Membrane prewetting by condensates promotes tight-junction belt formation.

Biomolecular condensates enable cell compartmentalization by acting as membraneless organelles1. How cells control the interactions of condensates with other cellular structures such as membranes to drive morphological transitions remains poorly understood. We discovered that formation of a tight-junction belt, which is essential for sealing epithelial tissues, is driven by a wetting phenomenon that promotes the growth of a condensed ZO-1 layer2 around the apical membrane interface. Using temporal proximity proteomics in combination with imaging and thermodynamic theory, we found that the polarity protein PATJ mediates a transition of ZO-1 into a condensed surface layer that elongates around the apical interface. In line with the experimental observations, our theory of condensate growth shows that the speed of elongation depends on the binding affinity of ZO-1 to the apical interface and is constant. Here, using PATJ mutations, we show that ZO-1 interface binding is necessary and sufficient for tight-junction belt formation. Our results demonstrate how cells exploit the collective biophysical properties of protein condensates at membrane interfaces to shape mesoscale structures.

MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via protein kinase C.

Glycosylated mucin proteins contribute to the essential barrier function of the intestinal epithelium. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in human intestinal epithelial monolayers, MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TEER buildup in ΔMUC13 cells could be prevented by addition of MLCK, ROCK or protein kinase C (PKC) inhibitors. The levels of TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not affect TEER. The increased buildup of TJ complexes in ΔMUC13 and MUC13-ΔCT cells was dependent on PKC. The responsible PKC member might be PKCδ (or PRKCD) based on elevated protein levels in the absence of full-length MUC13. Our results demonstrate for the first time that a mucin protein can negatively regulate TJ function and stimulate intestinal barrier permeability.

PALS1 is a key regulator of the lateral distribution of tight junction proteins in renal epithelial cells.

The evolutionarily conserved apical Crumbs (CRB) complex, consisting of the core components CRB3a (an isoform of CRB3), PALS1 and PATJ, plays a key role in epithelial cell-cell contact formation and cell polarization. Recently, we observed that deletion of one Pals1 allele in mice results in functional haploinsufficiency characterized by renal cysts. Here, to address the role of PALS1 at the cellular level, we generated CRISPR/Cas9-mediated PALS1-knockout MDCKII cell lines. The loss of PALS1 resulted in increased paracellular permeability, indicating an epithelial barrier defect. This defect was associated with a redistribution of several tight junction-associated proteins from bicellular to tricellular contacts. PALS1-dependent localization of tight junction proteins at bicellular junctions required its interaction with PATJ. Importantly, reestablishment of the tight junction belt upon transient F-actin depolymerization or upon Ca2+ removal was strongly delayed in PALS1-deficient cells. Additionally, the cytoskeleton regulator RhoA was redistributed from junctions into the cytosol under PALS1 knockout. Together, our data uncover a critical role of PALS1 in the coupling of tight junction proteins to the F-actin cytoskeleton, which ensures their correct distribution along bicellular junctions and the formation of tight epithelial barrier.

Tight junction protein occludin is an internalization factor for SARS-CoV-2 infection and mediates virus cell-to-cell transmission.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads efficiently by spike-mediated, direct cell-to-cell transmission. However, the underlying mechanism is poorly understood. Herein, we demonstrate that the tight junction protein occludin (OCLN) is critical to this process. SARS-CoV-2 infection alters OCLN distribution and expression and causes syncytium formation that leads to viral spread. OCLN knockdown fails to alter SARS-CoV-2 binding but significantly lowers internalization, syncytium formation, and transmission. OCLN overexpression also has no effect on virus binding but enhances virus internalization, cell-to-cell transmission, and replication. OCLN directly interacts with the SARS-CoV-2 spike, and the endosomal entry pathway is involved in OCLN-mediated cell-to-cell fusion rather than in the cell surface entry pathway. All SARS-CoV-2 strains tested (prototypic, alpha, beta, gamma, delta, kappa, and omicron) are dependent on OCLN for cell-to-cell transmission, although the extent of syncytium formation differs between strains. We conclude that SARS-CoV-2 utilizes OCLN as an internalization factor for cell-to-cell transmission.

A Direct Link Implicating Loss of SLC26A6 to Gut Microbial Dysbiosis, Compromised Barrier Integrity, and Inflammation.

