Intercellular Junctions Research Articles

Mechanism of intestinal injury induced by acute diquat poisoning in rats

To investigate the effects of diquat (DQ) on the expression of intestinal pyroptosis-related proteins and tight junction proteins in rats,and to analyze the role of pyroptosis in the intestinal injury of rats with acute DQ poisoning. A total of 36 Wistar male rats were randomly divided into control group, and 3 hours, 12 hours, 36 hours and 3 days exposure groups, with 6 rats in each group. Each exposure group was given 1/2 median lethal dose (LD50) of 115.5 mg/kg DQ by one-time gavage. The control group was given the same amount of normal saline by gavage. The control group was anesthetized at 3 hours after DQ gavage to take jejunal tissues; each exposure group was anesthetized at 3 hours, 12 hours, 36 hours, and 3 days after DQ gavage to take jejunal tissues, respectively. The general conditions of the rats were recorded. The pathological changes of jejunum tissue were observed by hematoxylin-eosin (HE) staining. The expression of intestinal pyroptosis-related proteins [NOD-like receptor protein 3 (NLRP3), cysteine aspartate-specific protease 1 (caspase-1), Gasdemin D (GSDMD)] in the intestinal tissues was observed by immunohistochemical staining. Western blotting was used to detect the expression of intestinal pyroptosis-related proteins and intestinal tight junction proteins (Occludin and Claudin-1). Light microscopy showed that pathological changes occurred in jejunum tissue at the early stage of exposure (3 hours), and the injury was the most serious in the 12 hours exposure group, with a large number of inflammatory cells infiltrating in the tissue, and the damage was significantly reduced after 3 days exposure. Immunohistochemical results showed that NLRP3, caspase-1 and GSDMD were expressed in the jejunal mucosa of the control group and the exposure groups, and the positive cells in the control group were less expressed with light staining. The expression of the above proteins in the exposed group was increased significantly and the staining was deep. Western blotting results showed that compared with the control group, the expression of NLRP3 protein in jejunum tissues of all groups was increased, with the most significant increase in the 36 hours group (NLRP3/β-actin: 1.47±0.06 vs. 0.43±0.14, P < 0.01). Compared with the control group, the expression of GSDMD protein in the 3 hours, 12 hours and 36 hours exposure groups increased, and the expression of GSDMD protein in the 3 hours and 12 hours exposure groups increased significantly (GSDMD/β-actin: 1.04±0.40, 1.25±0.15 vs. 0.65±0.25, both P < 0.05). The expression of caspase-1 protein was increased in 36 hours exposure group compared with the control group (caspase-1/β-actin: 1.44±0.34 vs. 0.98±0.19, P > 0.05). Compared with the control group, the expression of Occludin and Claudin-1 proteins in each exposure group decreased, and the expression of Occludin proteins was significantly decreased in the 3 hours, 12 hours, and 36 hours exposure groups decreased significantly (Occludin/β-actin: 0.74±0.17, 0.91±0.20, 0.79±0.23 vs. 1.41±0.08, all P < 0.05). Although the protein expression of Claudin-1 decreased in each exposure group, the difference was not statistically significant. The intestinal injury caused by acute DQ poisoning may be related to the activation of pyroptosis pathway of small intestinal cells and the reduction of the density of intercellular junctions.

Abstract PR008: Location, location, location: Why cellular localization of mutant β-catenin matters in endometrial cancer

