasing the TNF-driven increase in total ERK-1,2. This study suggests RA could be effective as a prophylactic agent in minimizing airway barrier leak and as a therapeutic in preventing leak triggered by inflammatory cascades. Given the growing literature suggesting a “cytokine storm” may be related to COVID-19 morbidity, RA may be a useful adjuvant for use with anti-viral therapies.

Using the 16HBE 14o‐ human airway epithelial cell culture model, calcitriol (Vitamin D) was shown to improve barrier function by two independent metrics – increased transepithelial electrical resistance (TER) and reduced transepithelial diffusion of 14C‐D‐mannitol (Jm). Both effects were concentration dependent and active out to 168 h post‐treatment. Barrier improvement associated with changes in the abundance of specific tight junctional (TJ) proteins in detergent‐soluble fractions, most notably decreased claudin‐2. TNF‐α‐induced compromise of barrier function could be attenuated by calcitriol with a concentration dependence similar to that observed for improvement of control barrier function. TNF‐α‐induced increases in claudin‐2 were partially reversed by calcitriol. The ERK 1,2 inhibitor, U0126, itself improved 16HBE barrier function indicating MAPK pathway regulation of 16HBE barrier function. Calcitriol's action was additive to the effect of U0126 in reducing TNF‐ α ‐induced barrier compromise, suggesting that calcitriol may be acting through a non‐ERK pathway in its blunting of TNF‐ α – induced barrier compromise. This was supported by calcitriol being without effect on pERK levels elevated by the action of TNF‐α. Lack of effect of TNF‐ α on the death marker, caspase‐3, and the inability of calcitriol to decrease the elevated LC3B II level caused by TNF‐α, suggest that calcitriol's barrier improvement does not involve a cell death pathway. Calcitriol's improvement of control barrier function was not additive to barrier improvement induced by retinoic acid (Vitamin A). Calcitriol improvement and protection of airway barrier function could in part explain Vitamin D's reported clinical efficacy in COVID‐19 and other airway diseases. Vitamin D utility in COVID‐19 infection may be due in part to improvement of airway epithelial barrier function and support of airway epithelial barrier function in \"cytokine storm\" situations. Remodeling of tight junctions is part of this process. Although the ERK‐1,2 pathway is involved in regulation of airway barrier function, Vitamin D is seemingly acting through a non‐ERK‐1,2 pathway to achieve its epithelial barrier support here.

This study focuses on the issue of whether orally administered zinc (gluconate) (26 mg BID) can induce the remodeling of gastrointestinal barrier function and reduce passive leak across the human intestinal mucosal barrier in situ. Increased transmucosal leak has been implicated in diseases as diverse and seemingly unconnected as Inflammatory Bowel Disease (IBD), Celiac Disease, Autism Spectrum Disorders and Alzheimer’s Dementia. Our current investigation represents the first patient-based study to examine the effect of zinc on gastrointestinal epithelial tight junctions and gastrointestinal barrier leak in otherwise healthy test subjects. Using independent test subject groups for each endpoint, three separate molecular analyses indicated that zinc treatment can achieve a positive outcome: (1) RNA-seq analyses of intestinal biopsies showed salutary patterns of gene transcription changes dealing with not only transcripts of junctional proteins but also transcripts mitigating the proinflammatory state, as well as dedifferentiation (both modulators of tight junction permeability); (2) Western immunoblot analyses of intestinal tissue indicated that tight junctional protein expression was being modified by the administered zinc, most notably Claudin-2 and Tricellulin; (3) zinc treatment induced a reduction in serum levels of a functional marker of passive intestinal leak, namely the GI microbiome metabolite D-Lactate. The data collectively suggest that orally administered zinc can induce remodeling of the intestinal epithelial barrier, resulting in the reduction in GI barrier leak. The overall safety and economy of supplement levels of zinc suggest that this micronutrient could be efficacious as an adjuvant therapy to reduce the condition known as leaky gut, and possibly therefore be protective regarding diseases postulated to involve leaky gut.