BACKGROUND & AIMS: Putative anion transporter-1 (PAT1, SLC26A6) plays a key role in intestinal oxalate and bicarbonate secretion. PAT1 knockout (PKO) mice exhibit hyperoxaluria and nephrolithiasis. Notably, diseases such as inflammatory bowel disease are also associated with higher risk of hyperoxaluria and nephrolithiasis. However, the potential role of PAT1 deficiency in gut-barrier integrity and susceptibility to colitis is currently elusive. METHODS: Age-matched PKO and wild-type littermates were administered 3.5% dextran sulfate sodium in drinking water for 6 days. Ileum and colon of control and treated mice were harvested. Messenger RNA and protein expression of tight junction proteins were determined by reverse transcription polymerase chain reaction and western blotting. Severity of inflammation was assessed by measuring diarrheal phenotype, cytokine expression, and hematoxylin and eosin staining. Gut microbiome and associated metabolome were analyzed by 16S ribosomal RNA sequencing and mass spectrometry, respectively. RESULTS: PKO mice exhibited significantly higher loss of body weight, gut permeability, colonic inflammation, and diarrhea in response to dextran sulfate sodium treatment. In addition, PKO mice showed microbial dysbiosis and significantly reduced levels of butyrate and butyrate-producing microbes compared with controls. Co-housing wild-type and PKO mice for 4 weeks resulted in PKO-like signatures on the expression of tight junction proteins in the colons of wild-type mice. CONCLUSIONS: Our data demonstrate that loss of PAT1 disrupts gut microbiome and related metabolites, decreases gut-barrier integrity, and increases host susceptibility to intestinal inflammation. These findings, thus, highlight a novel role of the oxalate transporter PAT1 in promoting gut-barrier integrity, and its deficiency appears to contribute to the pathogenesis of inflammatory bowel diseases.

Tight Junction Proteins as Therapeutic Targets to Treat Liver Fibrosis and Hepatocellular Carcinoma.

In the last decade tight junction proteins exposed at the surface of liver or cancer cells have been uncovered as mediators of liver disease biology: Claudin-1 and Occludin are host factors for hepatitis C virus entry and Claudin-1 has been identified as a driver for liver fibrosis and hepatocellular carcinoma (HCC). Moreover, Claudins have emerged as therapeutic targets for liver disease and HCC. CLDN1 expression is upregulated in liver fibrosis and HCC. Monoclonal antibodies (mAbs) targeting Claudin-1 have completed preclinical proof-of-concept studies for treatment of liver fibrosis and HCC and are currently in clinical development for advanced liver fibrosis. Claudin-6 overexpression is associated with an HCC aggressive phenotype and treatment resistance. Claudin-6 mAbs or chimeric antigen receptor-T cells therapies are currently being clinically investigated for Claudin-6 overexpressing tumors. In conclusion, targeting Claudin proteins offers a novel clinical opportunity for the treatment of patients with advanced liver fibrosis and HCC.

Hypoxia impairs urothelial barrier function by inhibiting the expression of tight junction proteins in SV-HUC-1 cells.

Hypoxia plays an important role in the pathological process of bladder outlet obstruction. Previous research has mostly focused on the dysfunction of bladder smooth muscle cells, which are directly related to bladder contraction. This study delves into the barrier function changes of the urothelial cells under exposure to hypoxia. Results indicated that after a 5-day culture, SV-HUC-1 formed a monolayer and/or bilayer of cell sheets, with tight junction formation, but no asymmetrical unit membrane was observed. qPCR and western blotting revealed the expression of TJ-associated proteins (occludin, claudin1 and ZO-1) was significantly decreased in the hypoxia group in a time-dependent manner. No expression changes were observed in uroplakins. When compared to normoxic groups, immunofluorescent staining revealed a reduction in the expression of TJ-associated proteins in the hypoxia group. Transepithelial electrical resistance (TEER) revealed a statistically significant decrease in resistance in the hypoxia group. Fluorescein isothiocyanate-conjugated dextran assay was inversely proportional to the results of TEER. Taken together, hypoxia down-regulates the expression of TJ-associated proteins and breaks tight junctions, thus impairing the barrier function in human urothelial cells.

Porous Se@SiO2 nanospheres alleviate diabetic retinopathy by inhibiting excess lipid peroxidation and inflammation.

