Intercellular Junctions Research Articles

Effect of opioid receptor antagonist on mitigating tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-induced apoptolysis in pemphigus pathogenesis

Pemphigus is a severe autoimmune blistering disease characterized by acantholysis triggered by autoantibodies against desmoglein 1 and 3 (DSG1/3). Apoptosis plays a pivotal role in facilitating acantholysis, yet the precise underlying mechanism remains obscure. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is known to promote apoptosis and disrupt cell junctions, although its involvement in pemphigus pathogenesis remains ambiguous. Our study observed decreased DSG1/3 expression alongside increased TWEAK/fibroblast growth factor-inducible 14 (Fn14) expression and keratinocyte apoptosis in both lesional and perilesional skin. In vitro experiments revealed that TWEAK-stimulated keratinocytes exhibited enhanced apoptosis, STAT1 phosphorylation, and reduced intercellular DSG1/3 expression. Notably, bulk-RNA sequencing unveiled that CASPASE-3 was responsible for mediating the DSG1/3 depletion, as confirmed by direct interaction with DSG1/3 in a co-immunoprecipitation assay. Naloxone, known for preserving cellular adhesion and preventing cell death, effectively reduced apoptosis and restored DSG1/3 levels in TWEAK-stimulated keratinocytes. The anti-apoptotic properties of naloxone were further validated in a murine pemphigus model. Our findings elucidate that TWEAK facilitates keratinocyte apoptosis by augmenting caspase-3 activity, leading to DSG1/3 depletion and apoptosis in pemphigus. Importantly, naloxone can counter TWEAK-induced apoptosis in pemphigus pathogenesis, offering a potential therapeutic intervention.

The Protective Role of L-Cysteine in the Regulation of Blood-Testis Barrier Functions-A Brief Review.

Blood-testis barrier (BTB) genes are crucial for the cellular mechanisms of spermatogenesis as they protect against detrimental cytotoxic agents, chemicals, and pathogens, thereby maintaining a sterile environment necessary for sperm development. BTB proteins predominantly consist of extensive tight and gap junctions formed between Sertoli cells. These junctions form a crucial immunological barrier restricting the intercellular movement of substances and molecules within the adluminal compartment. Epithelial tight junctions are complex membrane structures composed of various integral membrane proteins, including claudins, zonula occludens-1, and occludin. Inter-testicular cell junction proteins undergo a constant process of degradation and renewal. In addition, the downregulation of genes crucial to the development and preservation of cell junctions could disrupt the functionality of the BTB, potentially leading to male infertility. Oxidative stress and inflammation may contribute to disrupted spermatogenesis, resulting in male infertility. L-cysteine is a precursor to glutathione, a crucial antioxidant that helps mitigate damage and inflammation resulting from oxidative stress. Preclinical research indicates that L-cysteine may offer protective benefits against testicular injury and promote the expression of BTB genes. This review emphasizes various BTB genes essential for preserving its structural integrity and facilitating spermatogenesis and male fertility. Furthermore, it consolidates various research findings suggesting that L-cysteine may promote the expression of BTB-associated genes, thereby aiding in the maintenance of testicular functions.

Paracellular barriers: Advances in assessing their contribution to renal epithelial function

Regulation of salt and water balance occupies a dominant role in the physiology of many animals and often relies on the function of the renal system. In the mammalian kidney, epithelial ion and water transport requires high degree of coordination between the transcellular and paracellular pathways, the latter being defined by the intercellular tight junctions (TJs). TJs seal the paracellular pathway in a highly specialized manner, either by forming a barrier against the passage of solutes and/or water or by allowing the passage of ions and/or water through them. This functional TJ plasticity is now known to be provided by the members of the claudin family of tetraspan proteins. Unlike mammalian nephron, the renal structures of insects, the Malpighian tubules, lack TJs and instead have smooth septate junctions (sSJs) as paracellular barrier forming junctions. Many questions regarding the molecular and functional properties of sSJs remain open but research on model species have begun to inform our understanding. The goal of this commentary is to highlight key concepts and most recent findings that have emerged from the molecular and functional dissection of paracellular barriers in the mammalian and insect renal epithelia.