Retinoic acid (RA) has been shown to improve epithelial and endothelial barrier function and development and even suppress damage inflicted by inflammation on these barriers through regulating immune cell activity. This paper thus sought to determine whether RA could improve baseline barrier function and attenuate TNF-[alpha]-induced barrier leak in the human bronchial epithelial cell culture model, 16HBE14o- (16HBE). We show for the first time that RA increases baseline barrier function of these cell layers indicated by an 89% increase in transepithelial electrical resistance (TER) and 22% decrease in .sup.14 C-mannitol flux. A simultaneous, RA-induced 70% increase in claudin-4 attests to RA affecting the tight junctional (TJ) complex itself. RA was also effective in alleviating TNF-[alpha]-induced 16HBE barrier leak, attenuating 60% of the TNF-[alpha]-induced leak to .sup.14 C-mannitol and 80% of the leak to .sup.14 C-inulin. Interleukin-6-induced barrier leak was also reduced by RA. Treatment of 16HBE cell layers with TNF-[alpha] resulted in dramatic decrease in immunostaining for occludin and claudin-4, as well as a downward \"band-shift\" in occludin Western immunoblots. The presence of RA partially reversed TNF-[alpha]'s effects on these select TJ proteins. Lastly, RA completely abrogated the TNF-[alpha]-induced increase in ERK-1,2 phosphorylation without significantly decreasing the TNF-driven increase in total ERK-1,2. This study suggests RA could be effective as a prophylactic agent in minimizing airway barrier leak and as a therapeutic in preventing leak triggered by inflammatory cascades. Given the growing literature suggesting a \"cytokine storm\" may be related to COVID-19 morbidity, RA may be a useful adjuvant for use with anti-viral therapies.

The published literature makes a very strong case that a wide range of disease morbidity associates with and may in part be due to epithelial barrier leak. An equally large body of published literature substantiates that a diverse group of micronutrients can reduce barrier leak across a wide array of epithelial tissue types, stemming from both cell culture as well as animal and human tissue models. Conversely, micronutrient deficiencies can exacerbate both barrier leak and morbidity. Focusing on zinc, Vitamin A and Vitamin D, this review shows that at concentrations above RDA levels but well below toxicity limits, these micronutrients can induce cell- and tissue-specific molecular-level changes in tight junctional complexes (and by other mechanisms) that reduce barrier leak. An opportunity now exists in critical care—but also medical prophylactic and therapeutic care in general—to consider implementation of select micronutrients at elevated dosages as adjuvant therapeutics in a variety of disease management. This consideration is particularly pointed amidst the COVID-19 pandemic.

Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2-induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway and/or gastrointestinal barrier damage and mitigate virus spread.

Introduction: The chemopreventive action of zinc (Zn) regarding experimentally-induced squamous cell carcinoma in animal models has been well documented, as has the cancer promotional state associated with nutritional Zn deficiency (Fong, LY et al., 2016). This study examined the effect of orallyadministered Zn in humans on Barretts tissue. Methods: In a prospective clinical study involving 120 Barretts Esophagus patients, patients were treated with Zn gluconate (26 mg Zn, BID) or sodium gluconate (placebo) in slow-dissolving lozenge form, for 14 days prior to a surveillance endoscopy. Barretts biopsy samples were frozen on dry ice, and then analyzed by microarray analyses for miRNA and mRNA, as well as PAGE and western immunoblot protein analyses. Results: Zn-treated patients exhibited molecular level changes in their Barretts tissue consistent with efficient Zn delivery to the Barretts epithelia in a pharmacologically effective dose. Specifically, we observed statistically significant 70% and 50% decreases in the known Zn-regulated proteins, Znt-1 and Protein Kinase C-a. Concerning possible cancer chemopreventive action in the Zn treatment group compared to a matched placebo (sodium gluconate) group, statistically significant increases in six microRNA species with documented tumor suppressor activity were observed in Barretts biopsies: miR 125b-5p, 132-3p, 548z, 551a, 504 and 518b. Decreases of microRNA species, miR-21 and miR-31 were also observed. In western immunoblot studies of Barretts tissue, a 30% decrease in VEGF-R2 and a 35% decrease in COX-2 were observed. In mRNA microarray analyses of Barretts tissue, 6- and 3-fold downregulation of claudins -3 and -4 (P < 0.01), along with 2.3 fold downregulation of cyclin D2 (P < 0.005), and 1.6-fold downregulation of LYN (P < 0.01), MAPKK (P < 0.05) and STAT5A (P < 0.01) in the Zn-treatment group also suggest a potential chemopreventive action of Zn in Barretts Esophagus. This is supported by statistically significant Zn-mediated upregulations of mRNA transcripts for protein kinase A (catalytic subunit) (2.7-fold, P < 0.01), trefoil factor 1 (1.4-fold, P < 0.05), and keratin-5 (8-fold, NS). Conclusion: The combined lines of evidence from miRNA arrays, protein immunoblots and mRNA arrays suggest that orally-administered Zn could have a cancer-preventive action regarding Barretts progression to esophageal adenocarcinoma, and argue for future prospective clinical studies.

Abstract Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2-induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe respiratory dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway barrier damage and mitigate virus spread. Competing Interest Statement The authors have declared no competing interest.