BACKGROUND: Lipid peroxidation is a characteristic metabolic manifestation of diabetic retinopathy (DR) that causes inflammation, eventually leading to severe retinal vascular abnormalities. Selenium (Se) can directly or indirectly scavenge intracellular free radicals. Due to the narrow distinction between Se's effective and toxic doses, porous Se@SiO2 nanospheres have been developed to control the release of Se. They exert strong antioxidant and anti-inflammatory effects. METHODS: The effect of anti-lipid peroxidation and anti-inflammatory effects of porous Se@SiO2 nanospheres on diabetic mice were assessed by detecting the level of Malondialdehyde (MDA), glutathione peroxidase 4 (GPX4), decreased reduced/oxidized glutathione (GSH/GSSG) ratio, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL) -1ß of the retina. To further examine the protective effect of porous Se@SiO2 nanospheres on the retinal vasculopathy of diabetic mice, retinal acellular capillary, the expression of tight junction proteins, and blood-retinal barrier destruction was observed. Finally, we validated the GPX4 as the target of porous Se@SiO2 nanospheres via decreased expression of GPX4 and detected the level of MDA, GSH/GSSG, TNF-α, IFN-γ, IL -1ß, wound healing assay, and tube formation in high glucose (HG) cultured Human retinal microvascular endothelial cells (HRMECs). RESULTS: The porous Se@SiO2 nanospheres reduced the level of MDA, TNF-α, IFN-γ, and IL -1ß, while increasing the level of GPX4 and GSH/GSSG in diabetic mice. Therefore, porous Se@SiO2 nanospheres reduced the number of retinal acellular capillaries, depletion of tight junction proteins, and vascular leakage in diabetic mice. Further, we identified GPX4 as the target of porous Se@SiO2 nanospheres as GPX4 inhibition reduced the repression effect of anti-lipid peroxidation, anti-inflammatory, and protective effects of endothelial cell dysfunction of porous Se@SiO2 nanospheres in HG-cultured HRMECs. CONCLUSION: Porous Se@SiO2 nanospheres effectively attenuated retinal vasculopathy in diabetic mice via inhibiting excess lipid peroxidation and inflammation by target GPX4, suggesting their potential as therapeutic agents for DR.

Irisin links Claudin-5 preservation and Mfn2-mediated mitochondrial dynamics to resist doxorubicin-induced cardiac endothelial damage.

Irisin is protective in the cardiac microenvironment and can resist doxorubicin-induced cardiotoxicity. The purpose of this study was to investigate the connection between Irisin, endothelial cell integrity, and mitochondrial dynamics. Primary cardiac microvascular endothelial cells (CMECs) were used to explore the regulatory effects of Irisin on tight junction proteins, mitochondrial dynamics, ß-catenin expression, and transcriptional activity. Results showed that Irisin can suppress doxorubicin-induced upregulation of MMP2 and MMP9, thereby reducing the degradation of tight junction proteins (ZO-1 and Claudin-5) and VE-cadherin. The preservation of Claudin-5 contributes to maintaining Mfn2 expression, which in turn supports mitochondrial fusion. Although Irisin restores doxorubicin-induced downregulation of ß-catenin, it concurrently limits ß-catenin transcriptional activity via Mfn2-mediated sulfenylation. Therefore, this study revealed a novel mechanism linking the protective effects of Irisin on the tight junction proteins and mitochondrial dynamics upon doxorubicin exposure.

Modulation of intestinal epithelial permeability by chronic small intestinal helminth infections.

Increased permeability of the intestinal epithelial layer is linked to the pathogenesis and perpetuation of a wide range of intestinal and extra-intestinal diseases. Infecting humans with controlled doses of helminths, such as human hookworm (termed hookworm therapy), is proposed as a treatment for many of the same diseases. Helminths induce immunoregulatory changes in their host which could decrease epithelial permeability, which is highlighted as a potential mechanism through which helminths treat disease. Despite this, the influence of a chronic helminth infection on epithelial permeability remains unclear. This study uses the chronically infecting intestinal helminth Heligmosomoides polygyrus to reveal alterations in the expression of intestinal tight junction proteins and epithelial permeability during the infection course. In the acute infection phase (1 week postinfection), an increase in intestinal epithelial permeability is observed. Consistent with this finding, jejunal claudin-2 is upregulated and tricellulin is downregulated. By contrast, in the chronic infection phase (6 weeks postinfection), colonic claudin-1 is upregulated and epithelial permeability decreases. Importantly, this study also investigates changes in epithelial permeability in a small human cohort experimentally challenged with the human hookworm, Necator americanus. It demonstrates a trend toward small intestinal permeability increasing in the acute infection phase (8 weeks postinfection), and colonic and whole gut permeability decreasing in the chronic infection phase (24 weeks postinfection), suggesting a conserved epithelial response between humans and mice. In summary, our findings demonstrate dynamic changes in epithelial permeability during a chronic helminth infection and provide another plausible mechanism by which chronic helminth infections could be utilized to treat disease.