Transendothelial migration of the Lyme disease spirochete involves spirochete internalization as an intermediate step through a transcellular pathway that involves Cdc42 and Rac1.

Lyme borreliosis is caused by Borrelia burgdorferi and related bacteria. It is the most common tick-transmitted illness in the Northern Hemisphere. The ability of this pathogen to spread to a wide variety of locations results in a diverse set of clinical manisfestations, yet little is known regarding vascular escape of the spirochete, an important pathway for dissemination. Our current work has studied the traversal of B. burgdorferi across a monolayer of microvascular endothelial cells grown in culture. We show that this occurs by passage of the spirochetes directly through these cells rather than at cellular junctions and that internalization of B. burgdorferi is an intermediate step in the transmigration process. We also identify the first two host proteins, Cdc42 and Rac1, that are used by the spirochetes to promote traversal of the cellular monolayer. Our new experimental system also provides a new avenue for further studies of this important process.

Multi-omics analysis reveals the effects of host-rumen microbiota interactions on growth performance in a goat model.

The growth rate of young ruminants has been associated with production performance in later life, with recent studies highlighting the importance of rumen microbes in supporting the health and growth of ruminants. However, the specific role of rumen epithelium bacteria and microbiota-host interactions in influencing the early life growth rate of ruminants remains poorly understood. In this study, we investigated the rumen fermentation pattern, microbiota characteristics, and global gene expression profiles of the rumen epithelium in 6-month-old goats with varying growth rates. Our results showed that goats with high average daily gain (HADG) exhibited higher rumen propionate concentrations. Goats with low average daily gain (LADG) had the higher relative abundances of rumen epithelium bacteria genera U29-B03 and Quinella, while exhibiting a lower relative abundance of Lachnospiraceae UCG-009. In the rumen fluid, the relative abundances of bacteria genus Alloprevotella were lower and Desulfovibrio were higher in LADG goats compared to HADG goats. Additionally, the relative abundance of fungal genus Symmetrospora was lower in LADG goats compared to HADG goats. Transcriptome analysis showed that 415 genes were differentially expressed between LADG and HADG goats, which were enriched in functions related to cell junction and cell adhesion, etc. Correlation analysis revealed that rumen epithelium bacteria genera UCG-005 and Candidatus Saccharimonas were negatively associated, while Lachnospiraceae NK3A20 group and Oscillospiraceae NK4A214 group were positively associated with average daily gain (ADG) and genes related to barrier function. The rumen fluid bacteria genus Alloprevotella was positively correlated, while Desulfovibrio was negatively correlated with rumen propionate and ammoniacal nitrogen (NH3-N) concentrations, as well as genes related to barrier function and short chain fatty acids (SCFAs) transport. In summary, our study reveals that the higher ruminal fermentation efficiency, improved rumen epithelial barrier functions, and enhanced SCFAs transport in HADG goats could be attributed to the rumen microbiota, particularly the rumen epithelium bacteria, such as Lachnospiraceae and Oscillospiraceae NK4A214 group.

-catenin phosphorylation is elevated during mitosis to resist apical rounding and epithelial barrier leak.

Epithelial cell cohesion and barrier function critically depend on -catenin, an actin-binding protein and essential constituent of cadherin-catenin-based adherens junctions. -catenin undergoes actomyosin force-dependent unfolding of both actin-binding and middle domains to strongly engage actin filaments and its various effectors, where this mechanosensitivity is critical for adherens junction function. We previously showed that -catenin is highly phosphorylated in an unstructured region that links mechanosensitive middle- and actin-binding domains (known as the P-linker region), but the cellular processes that promote -catenin phosphorylation have remained elusive. Here, we leverage a previously published phosphor-proteomic data set to show that the -catenin P-linker region is maximally phosphorylated during mitosis. By reconstituting -catenin Crispr KO MDCK with wild-type, phospho- mutant and mimic forms of -catenin, we show that full phosphorylation restrains mitotic cell rounding in the apical direction, strengthening interactions between dividing and non-dividing neighbors to limit epithelial barrier leak. Since major scaffold components of adherens junctions, tight junctions and desmosomes are also differentially phosphorylated during mitosis, we reason that epithelial cell division may be a tractable system to understand how junction complexes are coordinately regulated to sustain barrier function under tension-generating morphogenetic processes.