Abstract The present study aims to understand the mechanisms of mutant β-catenin in endometrial cancer (EC) patients with exon 3 CTNNB1 mutations. Endometrial cancer patients with exon 3 CTNNB1 mutations tend to be younger in age and have low cancer grades, but they exhibit worse recurrence-free survival than patients with similar clinical characteristics. It is unknown why EC patients with CTNNB1 mutations have worse survival. CTNNB1 encodes the protein β-catenin, which functions primarily at the cell membrane in the adherens junction complex and in the Wnt signaling pathway, where it localizes in the cytoplasm or nucleus. Exon 3 CTNNB1 mutations in EC are classically thought to promote nuclear localization of β-catenin. In this study, we conducted immunohistochemistry for β-catenin localization on 53 EC cases with an exon 3 CTNNB1 mutation from the UNCseq project. Surprisingly, 32 cases had primarily non-nuclear, either cytoplasmic or membranous, localization and 21 cases had primarily nuclear localization. Additionally, we identified 16 cases with intratumoral heterogeneity containing both nuclear and non-nuclear β-catenin localization. To investigate the functional consequences of this differential localization, we performed spatial transcriptomics using the Nanostring GeoMx Whole Transcriptome Analysis. Nuclear and non-nuclear regions of interest (ROI) were selected from 16 CTNNB1 mutant EC cases exhibiting heterogenous β-catenin localization within the same tissue (n=95 total ROIs). We hypothesize that β-catenin nuclear and non-nuclear ROIs will have differing transcriptional outputs in CTNNB1 mutant EC. Differential expression analysis of all ROIs yielded distinct transcriptional profiles based on localization of mutant β-catenin. Gene Set Enrichment Analysis revealed enrichment for Wnt-signaling and the epithelial-to-mesenchymal transition pathways in nuclear ROIs and enrichment for estrogen and androgen signaling pathways in non-nuclear ROIs. Hierarchical clustering of all ROIs yielded two genetically distinct clusters comprised of entirely nuclear or non-nuclear ROIs, with the exception of four ROIs. Higher expression of TACSTD2, encoding the protein Trop2, was enriched in the nuclear ROI cluster. Trop2 is the target of Sacituzumab Govitecan (SG), a drug conjugate with recent success in metastatic triple negative breast cancer patients. Additionally, studies have shown Trop2 to induce nuclear localization of β-catenin in other cancer types. Therefore, we hypothesize that CTNNB1 mutant EC patients with nuclear localization of β-catenin will respond better to SG treatment. To conclude, we established gene expression profiles for areas of nuclear and non-nuclear β-catenin in CTNNB1 mutant EC tissues and identified a novel gene with implications in mechanisms of β-catenin localization which may allow therapeutic targeting of advanced disease. Citation Format: Molly L. Parrish, Andrew B. Gladden, Russell R. Broaddus. Location, location, location: Why cellular localization of mutant β-catenin matters in endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr PR008.

First person – Michelle Ly

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Michelle Ly is first author on ‘ Integrin-based adhesions promote cell–cell junction and cytoskeletal remodelling to drive embryonic wound healing’, published in JCS. Michelle conducted the research described in this article while a MASc student in Rodrigo Fernandez-Gonzalez's lab at University of Toronto, Canada where she investigated the molecular mechanisms underlying cell migration and cell–extracellular matrix adhesion, and their roles in embryology and developmental processes. She is now a Research Assistant in the lab of Stewart Russel at the CReATe Fertility Centre, Toronto, Canada, working to pinpoint biomarkers associated with successful early embryo development.

Plasmodesmata: Channels Under Pressure.

Multicellularity has emerged multiple times in evolution, enabling groups of cells to share a living space and reducing the burden of solitary tasks. While unicellular organisms exhibit individuality and independence, cooperation among cells in multicellular organisms brings specialization and flexibility. However, multicellularity also necessitates intercellular dependence and relies on intercellular communication. In plants, this communication is facilitated by plasmodesmata: intercellular bridges that allow the direct (cytoplasm-to-cytoplasm) transfer of information between cells. Plasmodesmata transport essential molecules that regulate plant growth, development, and stress responses. They are embedded in the extracellular matrix but exhibit flexibility, adapting intercellular flux to meet the plant's needs.In this review, we delve into the formation and functionality of plasmodesmata and examine the capacity of the plant communication network to respond to developmental and environmental cues. We illustrate how environmental pressure shapes cellular interactions and aids the plant in adapting its growth.

Membranes are functionalized by a proteolipid code.

Membranes are protein and lipid structures that surround cells and other biological compartments. We present a conceptual model wherein all membranes are organized into structural and functional zones. The assembly of zones such as receptor clusters, protein-coated pits, lamellipodia, cell junctions, and membrane fusion sites is explained to occur through a protein-lipid code. This challenges the theory that lipids sort proteins after forming stable membrane subregions independently of proteins.