Tauroursodeoxycholic Acid Reverses Dextran Sulfate Sodium-Induced Colitis in Mice via Modulation of Intestinal Barrier Dysfunction and Microbiome Dysregulation.

Ulcerative colitis (UC) is an immune-mediated inflammatory disease that can lead to persistent damage and even cancer without any intervention. Conventional treatments can alleviate UC symptoms but are costly and cause various side effects. Tauroursodeoxycholic acid (TUDCA), a secondary bile acid derivative, possesses anti-inflammatory and cytoprotective properties for various diseases, but its potential therapeutic benefits in UC have not been fully explored. Mice were subjected to colitis induction using 3% dextran sulfate sodium (DSS). The therapeutic effect of TUDCA was evaluated by body weight loss, disease activity index (DAI), colon length, and spleen weight ratio. Tissue pathology was assessed using H&E staining, while the levels of pro-inflammatory and anti-inflammatory cytokines in colonic tissue were quantified via ELISA. Tight junction proteins were detected by immunoblotting and intestinal permeability was assessed using fluorescein isothiocyanate (FITC)-dextran. Moreover, the gut microbiota was profiled using high-throughput sequencing of the 16S rDNA gene. TUDCA alleviated the colitis in mice, involving reduced DAI, attenuated colon and spleen enlargement, ameliorated histopathological lesions, and normalized levels of pro-inflammatory and anti-inflammatory cytokines. Furthermore, TUDCA treatment inhibited the downregulation of intestinal barrier proteins, including zonula occludens-1 and occludin, thus reducing intestinal permeability. The analysis of gut microbiota suggested that TUDCA modulated the dysbiosis in mice with colitis, especially for the remarkable rise in Akkermansia TUDCA exerted a therapeutic efficacy in DSS-induced colitis by reducing intestinal inflammation, protecting intestinal barrier integrity, and restoring gut microbiota balance. SIGNIFICANCE STATEMENT: This study demonstrates the potential therapeutic benefits of Tauroursodeoxycholic acid (TUDCA) in ulcerative colitis. TUDCA effectively alleviated colitis symptoms in mice, including reducing inflammation, restoring intestinal barrier integrity and the dysbiosis of gut microbiota. This work highlights the promising role of TUDCA as a potentially alternative treatment, offering new insights into managing this debilitating condition.

Glaesserella parasuis serotype 5 breaches the porcine respiratory epithelial barrier by inducing autophagy and blocking the cell membrane Claudin-1 replenishment.

Glaesserella parasuis (G. parasuis), the primary pathogen of Glässer's disease, colonizes the upper respiratory tract and can break through the epithelial barrier of the respiratory tract, leading to lung infection. However, the underlying mechanisms for this adverse effect remain unclear. The G. parasuis serotype 5 SQ strain (HPS5-SQ) infection decreased the integrity of piglets' lung Occludin and Claudin-1. Autophagy regulates the function of the epithelial barrier and tight junction proteins (TJs) expression. We tested the hypothesis that HPS5-SQ breaking through the porcine respiratory epithelial barrier was linked to autophagy and Claudin-1 degradation. When HPS5-SQ infected swine tracheal epithelial cells (STEC), autophagosomes encapsulated, and autolysosomes degraded oxidatively stressed mitochondria covered with Claudin-1. Furthermore, we found that autophagosomes encapsulating mitochondria resulted in cell membrane Claudin-1 being unable to be replenished after degradation and damaged the respiratory tract epithelial barrier. In conclusion, G. parasuis serotype 5 breaks through the porcine respiratory epithelial barrier by inducing autophagy and interrupting cell membrane Claudin-1 replenishment, clarifying the mechanism of the G. parasuis infection and providing a new potential target for drug design and vaccine development.