Tensions on the actin cytoskeleton and apical cell junctions in the C. elegans spermatheca are influenced by spermathecal anatomy, ovulation state and activation of myosin.

Cells generate mechanical forces mainly through myosin motor activity on the actin cytoskeleton. In C. elegans , actomyosin stress fibers drive contractility of the smooth muscle-like cells of the spermatheca, a distensible, tube-shaped tissue in the hermaphrodite reproductive system and the site of oocyte fertilization. Stretching of the spermathecal cells by oocyte entry triggers activation of the small GTPase Rho. In this study, we asked how forces are distributed in vivo using the spermatheca, and explored how this tissue responds to alterations in myosin activity. Using laser ablation, we show that the basal actomyosin fibers are under tension in the occupied spermatheca. Reducing actomyosin contractility by depletion of the phospholipase C-ε/PLC-1 or non-muscle myosin II/NMY-1, leads to distended spermathecae occupied by one or more embryos, but does not alter tension on the basal actomyosin fibers. This suggests that much of the tension on the basal actin fibers in the occupied spermatheca is due to the presence of the embryo. However, activating myosin through depletion of the Rho GAP SPV-1 increases tension on the actomyosin fibers, consistent with earlier studies showing Rho drives spermathecal contractility. On the inner surface of the spermathecal tube, tension on the apical junctions is decreased by depletion of PLC-1 and NMY-1. Surprisingly, when basal contractility is increased through SPV-1 depletion, the tension on apical junctions also decreases, with the most significant effect on the junctions aligned in perpendicular to the axis of the spermatheca. This suggests tension on the outer basal surface may compress the apical side, and suggests the three-dimensional shape of the spermatheca plays a role in force distribution and contractility during ovulation.

A robust deep learning attack immune MRAM-based physical unclonable function

The ubiquitous presence of electronic devices demands robust hardware security mechanisms to safeguard sensitive information from threats. This paper presents a physical unclonable function (PUF) circuit based on magnetoresistive random access memory (MRAM). The circuit utilizes inherent characteristics arising from fabrication variations, specifically magnetic tunnel junction (MTJ) cell resistance, to produce corresponding outputs for applied challenges. In contrast to Arbiter PUF, the proposed effectively satisfies the strict avalanche criterion (SAC). Additionally, the grid-like structure of the proposed circuit preserves its resistance against machine learning-based modeling attacks. Various machine learning (ML) attacks employing multilayer perceptron (MLP), linear regression (LR), and support vector machine (SVM) networks are simulated for two-array and four-array architectures. The MLP-attack prediction accuracy was 53.61% for a two-array circuit and 49.87% for a four-array circuit, showcasing robust performance even under the worst-case process variations. In addition, deep learning-based modeling attacks in considerable high dimensions utilizing multiple networks such as convolutional neural network (CNN), recurrent neural network (RNN), MLP, and Larq are used with the accuracy of 50.31%, 50.25%, 50.31%, and 50.31%, respectively. The efficiency of the proposed circuit at the layout level is also investigated for simplified two-array architecture. The simulation results indicate that the proposed circuit offers intra and inter-hamming distance (HD) with a mean of 0.98% and 49.96%, respectively, and a mean diffuseness of 49.09%.

Hyperlipidemia negatively impacts implantation by dysregulating tight junction and Claudin-3 and Claudin-4 expression in the endometrium