Carnosol alleviates sepsis-induced pulmonary endothelial barrier dysfunction by targeting nuclear factor erythroid2-related factor 2/sirtuin-3 signaling pathway to attenuate oxidative damage.

Excessive reactive oxygen species production during acute lung injury (ALI) will aggravate the inflammatory process and endothelial barrier dysfunction. Carnosol is a natural phenolic diterpene with antioxidant and anti-inflammatory properties, but its role in treating sepsis-induced ALI remains unclear. This study aims to explore the protective effects and underlying mechanisms of carnosol in sepsis-induced ALI. C57BL/6 mouse were preconditioned with carnosol for 1 h, then the model of lipopolysaccharide (LPS)-induced sepsis was established. The degree of pulmonary edema, oxidative stress, and inflammation were detected. Endothelial barrier function was evaluated by apoptosis and cell junctions. In vitro, Mito Tracker Green probe, JC-1 staining, and MitoSOX staining were conducted to investigate the effect of carnosol on mitochondria. Finally, we investigated the role of nuclear factor-erythroid 2-related factor (Nrf2)/sirtuin-3 (SIRT3) in carnosol against ALI. Carnosol alleviated LPS-induced pulmonary oxidative stress and inflammation by inhibiting excess mitochondrial reactive oxygen species production and maintaining mitochondrial homeostasis. Furthermore, carnosol also attenuated LPS-induced endothelial cell barrier damage by reducing vascular endothelial cell apoptosis and restoring occludin, ZO-1, and vascular endothelial-Cadherin expression in vitro and in vivo. In addition, carnosol increased Nrf2 nuclear translocation to promote SIRT3 expression. The protective effects of carnosol on ALI were largely abolished by inhibition of Nrf2/SIRT3. Our study has provided the first evidence that the Nrf2/SIRT3 pathway is a protective target of the endothelial barrier in ALI, and carnosol can serve as a potential therapeutic candidate for ALI by utilizing its ability to target this pathway.

Long-Term Tracking of the Effects of Colostrum-Derived Lacticaseibacillus rhamnosus Probio-M9 on Gut Microbiota in Mice with Colitis-Associated Tumorigenesis.

Intestinal bacteria play important roles in the progression of colitis-associated carcinogenesis. Colostrum-derived Lacticaseibacillus rhamnosus Probio-M9 (Probio-M9) has shown a protective effect in a colitis-associated cancer (CAC) model, but detailed metagenomic analysis had not been performed. Here, we investigated the preventive effects of the probiotic Probio-M9 on CAC-model mice, tracking the microbiota. Feces were obtained at four time points for evaluation of gut microbiota. The effect of Probio-M9 on tight junction protein expression was evaluated in co-cultured Caco-2 cells. Probio-M9 treatment decreased the number of tumors as well as stool consistency score, spleen weight, inflammatory score, and macrophage expression in the CAC model. Probio-M9 accelerated the recovery of the structure, composition, and function of the intestinal microbiota destroyed by azoxymethane (AOM)/dextran sulfate sodium (DSS) by regulating key bacteria (including Lactobacillus murinus, Muribaculaceae bacterium DSM 103720, Muribaculum intestinale, and Lachnospiraceae bacterium A4) and pathways from immediately after administration until the end of the experiment. Probio-M9 co-culture protected against lipopolysaccharide-induced impairment of tight junctions in Caco-2 cells. This study provides valuable insight into the role of Probio-M9 in correcting gut microbiota defects associated with inflammatory bowel disease carcinogenesis and may have clinical application in the treatment of inflammatory carcinogenesis.