Fentanyl dysregulates neuroinflammation and disrupts blood-brain barrier integrity in HIV-1 Tat transgenic mice.

Opioid overdose deaths have dramatically increased by 781% from 1999 to 2021. In the setting of HIV, opioid drug abuse exacerbates neurotoxic effects of HIV in the brain, as opioids enhance viral replication, promote neuronal dysfunction and injury, and dysregulate an already compromised inflammatory response. Despite the rise in fentanyl abuse and the close association between opioid abuse and HIV infection, the interactive comorbidity between fentanyl abuse and HIV has yet to be examined in vivo. The HIV-1 Tat-transgenic mouse model was used to understand the interactive effects between fentanyl and HIV. Tat is an essential protein produced during HIV that drives the transcription of new virions and exerts neurotoxic effects within the brain. The Tat-transgenic mouse model uses a glial fibrillary acidic protein (GFAP)-driven tetracycline promoter which limits Tat production to the brain and this model is well used for examining mechanisms related to neuroHIV. After 7 days of fentanyl exposure, brains were harvested. Tight junction proteins, the vascular cell adhesion molecule, and platelet-derived growth factor receptor-ß were measured to examine the integrity of the blood brain barrier. The immune response was assessed using a mouse-specific multiplex chemokine assay. For the first time in vivo, we demonstrate that fentanyl by itself can severely disrupt the blood-brain barrier and dysregulate the immune response. In addition, we reveal associations between inflammatory markers and tight junction proteins at the blood-brain barrier.

ß-defensin 2 protects against Escherichia coli-induced acute urinary tract infection by downregulating ß-catenin.

ß-defensin 2 (BD2) is a small cationic peptide that exerts a critical role in host defense against bacterial infections. Here, we aimed to investigate the role of BD2 in protecting against acute urinary tract infection (AUTI) caused by Escherichia coli (UPEC). Here, LPS-induced human urinary bladder epithelial cell (HCV-29) model and UPEC-induced mice model were used for assessing AUTI. Visceral organ lesions of mice following treatment was assessed by HE staining. Cell viability was determined by CCK-8 assay. Permeability in HCV-29 cells was analyzed in Transwell assay. Expression of inflammatory factors (IL-1ß, IL-6, and TNF-α) was measured by ELISA assay. The levels of BD2, ß-catenin and tight-junction proteins (ZO-1, Occludin, and Claudin-1) were detected by RT-qPCR, western blotting, immunofluorescence or immunohistochemistry. Our results showed that BD2 was lowly expressed and ß-catenin showed the reverse trend in response to bacterial infection in vitro and in vivo. BD2 overexpression alleviated the decreased cell viability, increased cell permeability, upregulation of inflammatory factors, downregulation of tight-junction protein and high ß-catenin expression in LPS-induced HCV-29 cells, which may contribute to the negative regulation of ß-catenin expression. Furthermore, BD2 overexpression attenuated the bacterial infection of tissues, high levels of inflammatory factors and ß-catenin, and low levels of tight-junction proteins in mice stimulated with UPEC. This study showed that BD2 played a crucial role in protecting against AUTI caused by gram-negative bacteria through suppressing ß-catenin expression. Targeting BD2 may provide a potential therapeutic approach for the prevention and treatment of AUTI.

Gallic acid mitigates intestinal inflammation and loss of tight junction protein expression using a 2D-Caco-2 and RAW 264.7 co-culture model.