Clinical observational studies have suggested hyperlipidemia may disturb embryo implantation through endometrium; however, the mechanism has been unclear. With its profound implications for reproductive health, the present study aims to investigate whether hyperlipidemia affects endometrial epithelial cell tight junctions for implantation failures. By constructing hyperlipidemia mice model, the number and distribution of embryo implantation status were investigated after both natural mating and in vitro fertilization and embryo transfer (IVF-ET). Transmission electron microscopy (TEM) was used to compare the ultrastructure of tight junctions in endometrial endothelial cells. Western blot and immunofluorescence were used to explore the expression and localization of tight junction proteins, such as Claudin (CDLN)3, CLDN4, occludin (OCLN), and zonula occludens-1 (ZO1). For women with reproductive failure, mid-luteal phase endometrial tissues were collected, and gene expression of tight junction proteins was investigated using RNA sequencing and qRT-PCR. In hyperlipidemic mice, the number of embryo implantation sites significantly decreased with uneven distribution after natural mating and IVF-ET. Disrupted tight junctions were found, characterized by a decreased number of tight junctions by TEM, downregulated expressions of CDLN4, OCLN, and ZO1, and an increased expression of CLDN3 by western blot. In hyperlipidemic women with reproductive failure, the dysregulated expression of CLDN3 and CLDN4 was also present in the luteal phase endometrium. In this study, evaluation of both animal models and infertile women in vivo demonstrated that hyperlipidemia reduced female fertility, accompanied by disruption of tight junction structures and dysregulation of CLDN3 and CLDN4 expression in the endothelial cells of luteal phase endometrium.

Multi-Omics Profiles of Small Intestine Organoids in Reaction to Breast Milk and Different Infant Formula Preparations.

Ensuring optimal infant nutrition is crucial for the health and development of children. Many infants aged 0-6 months are fed with infant formula rather than breast milk. Research on cancer cell lines and animal models is limited to examining the nutrition effects of formula and breast milk, as it does not comprehensively consider absorption, metabolism, and the health and social determinants of the infant and its physiology. Our study utilized small intestine organoids induced from human embryo stem cell (ESC) to compare the nutritional effects of breast milk from five donors during their postpartum lactation period of 1-6 months and three types of Stage 1 infant formulae from regular retail stores. Using transcriptomics and untargeted metabolomics approaches, we focused on the differences such as cell growth and development, cell junctions, and extracellular matrix. We also analyzed the roles of pathways including AMPK, Hippo, and Wnt, and identified key genes such as ALPI, SMAD3, TJP1, and WWTR1 for small intestine development. Through observational and in-vitro analysis, our study demonstrates ESC-derived organoids might be a promising model for exploring nutritional effects and underlying mechanisms.

Sialyl Lewis X decorated integrin α3 on small extracellular vesicles promotes metastasis of bladder cancer via enhancing vascular permeability.

The permeability of blood vessels plays a crucial role in the spread of cancer cells, facilitating their metastasis at distant sites. Small extracellular vesicles (sEVs) are known to contribute to the metastasis of various cancers by crossing the blood vessel wall. However, the role of abnormal glycoconjugates on sEVs in tumor blood vessels remains unclear. Our study found elevated levels of fucosyltransferase VII (FUT7) and its product sialyl Lewis X (sLeX) in muscle-invasive bladder cancer (BLCA), with high levels of sLeX promoting the growth and invasion of BLCA cells. Further investigation revealed that sLeX was enriched in sEVs derived from BLCA. sLeX-decorated sEVs increased blood vessel permeability by disrupting the tight junctions of human umbilical vein endothelial cells (HUVECs). Using the glycoproteomics approach, we identified integrin α3 (ITGA3) as a sLeX-bearing glycoprotein in BLCA cells and their sEVs. Mechanically, sLeX modification stabilized ITGA3 by preventing its degradation in lysosomes. sEVs carrying sLeX-modified ITGA3 can be effectively internalized by HUVECs, leading to a decrease in the expression of tight junction protein. Conversely, silencing ITGA3 in sLeX-decorated sEVs restored tight junction proteins and reduced blood vessel permeability by inhibiting the MAPK pathway. Moreover, sLeX-modification of ITGA3 at Asn 265 in HUVECs promoted occludin dephosphorylation at Ser/Thr residues, followed by inducing its importin α1-mediated nuclear translocation, which resulted in the disruption of tight junctions. Our findings suggest a potential strategy for disrupting the formation of a metastatic microenvironment and preventing the spread of malignant bladder cancer.