A neuroligin-2-YAP axis regulates progression of pancreatic intraepithelial neoplasia

Pancreatic ductal adenocarcinoma (PDAC) is a tumor with a dismal prognosis that arises from precursor lesions called pancreatic intraepithelial neoplasias (PanINs). Progression from low- to high-grade PanINs is considered as tumor initiation, and a deeper understanding of this switch is needed. Here, we show that synaptic molecule neuroligin-2 (NLGN2) is expressed by pancreatic exocrine cells and plays a crucial role in the regulation of contact inhibition and epithelial polarity, which characterize the switch from low- to high-grade PanIN. NLGN2 localizes to tight junctions in acinar cells, is diffusely distributed in the cytosol in low-grade PanINs and is lost in high-grade PanINs and in a high percentage of advanced PDACs. Mechanistically, NLGN2 is necessary for the formation of the PALS1/PATJ complex, which in turn induces contact inhibition by reducing YAP function. Our results provide novel insights into NLGN2 functions outside the nervous system and can be used to model PanIN progression.

CRB1-associated retinal degeneration is dependent on bacterial translocation from the gut

The Crumbs homolog 1 (CRB1) gene is associated with retinal degeneration, most commonly Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Here, we demonstrate that murine retinas bearing the Rd8 mutation of Crb1 are characterized by the presence of intralesional bacteria. While normal CRB1 expression was enriched in the apical junctional complexes of retinal pigment epithelium and colonic enterocytes, Crb1 mutations dampened its expression at both sites. Consequent impairment of the outer blood retinal barrier and colonic intestinal epithelial barrier in Rd8 mice led to the translocation of intestinal bacteria from the lower gastrointestinal (GI) tract to the retina, resulting in secondary retinal degeneration. Either the depletion of bacteria systemically or the reintroduction of normal Crb1 expression colonically rescued Rd8-mutation-associated retinal degeneration without reversing the retinal barrier breach. Our data elucidate the pathogenesis of Crb1-mutation-associated retinal degenerations and suggest that antimicrobial agents have the potential to treat this devastating blinding disease.

A Modified Tridecapeptide Probe for Imaging Cell Junction.

Cell junctions, which are typically associated with dynamic cytoskeletons, are essential for a wide range of cellular activities, including cell migration, cell communication, barrier function and signal transduction. Observing cell junctions in real-time can help us understand the mechanisms by which they regulate these cellular activities. This study examined the binding capacity of a modified tridecapeptide from Connexin 43 (Cx43) to the cell junction protein zonula occludens-1 (ZO-1). The goal was to create a fluorescent peptide that can label cell junctions. A cell-penetrating peptide was linked to the modified tridecapeptide. The heterotrimeric peptide molecule was then synthesized. The binding of the modified tridecapeptide was tested using pulldown and immunoprecipitation assays. The ability of the peptide to label cell junctions was assessed by adding it to fixed or live Caco-2 cells. The testing assays revealed that the Cx43-derived peptide can bind to ZO-1. Additionally, the peptide was able to label cell junctions of fixed cells, although no obvious cell junction labeling was observed clearly in live cells, probably due to the inadequate affinity. These findings suggest that labeling cell junctions using a peptide-based strategy is feasible. Further efforts to improve its affinity are warranted in the future.

Extracting interpretable features for pathologists using weakly supervised learning to predict p16 expression in oropharyngeal cancer

One drawback of existing artificial intelligence (AI)-based histopathological prediction models is the lack of interpretability. The objective of this study is to extract p16-positive oropharyngeal squamous cell carcinoma (OPSCC) features in a form that can be interpreted by pathologists using AI model. We constructed a model for predicting p16 expression using a dataset of whole-slide images from 114 OPSCC biopsy cases. We used the clustering-constrained attention-based multiple-instance learning (CLAM) model, a weakly supervised learning approach. To improve performance, we incorporated tumor annotation into the model (Annot-CLAM) and achieved the mean area under the receiver operating characteristic curve of 0.905. Utilizing the image patches on which the model focused, we examined the features of model interest via histopathologic morphological analysis and cycle-consistent adversarial network (CycleGAN) image translation. The histopathologic morphological analysis evaluated the histopathological characteristics of image patches, revealing significant differences in the numbers of nuclei, the perimeters of the nuclei, and the intercellular bridges between p16-negative and p16-positive image patches. By using the CycleGAN-converted images, we confirmed that the sizes and densities of nuclei are significantly converted. This novel approach improves interpretability in histopathological morphology-based AI models and contributes to the advancement of clinically valuable histopathological morphological features.