A 2D-intestinal epithelial Caco-2/RAW 264.7 macrophage co-culture model was developed to demonstrate the relative efficacy of different phenolic acids to mitigate changes in Caco-2 epithelial cell redox state initiated both directly by autoxidation products, H2O2, and indirectly through cell communication events originating from cytokine stimulated macrophage. An inducer cocktail (lipopolysaccharide + interferon gamma) was used to activate RAW 264.7 cells in the 2D- Caco-2/RAW co-culture and intracellular changes in Caco-2 cell redox signaling occurred in response to positive changes (p < 0.05) in inflammatory biomarkers derived in macrophage that included IL-6, TNF-α, nitric oxide and peroxynitrite, respectively. Phenolic acids varied in relative capacity to reduce NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in cocktail inflamed induced macrophage. This response in addition to the relative predisposition of gallic acid (GA) to undergo autoxidation to generate H2O2 activity (p < 0.05), culminated in downstream cell signaling in Caco-2 nuclear factor erythroid 2-related factor (Nrf2) activity (increase 26.9 %), altered monolayer integrity (increase 33.7 %), and release of interleukin 8 (IL-8) (decrease 80.5 %) (p < 0.05). It can be concluded that the co-culture model described herein was useful to assess the importance of communication between cytokine stimulated macrophage and intestinal cells. Moreover, the relative unique efficacy of GA, compared to other phenolic acids tested to protect against activated macrophage induced changes related to intestinal dysfunction were particularly relevant to epithelial redox signaling, intestinal permeability and regulation of tight junction proteins. This study concludes that phenolic acids are not equal in the capacity to protect against intestinal cell dysfunction despite some indication of biological activity.

Yingxiang Acupoint Embedding Improves Mucosal Barrier Function in Rats with Local Allergic Rhinitis.

INTRODUCTION: Epithelial barrier disruption is the initial cause of various diseases. We previously reported that acupoint catgut embedding (AE) improves tight junction proteins (TJs) in rats with allergic rhinitis. However, whether AE improves the epithelial barrier in local allergic rhinitis (LAR) remains unknown. METHODS: A total of 36 Sprague Dawley (SD) male rats aged 5-7 weeks were divided into 6 groups with 6 rats each: control group, LAR model group, false acupoint embedding + LAR group, acupoint embedding + LAR group, capsaicin + LAR group, and tunicamycin + acupoint embedding + LAR group. Behavioral observation, ELISA to detect inflammatory factors in nasal lavage fluid and serum IgE, nasal mucosal permeability test, hematoxylin-eosin staining, PCR to detect Substance P (SP), Western blot, and immunofluorescence to detect endoplasmic reticulum stress (ERS) index and TJs were used to investigate the mechanism of AE in LAR. RESULTS: AE improved the symptoms and pathological features of nasal mucosa of LAR rats, reduced the inflammatory factors (IL4, IL5, IL13) of nasal lavage fluid, and showed no significant change in serum IgE levels in all groups. In addition, AE decreased the expression of SP in nasal mucosa of LAR rats, inhibited ERS, increased the expression of tight junction protein, reduced the permeability of nasal mucosa, and improved the function of nasal mucosal barrier. CONCLUSION: This study confirms that AE can improve the nasal mucosal barrier function of LAR by reducing the expression of SP, inhibiting ERS and increasing the expression of TJs, thus enhancing the nasal mucosal barrier function.

Tight junction proteins and biliary diseases.

PURPOSE OF REVIEW: In the pathophysiological context of cholangiopathies and more broadly of hepatopathies, while it is conceptually clear that the maintenance of inter-cholangiocyte and inter-hepatocyte tight junction integrity would be crucial for liver protection, only scarce studies have been devoted to this topic. Indeed, in the liver, alteration of tight junctions, the intercellular adhesion complexes that control paracellular permeability would result in leaky bile ducts and bile canaliculi, allowing bile reflux towards hepatic parenchyma, contributing to injury during the disease process. RECENT FINDINGS: Last decades have provided a great deal of information regarding both tight junction structural organization and signaling pathways related to tight junctions, providing clues about potential intervention to modulate paracellular permeability during cholangiopathies pathogenesis. Interestingly, several liver diseases have been reported to be associated with abnormal expression of one or several tight junction proteins. However, the question remains unanswered if these alterations would be primarily involved in the disease pathogenesis or if they would occur secondarily in the pathological course. SUMMARY: In this review, we provide an overview of tight junction disruptions described in various biliary diseases that should pave the way for defining new therapeutic targets in this field.

Endothelial BACE1 Impairs Cerebral Small Vessels via Tight Junctions and eNOS.