Persistent Desmoglein-1 downregulation and Periostin accumulation in histologic remission of Eosinophilic Esophagitis

BackgroundPatients with Eosinophilic esophagitis (EoE) require long-lasting resolution of inflammation to prevent fibrostenosis and dysphagia. However, the dissociation between symptoms and histologic improvement suggests persistent molecular drivers despite histologic remission. ObjectiveTo characterize persisting molecular alterations in pediatric patients with EoE using tissue transcriptomics and proteomics. MethodsEsophageal biopsies (n=247) collected prospectively during 189 endoscopies from pediatric patients with EoE (N=36, up to 11 follow-up endoscopies) and pediatric controls (N=44, single endoscopies) were subjected to bulk transcriptomics (n=96) and proteomics (n=151). Intercellular junctions (Desmoglein-1/-3, Desmoplakin, E-cadherin) and epithelial-to-mesenchymal transition (EMT, Vimentin:E-cadherin ratio) were assessed by immunofluorescence staining. ResultsActive EoE (≥15 eosinophils/hpf), inactive EoE (<15 eosinophils/hpf) and deep remission EoE (0 eosinophils/hpf) were diagnosed in 107/185, 78/185 and 41/185 biopsies, respectively. Among the dysregulated genes (up-/downregulated 310/112) and proteins (up-/downregulated 68/16) between active EoE and controls, 17 genes and 6 proteins remained dysregulated in inactive EoE. Using persistently upregulated genes (n=9) and proteins (n=3) only, such as ALOX15, CXCL1, CXCL6, CTSG, CDH26, PRRX1, CLC, EPX, and POSTN was sufficient to separate inactive EoE, as well as deep remission biopsies from control tissue. While 32 differentially expressed genes persisted in deep remission of EoE compared to controls, the proteome normalized except for persistently upregulated Periostin (POSTN). Epithelial-mesenchymal transition normalized in inactive EoE, whereas desmosome recovery remained impaired due to Desmoglein-1 downregulation. ConclusionThe analysis of molecular changes shows persistent EoE-associated esophageal dysregulation despite histologic remission. These data expand our understanding of inflammatory processes and possible mechanisms that underlie tissue remodeling in EoE.

Noncoding Vault RNA1-1 Impairs Intestinal Epithelial Renewal and Barrier Function by Interacting With CUG-binding Protein 1

Small noncoding vault RNAs (vtRNAs) are involved in many cell processes important for health and disease, but their pathobiological functions in the intestinal epithelium are underexplored. Here, we investigated the role of human vtRNA1-1 in regulating intestinal epithelial renewal and barrier function. Studies were conducted in vtRNA1-1 transgenic (vtRNA1-1Tg) mice, primary enterocytes, and Caco-2 cells. Extracellular vesicles (EVs) were isolated from the serum of shock patients and septic mice. Intestinal organoids (enteroids) were prepared from vtRNA1-1Tg and littermate mice. Mucosal growth was measured by Ki67 immunostaining or BrdU incorporation, and gut permeability assessed using the FITC-dextran assay. Intestinal tissues recovered from shock patients and septic mice evidenced mucosal injury and gut barrier dysfunction; vtRNA levels were elevated in EVs isolated from their sera. In mice, intestinal epithelial-specific transgenic expression of vtRNA1-1 inhibited mucosal growth, reduced Paneth cell numbers and intercellular junction (IJ) protein expression, and increased gut barrier vulnerability to lipopolysaccharide exposure. Conversely, in vitro silencing of vtRNA1-1 increased IJ protein levels and enhanced epithelial barrier function. Exposing enteroids to vtRNA1-1-rich EVs augmented paracellular permeability. Mechanistically, vtRNA1-1 interacted with CUG-binding protein 1 (CUGBP1) and increased CUGBP1 association with claudin-1 and occludin mRNAs, thereby inhibiting their expression. These findings indicate that elevated levels of vtRNA1-1 in EVs and mucosal tissues repress intestinal epithelial renewal and barrier function. Notably, this work reveals a novel role for dysregulation of the vtRNA1-1/CUGBP1 axis in the pathogenesis of gut mucosal disruption in critical illness.

Aging-Associated Molecular Changes in Human Alveolar Type I Cells.