Targeting PKM2 signaling cascade with salvianic acid A normalizes tumor blood vessels to facilitate chemotherapeutic drug delivery

Aberrant tumor blood vessels are prone to propel the malignant progression of tumors, and targeting abnormal metabolism of tumor endothelial cells emerges as a promising option to achieve vascular normalization and antagonize tumor progression. Herein, we demonstrated that salvianic acid A (SAA) played a pivotal role in contributing to vascular normalization in the tumor-bearing mice, thereby improving delivery and effectiveness of the chemotherapeutic agent. SAA was capable of inhibiting glycolysis and strengthening endothelial junctions in the human umbilical vein endothelial cells (HUVECs) exposed to hypoxia. Mechanistically, SAA was inclined to directly bind to the glycolytic enzyme PKM2, leading to a dramatic decrease in endothelial glycolysis. More importantly, SAA improved the endothelial integrity via activating the β-Catenin/Claudin-5 signaling axis in a PKM2-dependent manner. Our findings suggest that SAA may serve as a potent agent for inducing tumor vascular normalization.

Protective Effect of Vitamin K2 (MK-7) on Acute Lung Injury Induced by Lipopolysaccharide in Mice.

Vitamin K2 (MK-7) has been shown to cause significant changes in different physiological processes and diseases, but its role in acute lung injury (ALI) is unclear. Therefore, in this study, we aimed to evaluate the protective effects of VK2 against LPS-induced ALI in mice. The male C57BL/6J mice were randomly divided into six groups (n = 7): the control group, LPS group, negative control group (LPS + Oil), positive control group (LPS + DEX), LPS + VK2 (L) group (VK2, 1.5 mg/kg), and LPS + VK2 (H) group (VK2, 15 mg/kg). Hematoxylin-eosin (HE) staining of lung tissue was performed. Antioxidant superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) activities, and the Ca2+ level in the lung tissue were measured. The effects of VK2 on inflammation, apoptosis, tight junction (TJ) injury, mitochondrial dysfunction, and autophagy were quantitatively assessed using Western blot analysis. Compared with the LPS group, VK2 improved histopathological changes; alleviated inflammation, apoptosis, and TJ injury; increased antioxidant enzyme activity; reduced Ca2+ overload; regulated mitochondrial function; and inhibited lung autophagy. These results indicate that VK2 could improve tight junction protein loss, inflammation, and cell apoptosis in LPS-induced ALI by inhibiting the mitochondrial dysfunction and excessive autophagy, indicating that VK2 plays a beneficial role in ALI and might be a potential therapeutic strategy.

Regulation of Epithelial and Endothelial Barriers by Molecular Chaperones.

The integrity and permeability of epithelial and endothelial barriers depend on the formation of tight junctions, adherens junctions, and a junction-associated cytoskeleton. The establishment of this junction-cytoskeletal module relies on the correct folding and oligomerization of its protein components. Molecular chaperones are known regulators of protein folding and complex formation in different cellular compartments. Mammalian cells possess an elaborate chaperone network consisting of several hundred chaperones and co-chaperones. Only a small part of this network has been linked, however, to the regulation of intercellular adhesions, and the systematic analysis of chaperone functions at epithelial and endothelial barriers is lacking. This review describes the functions and mechanisms of the chaperone-assisted regulation of intercellular junctions. The major focus of this review is on heat shock protein chaperones, their co-chaperones, and chaperonins since these molecules are the focus of the majority of the articles published on the chaperone-mediated control of tissue barriers. This review discusses the roles of chaperones in the regulation of the steady-state integrity of epithelial and vascular barriers as well as the disruption of these barriers by pathogenic factors and extracellular stressors. Since cytoskeletal coupling is essential for junctional integrity and remodeling, chaperone-assisted assembly of the actomyosin cytoskeleton is also discussed.