BACKGROUND: Cerebral small vessel injury, including loss of endothelial tight junctions, endothelial dysfunction, and blood-brain barrier breakdown, is an early and typical pathology for Alzheimer's disease, cerebral amyloid angiopathy, and hypertension-related cerebral small vessel disease. Whether there is a common mechanism contributing to these cerebrovascular alterations remains unclear. Studies have shown an elevation of BACE1 (ß-site amyloid precursor protein cleaving enzyme 1) in cerebral vessels from cerebral amyloid angiopathy or Alzheimer's disease patients, suggesting that vascular BACE1 may involve in cerebral small vessel injury. METHODS: To understand the contribution of vascular BACE1 to cerebrovascular impairments, we combined cellular and molecular techniques, mass spectrometry, immunostaining approaches, and functional testing to elucidate the potential pathological mechanisms. RESULTS: We observe a 3.71-fold increase in BACE1 expression in the cerebral microvessels from patients with hypertension. Importantly, we discover that an endothelial tight junction protein, occludin, is a completely new substrate for endothelial BACE1. BACE1 cleaves occludin with full-length occludin reductions and occludin fragment productions. An excessive cleavage by elevated BACE1 induces membranal accumulation of caveolin-1 and subsequent caveolin-1-mediated endocytosis, resulting in lysosomal degradation of other tight junction proteins. Meanwhile, membranal caveolin-1 increases the binding to eNOS (endothelial nitric oxide synthase), together with raised circulating Aß (ß-amyloid peptides) produced by elevated BACE1, leading to an attenuation of eNOS activity and resultant endothelial dysfunction. Furthermore, the initial endothelial damage provokes chronic reduction of cerebral blood flow, blood-brain barrier leakage, microbleeds, tau hyperphosphorylation, synaptic loss, and cognitive impairment in endothelial-specific BACE1 transgenic mice. Conversely, inhibition of aberrant BACE1 activity ameliorates tight junction loss, endothelial dysfunction, and memory deficits. CONCLUSIONS: Our findings establish a novel and direct relationship between endothelial BACE1 and cerebral small vessel damage, indicating that abnormal elevation of endothelial BACE1 is a new mechanism for cerebral small vessel disease pathogenesis.

Dietary methanotroph bacteria meal alleviates soybean meal-induced enteritis by improving immune tolerance and intestinal flora profile of juvenile turbot (Scophthalmus maximus L.).

An 8-week growth trial was performed to investigate the protective effects of methanotroph bacteria meal (MBM) produced from methane against soybean meal-induced enteritis (SBMIE) in juvenile turbot (Scophthalmus maximus L.). Five isonitrogenous and isolipidic diets were formulated: fishmeal-based diet (FM, the control group); FM with approximate 50% of fishmeal substituted by 399.4 g/kg soybean meal (SBM); SBM supplemented with 63.6, 127.2 and 190.8 g/kg MBM (named MBM1, MBM2 and MBM3), each diet was randomly assigned to triplicate fibreglass tanks. Results showed that fish fed with SBM exhibited enteritis, identified by reduced relative weight of intestine (RWI), as well as expanded lamina propria width and up-regulated gene expression of pro-inflammatory cytokines (tnf-α, il-6 and il-8) in intestine. While the above symptoms were reversed when diet SBM supplemented with MBM at the levels of 63.6 and 127.2 g/kg, as well as characterized by up-regulated gene expression of anti-inflammatory cytokines (tgf-ß and il-10) and tight junction protein (claudin3, claudin4 and claudin7) in intestine. Intestinal transcriptome analysis showed that the differentially expressed genes between groups FM and SBM predominantly enriched in the JAK-STAT signaling pathway, and the enrichment of differentially expressed genes between groups SBM and SBM supplemented with 63.6 g/kg MBM was in the inflammatory bowel disease (IBD) and JAK-STAT signaling pathway. To be specific, the expression of jak1, jak2b, stat1 and stat5a was significantly up-regulated when fish fed with SBM, suggested the activation of JAK-STAT signaling pathway, while the expression of these above genes was depressed by providing MBM to diet SBM, and the gene expression of toll-like receptors tlr2 and tlr5b showed a similar pattern. Moreover, intestinal flora analysis showed that community richness and abundance of beneficial bacteria (Cetobacterium and acillus_coagulans) were improved when fish fed with SBM supplemented with 63.6 g/kg MBM. Overall, methanotroph bacteria meal may alleviate SBMIE by regulating the expression of tight junction protein, toll-like receptors and JAK-STAT signaling pathway, as well as improving intestinal flora profile, which would be beneficial for enhancing the immune tolerance and utilization efficiency of turbot to dietary soybean meal.