Human alveolar type I (AT1) cells are specialized epithelial cells that line the alveoli in the lungs where gas exchange occurs. The primary function of AT1 cells is not only to facilitate efficient gas exchange between the air and the blood in the lungs, but also to contribute to the structural integrity of the alveoli to maintain lung function and homeostasis. Aging has notable effects on the structure, function, and regenerative capacity of human AT1 cells. However, our understanding of the molecular mechanisms driving these age-related changes in AT1 cells remains limited. Leveraging a recent single-cell transcriptomics dataset we generated on healthy human lungs, we identified a series of significant molecular alterations in AT1 cells from aged lungs. Notably, the aged AT1 cells exhibited increased cellular senescence and chemokine gene expression, alongside diminished epithelial features such as decreases in cell junctions, endocytosis, and pulmonary matrisome gene expression. Gene set analyses also indicated that aged AT1 cells were resistant to apoptosis, a crucial mechanism for turnover and renewal of AT1 cells, thereby ensuring alveolar integrity and function. Further research on these alterations is imperative to fully elucidate the impact on AT1 cells and is indispensable for developing effective therapies to preserve lung function and promote healthy aging.

Simulated microgravity-induced dysregulation of cerebrospinal fluid immune homeostasis by disrupting the blood-cerebrospinal fluid barrier.

The blood-cerebrospinal fluid barrier (BCSFB) comprises the choroid plexus epithelia. It is important for brain development, maintenance, function, and especially for maintaining immune homeostasis in the cerebrospinal fluid (CSF). Although previous studies have shown that the peripheral immune function of the body is impaired upon exposure to microgravity, no studies have reported changes in immune cells and cytokines in the CSF that reflect neuroimmune status. The purpose of this study is to investigate the alterations in cerebrospinal fluid (CSF) immune homeostasis induced by microgravity and its mechanisms. This research is expected to provide basic data for brain protection of astronauts during spaceflight. The proportions of immune cells in the CSF and peripheral blood (PB) of SMG rats were analyzed using flow cytometry. Immune function was evaluated by measuring cytokine concentrations using the Luminex method. The histomorphology and ultrastructure of the choroid plexus epithelia were determined. The concentrations of intercellular junction proteins in choroid plexus epithelial cells, including vascular endothelial-cadherin (VE-cadherin), zonula occludens 1 (ZO-1), Claudin-1 and occludin, were detected using western blotting and immunofluorescence staining to characterize BCSFB injury. We found that SMG caused significant changes in the proportion of CD4 and CD8 T cells in the CSF and a significant increase in the levels of cytokines (GRO/KC, IL-18, MCP-1, and RANTES). In the PB, there was a significant decrease in the proportion of T cells and NKT cells and a significant increase in cytokine levels (GRO/KC, IL-18, MCP-1, and TNF-α). Additionally, we observed that the trends in immune markers in the PB and CSF were synchronized within specific SMG durations, suggesting that longer SMG periods (≥21 days) have a more pronounced impact on immune markers. Furthermore, 21d-SMG resulted in ultrastructural disruption and downregulated expression of intercellular junction proteins in rat choroid plexus epithelial cells. We found that SMG disrupts the BCSFB and affects the CSF immune homeostasis. This study provides new insights into the health protection of astronauts during spaceflight.

Vangl2 regulates intercellular junctions by remodeling actin-based cytoskeleton through the Rock signaling pathway during spermatogenesis in Eriocheir sinensis