Oral squamous cell carcinoma: A case report with scanning electron microscopy, Mallory’s, Masson’s trichrome, orcein, and Papanicolaou staining

Squamous cell carcinoma is a malignant epithelial tumor with squamous differentiation. It is characterized by formation of keratin or presence of intercellular bridges. This neoplasm is also known as epidermoid carcinoma and is the second most common malignant neoplasm of the oral cavity. This neoplasm is seen more frequently in the lower lip, tongue, floor of the mouth, upper lip, buccal mucosa, gingiva, and in some cases, in the hard palate and maxillary sinus. Multiple reports on oral squamous cell carcinoma (OSCC) are found in the literature but very few report the various investigations done using special stains and scanning electron microscopy. There is a need for investigations using special stains as it is less expensive and may also help in identifying various characteristics of OSCC. Here, we present a case report of well-differentiated squamous cell carcinoma in a 60-year-old male patient with complaints of pain and ulcers on both cheeks for 2–3 months.

Ser194Leu DSG2 mutation, associated with arrhythmogenic left ventricular cardiomyopathy and ventricular tachycardia.

Arrhythmogenic cardiomyopathy (AC) is an inherited cardiomyopathy characterized by fibro-fatty replacement of cardiomyocytes, leading to life-threatening ventricular arrhythmia and heart failure. Pathogenic variants of desmoglein2 gene (DSG2) have been reported as genetic etiologies of AC. In contrast, many reported DSG2 variants are benign or variants of uncertain significance. Correct genetic variant classification is crucial for determining the best medical therapy for the patient and family members. Pathogenicity of the DSG2 Ser194Leu variant that was identified by whole exome sequencing in a patient, who presented with ventricular tachycardia and was diagnosed with AC, was investigated by electron microscopy and immunohistochemical staining of endomyocardial biopsy sample. Electron microscopy demonstrated a widened gap in the adhering junction and a less well-organized intercalated disk region in the mutated cardiomyocytes compared to the control. Immunohistochemical staining in the proband diagnosed with AC showed reduced expression of desmoglein 2 and connexin 43 and intercalated disc distortion. Reduced expression of DSG2 and Connexin 43 were observed in cellular cytoplasm and gap junctions. Additionally, we detected perinuclear accumulation of DSG2 and Connexin 43 in the proband sample. Ser194Leu is a missense pathogenic mutation of DSG2 gene associated with arrhythmogenic left ventricular cardiomyopathy.

Resveratrol protects dopaminergic neurons in a mouse model of Parkinson's disease by regulating the gut-brain axis via inhibiting the TLR4 signaling pathway

To investigate the protective effect of resveratrol on intestinal barrier in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) mouse models and its mechanism for regulating TLR4/MyD88/NF-κB signaling to protect dopaminergic neurons. Fifty-two C57BL/6J mice were randomized into control group (n= 12), MPTP group (n=14), MPTP + resveratrol (30 mg/kg) group (n=13), and MPTP + resveratrol (90 mg/kg) group (n=13), and mouse models were established by intraperitoneal MPTP (30 mg/kg) injection for 7 days in the latter 3 groups. Behavioral tests were conducted to evaluate the effect of resveratrol on motor symptoms of the mice. Western blotting was used to detect the expression of TH, α-syn, ZO-1, Claudin-1, TLR4, MyD88, and NF-κB in the brain tissues of the mice. Immunohistochemistry, immunofluorescence, ELISA and transmission electron microscopy were used to verify the effect of resveratrol for suppressing inflammation and protecting the intestinal barrier. Compared with those in the normal control group, the mice in MPTP group showed significant changes in motor function, number of dopaminergic neurons, neuroinflammation, levels of LPS and LBP, and expressions of tight junction proteins in the intestinal barrier. Resveratrol treatment significantly improved motor function of the PD mice (P < 0.01), increased the number of neurons and TH protein expression (P < 0.05), down-regulated the expressions of GFAP, Iba-1, and TLR4, lowered fecal and plasma levels of LPS and LBP (P < 0.05), restored the expression levels of ZO-1 and Claudin-1 (P < 0.01), and down-regulated the expressions of TLR4, MyD88, and NF-κB in the colon tissue (P < 0.05). The mice with resveratrol treatment at 30 mg/kg showed normal morphology of the tight junction complex with neatly and tightly arranged intestinal villi. Resveratrol repairs the intestinal barrier by inhibiting TLR4/MyD88/NF-κB signaling pathway-mediated inflammatory response, thereby improving motor function and neuropathy in mouse models of MPTP-induced PD.