As a key planar cell polarity protein, Van Gogh-like 2 (Vangl2) is essential for mammalian spermatogenesis. As a decapod crustacean, Eriocheir sinensis exhibits distinct spermatogenic processes due to its unique seminiferous tubule morphology and hemolymph-testis barrier (HTB). To determine whether Vangl2 performs analogous functions in E. sinensis, we identified the Es-Vangl2. Es-Vangl2 exhibited high expression and wide distribution in the testes, indicating its crucial involvement in spermatogenesis. Following targeted knockdown of Es-Vangl2in vivo, the structure of seminiferous tubules was disrupted, characterized by vacuolization of the germinal zone and obstruction of spermatozoon release. Concurrently, the integrity of the HTB was compromised, accompanied by reduced expression and aberrant localization of junction proteins. More importantly, the regulatory influence of Es-Vangl2 was manifested through modulating the organization of microfilaments, a process mediated by epidermal growth factor receptor pathway substrate 8 (Eps8). Further studies demonstrated that these phenotypes resulting from Es-Vangl2 knockdown were attributed to the inhibition of Rock signaling pathway activity, which was verified by the Es-Rock interference and Y27632 inhibition assays. In summary, the findings highlight the pivotal role of Es-Vangl2 in stabilizing HTB integrity by regulating Eps8-mediated actin remodeling through the Rock signaling pathway in the spermatogenesis of E. sinensis.

Melatonin Prevents Thioacetamide-Induced Gut Leakiness and Liver Fibrosis Through the Gut-Liver Axis via Modulating Sirt1-Related Deacetylation of Gut Junctional Complex and Hepatic Proteins.

Intestinal barrier dysfunction with high serum endotoxin is common in patients with liver fibrosis, but the mechanisms underlying liver fibrosis remain unclear. Melatonin is a well-recognized antioxidant and an anti-inflammatory agent that benefits multiple organs. However, the beneficial effects of melatonin on gut leakiness-associated liver fibrosis have not been systemically studied. Here, we investigated the protective mechanisms of melatonin against thioacetamide (TAA)-induced gut barrier dysfunction and hepatic fibrosis by focusing on posttranslational protein modifications through the gut-liver axis. Our results showed that gut leakiness markers, including decreased gut tight/adherens junction proteins (TJ/AJs) with increased intestinal deformation, apoptosis, and serum endotoxin, were observed early at 1 week after TAA exposure. Liver injury, apoptosis, and fibrosis were prominent at 2 and 4 weeks. Mechanistically, we found that gut TJ/AJs were hyper-acetylated, followed by ubiquitin-dependent proteolysis, leading to their degradation and gut leakiness. Gut dysbiosis, hepatic protein hyper-acetylation, and SIRT1 downregulation were also observed. Consistently, intestinal Sirt1 deficiency greatly enhanced protein hyper-acetylation, gut leakiness, endotoxemia, and liver fibrosis. Pretreatment with melatonin prevented or improved all these changes in both the gut and liver. Furthermore, melatonin blunted protein acetylation and injury in TAA-exposed T84 human intestinal and AML12 mouse liver cells. Overall, this study demonstrated novel mechanisms by which melatonin prevents gut leakiness and liver fibrosis through the gut-liver axis by attenuating the acetylation of intestinal and hepatic proteins. Thus, melatonin consumption can become a potentially safe supplement for liver fibrosis patients by preventing protein hyper-acetylation and gut leakiness.

FOXC1 and FOXC2 Ablation Causes Abnormal Valvular Endothelial Cell Junctions and Lymphatic Vessel Formation in Myxomatous Mitral Valve Degeneration.

Mitral valve (MV) disease including myxomatous degeneration is the most common form of valvular heart disease with an age-dependent frequency. Genetic evidence indicates that mutations of the human transcription factor FOXC1 are associated with MV defects, including MV regurgitation. In this study, we sought to determine whether murine Foxc1 and its closely related factor, Foxc2, are required in valvular endothelial cells (VECs) for the maintenance of MV leaflets, including VEC junctions and the stratified trilaminar ECM (extracellular matrix). Adult mice carrying tamoxifen-inducible, vascular endothelial cell (EC), and lymphatic EC-specific, compound Foxc1;Foxc2 mutations (ie, EC-Foxc-DKO and lymphatic EC-Foxc-DKO mice, respectively) were used to study the function of Foxc1 and Foxc2 in the maintenance of MVs. The EC and lymphatic EC mutations of Foxc1/c2 were induced at 7 to 8 weeks of age by tamoxifen treatment, and abnormalities in the MVs of these mutant mice were assessed via whole-mount immunostaining, immunohistochemistry/RNAscope, Movat pentachrome/Masson Trichrome staining, and Evans blue injection. EC deletions of Foxc1 and Foxc2 in mice resulted in abnormally extended and thicker MVs by causing defects in the regulation of ECM organization with increased proteoglycan and decreased collagen. Notably, reticular adherens junctions were found in VECs of control MV leaflets, and these reticular structures were severely disrupted in EC-Foxc-DKO mice. PROX1 (prospero homeobox protein 1), a key regulator in a subset of VECs on the fibrosa side of MVs, was downregulated in EC-Foxc1/c2 mutant VECs. Furthermore, we determined the precise location of lymphatic vessels in murine MVs, and these lymphatic vessels were aberrantly expanded and dysfunctional in EC-Foxc1/c2 mutant MVs. Lymphatic EC deletion of Foxc1/c2 also resulted in similar structural/ECM abnormalities as seen in EC-Foxc1/c2 mutant MVs. Our results indicate that Foxc1 and Foxc2 are required for maintaining the integrity of the MV, including VEC junctions, ECM organization, and lymphatic vessel formation/function to prevent myxomatous MV degeneration.

Bone morphogenetic protein 4 inhibits corneal neovascularization by blocking NETs-induced disruption to corneal epithelial barrier

Corneal neovascularization (CoNV) is the second leading cause of visual impairment worldwide, and current drugs have certain limitations. Inflammatory response is the core pathological process of CoNV. Neutrophil extracellular traps (NETs) are generated after neutrophil activation, which promotes neovascularization. Prior studies demonstrated that bone morphogenetic protein 4 (BMP4) could significantly reduce inflammation and CoNV formation, its exact molecular mechanism remains unclear. Therefore, we stimulated human peripheral blood neutrophils with phorbol myristate acetate (PMA) or deoxyribonuclease I (DNase I) to induce or inhibit NETs formation. By using corneal sutures and subconjunctival injections of NETs or DNase I, rat CoNV models were established. Compared with the suture group, NETs formation and inflammatory cell infiltration in the corneal stroma were significantly increased, corneal edema was aggravated, and the length, area and diameter of CoNV were significantly enhanced in the NETs group. Furthermore, by curetting the corneal epithelial apical junctional complexes (AJCs), a crucial component in preserving the function of the corneal epithelial barrier, we discovered that the damage of AJCs had a significant role in inducing CoNV formation. NETs could induce CoNV formation by injuring corneal epithelial AJCs. Finally, by comparing the aforementioned indicators after the intervention of BMP4, BMP4 inhibitor Noggin and NADPH oxidase (NOX) inhibitor, we finally demonstrated that BMP4 could inhibit NETs-induced inflammation and corneal epithelial AJC injury, repair corneal epithelial barrier function and eventually inhibit CoNV formation by blocking NOX-2-dependent NETs formation.

Rice BARENTSZ genes are required to maintain floral developmental stability against temperature fluctuations.

BARENTSZ (BTZ), a core component of the exon junction complex, regulates diverse developmental processes in animals. However, its evolutionary and developmental roles in plants remain elusive. Here, we revealed that three groups of paralogous BTZ genes existed in Poaceae, and Group 2 underwent loss-of-function mutations during evolution. They showed surprisingly low (~33%) sequence identities, implying functional divergence. Two genes retained in rice, OsBTZ1 and OsBTZ3, were edited; however, the resultant osbtz1 and osbtz3 mutants showed similar floral morphological and functional defects at a low frequency. When growing under low-temperature conditions, developmental abnormalities became pronounced, and new floral variations were induced. In particular, stamen and carpel functionality was impaired in these rice btz mutants. The double-gene mutant osbtz1/3 shared these floral defects with an increased frequency, which was further induced under low-temperature conditions. OsBTZs interacted with OsMADS7 and OsMADS8, and the floral expressions of the OsTGA10 and MADS-box genes were correlatively altered in these osbtz mutants and responded to low-temperature treatment. These novel findings demonstrate that two highly diverged OsBTZs are required to maintain floral developmental stability under low-temperature conditions, and play an integral role in male and female fertility, thus providing new insights into the indispensable roles of BTZ genes in plant development and adaptive evolution.