A system of reporters for comparative investigation of EJC-independent and EJC-enhanced nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a network of pathways that degrades transcripts that undergo premature translation termination. In mammals, NMD can be divided into the exon junction complex (EJC)-enhanced and EJC-independent branches. Fluorescence- and luminescence-based reporters have long been effective tools to investigate NMD, yet existing reporters largely focus on the EJC-enhanced pathway. Here, we present a system of reporters for comparative studies of EJC-independent and EJC-enhanced NMD. This system also enables the study of NMD-associated outcomes such as premature termination codon (PTC) readthrough and truncated protein degradation. These reporters are compatible with fluorescence or luminescence-based readouts via transient transfection or stable integration. Using this reporter system, we show that EJC-enhanced NMD RNA levels are reduced by 2- or 9-fold and protein levels are reduced by 7- or 12-fold compared to EJC-independent NMD, depending on the reporter gene used. Additionally, the extent of readthrough induced by G418 and an NMD inhibitor (SMG1i), alone and in combination, varies across NMD substrates. When combined, G418 and SMG1i increase readthrough product levels in an additive manner for EJC-independent reporters, while EJC-enhanced reporters show a synergistic effect. We present these reporters as a valuable toolkit to deepen our understanding of NMD and its associated mechanisms.

Listeria adhesion protein orchestrates caveolae-mediated apical junctional remodeling of epithelial barrier for Listeria monocytogenes translocation.

Listeria monocytogenes (Lm) is a foodborne pathogen with high mortality (20%-30%) and hospitalization rates (94%), particularly affecting vulnerable groups such as pregnant women, fetuses, newborns, seniors, and immunocompromised individuals. Invasive listeriosis involves Lm's internalin (InlA) protein binding to E-cadherin to breach the intestinal barrier. However, non-functional InlA variants have been identified in Lm isolates, suggesting InlA-independent pathways for translocation. Our study reveals that Listeria adhesion protein (LAP) and InlA cooperatively assist Lm entry into the gut lamina propria in a gerbil model, mimicking human listeriosis in early infection stages. LAP triggers caveolin-1-mediated endocytosis of critical junctional proteins, transporting them to early and recycling endosomes, facilitating Lm passage through enterocytes. Furthermore, LAP-Hsp60-mediated junctional protein endocytosis precedes InlA's interaction with basolateral E-cadherin, emphasizing LAP and InlA's cooperation in enhancing Lm intestinal translocation. This understanding is vital in combating the severe consequences of Lm infection, including sepsis, meningitis, encephalitis, and brain abscess.

Modeling dry eye with an air–liquid interface in corneal epithelium-on-a-chip

Dry eye syndrome (DES) is a complex ocular condition characterized by an unstable tear film and inadequate tear production, leading to tissue damage. Despite its common occurrence, there is currently no comprehensive in vitro model that accurately reproduce the cellular characteristics of DES. Here we modified a corneal epithelium-on-a-chip (CEpOC) model to recapitulate DES by subjecting HCE-T human corneal epithelial cells to an air–liquid (AL) interface stimulus. We then assessed the effects of AL stimulation both in the presence and absence of diclofenac (DCF), non-steroidal anti-inflammatory drug. Transcriptomic analysis revealed distinct gene expression changes in response to AL and AL_DCF, affecting pathways related to development, epithelial structure, inflammation, and extracellular matrix remodeling. Both treatments upregulated PIEZO2, linked to corneal damage signaling, while downregulating OCLN, involved in cell–cell junctions. They increased the expression of inflammatory genes (e.g., IL-6) and reduced mucin production genes (e.g., MUC16), reflecting dry eye characteristics. Metabolomic analysis showed increased secretion of metabolites associated with cell damage and inflammation (e.g., methyl-2-oxovaleric acid, 3-methyl-2-oxobutanoic acid, lauroyl-carnitine) in response to AL and even more with AL_DCF, indicating a shift in cellular metabolism. This study showcases the potential use of AL stimulus within the CEpOC to induce cellular characteristics relevant to DES.