Epithelial Protein Research Articles

DOP127 A novel epithelial junction gene associated with very early onset IBD: a mechanistic study using patient organoids and transgenic mouse models

Abstract Background Very early onset inflammatory bowel disease (VEO-IBD) is often associated with a severe disease course and unique characteristics including underlying monogenic etiologies in some cases. In this study, we mechanistically defined the function of CGNL1, an epithelial junctional protein gene whose pathogenic variants may contribute to VEO-IBD. Methods Whole exome sequencing (WES) was performed on the affected patient and all first-degree relatives. Intestinal epithelial organoids (IEOs) were generated from the patient’s mucosal biopsies and were characterized by microscopy, whole-mount immunostaining, transcriptomics and transmission electron microscopy (TEM). In addition, we generated a new Cgnl1-/- mice for dextran-sodium sulfate (DSS)-induced colitis and infection by Citrobacter rodentium which directly compromises intestinal epithelium. Integrated analysis was performed to determine CGNL1 expression in single-cell datasets. Results In this patient with VEO-IBD who required early total colectomy with pouch formation, coexistent primary sclerosing cholangitis (PSC) and celiac disease, WES identified two rare, novel, pathogenic variants in the CGNL1 gene. Both pathogenic variants were also present in the patient’s younger sister, who has a similar history including fulminant UC and PSC-autoimmune hepatitis overlap syndrome. Furthermore, multiple relatives of the patient suffer from IBD and other inflammatory conditions including celiac disease, eosinophilic esophagitis, and type-1 diabetes. The RNA and protein levels of CGNL1 were diminished in the patient’s intestinal epithelium compared to that of healthy individuals and other IBD patients. The patient’s IEOs from pouch body and pre-pouch ileum formed smaller 3D spheroids with altered cell composition and significantly impaired barrier function compared to those from normal controls (Figure 1). TEM demonstrated disrupted tight junctions and adherens junctions in the patient’s IEOs. Similarly, impaired junctional complexes in intestinal epithelial cells and elevated intestinal permeability were present in Cgnl1-/- mice, which also showed increased susceptibility to DSS-induced colitis (Figure 2) and C. rodentium infection. Single-cell analysis revealed diminished expression of CGNL1 in intestinal and hepatic epithelial cells from patients with IBD or PSC, respectively. Conclusion The pathogenic CGNL1 variants may contribute to VEO-IBD and other inflammatory GI disease by disrupting gut barrier integrity. This study expands our understanding of epithelial barrier impairment in the pathogenesis of VEO-IBD and suggests the intestinal epithelium as a therapeutic target for some IBD patients.

DOP107 Real-time intestinal barrier assessment by endocytoscopy and confocal laser endomicroscopy uniquely correlates with multiple barrier protein expression and reflects the gut-brain axis

Abstract Background The intestinal barrier is gaining recognition as a key indicator of mucosal healing in IBD. Nonetheless, its assessment remains challenging. This study evaluates advanced endoscopic tools for real-time gut barrier assessment, examining their correlation with automated epithelial and vascular barrier markers and exploring their potential to elucidate the gut-brain axis. Methods IBD patients undergoing endoscopic assessment or surveillance and healthy controls were included. The intestinal mucosa was assessed with advanced imaging, i.e. ultra-high magnification endocytoscopy (ECS) or confocal laser endomicroscopy (CLE), using newly developed scores for barrier evaluation.1,2 Barrier healing was defined as ECS ≤ 1 -ileum and colon- and CLE ≤ 4 -colon- or ≤ 3 -ileum-. Biopsies were collected from different bowel segments, including representative inflamed and non-inflamed areas. Epithelial and vascular barrier markers, including ZO-1, Claudin-2, E-cadherin, PV-1 and CD-31, were analysed through confocal microscopy, and their expression was automatically quantified using QuPath. The validated IBD disk, focusing on energy, sleep and emotional components, was employed to evaluate neuropsychological functional impairment and indirectly assess gut-brain interaction.3 A per-biopsy-based linear regression model was used for inferential analysis using R software. Results A cohort of 33 IBD patients (15 CD and 18 UC) and 2 healthy controls were recruited (65 biopsies; 3.8 X106 cells). 9/16 (56%) patients who underwent ECS in the colon and 6/9 (67%) in the ileum had ECS scores indicative of barrier healing. CLE assessment revealed features suggestive of barrier healing in 3/11 patients (27%) in the colon and 1/14 (7%) in the ileum. ECS and CLE sub- and total scores demonstrated unique correlation with epithelial and vascular barrier proteins. For the epithelial barrier, CLE total scores in colon and ileum and villi architecture at ECS significantly correlated with Claudin-2 mean expression (p<0.01). Regarding the vascular barrier, a significant correlation was found between ECS-assessed vascular architecture in ileum and colon and PV-1 mean expression (p<0.01). In contrast, CLE-assessed blood flow in the colon was significantly correlated with CD-31 expression (p=0.03). Interestingly, patients with higher levels of sleeping difficulties based on IBD disk showed higher Claudin-2 and lower E-cadherin expression, while those with altered CD-31 expression reported lower energy levels. Conclusion CLE and ECS may offer a unique ability for real-time assessment of barrier impairment in IBD, showing promises for targeting real-time barrier healing and unravelling the complexities of gut-brain axis. References 1Iacucci M, Jeffery L, Acharjee A, et al. Computer-Aided Imaging Analysis of Probe-Based Confocal Laser Endomicroscopy With Molecular Labeling and Gene Expression Identifies Markers of Response to Biological Therapy in IBD Patients: The Endo-Omics Study. Inflamm Bowel Dis. 2023;29(9):1409-1420. doi:10.1093/ibd/izac233 2Majumder S, Santacroce G, Maeda Y, et al. Endocytoscopy with automated multispectral intestinal barrier pathology imaging for assessment of deep healing to predict outcomes in ulcerative colitis. Gut. 2024;73(10):1603-1606. Published 2024 Sep 9. doi:10.1136/gutjnl-2024-332894 3Le Berre C, Flamant M, Bouguen G, et al. VALIDation of the IBD-Disk Instrument for Assessing Disability in Inflammatory Bowel Diseases in a French Cohort: The VALIDate Study. J Crohns Colitis. 2020;14(11):1512-1523. doi:10.1093/ecco-jcc/jjaa100

P0560 Levels of disease-associated anti-integrin αVβ6 are substantially reduced after proctocolectomy in ulcerative colitis but persist in primary sclerosing cholangitis

Abstract Background Recently, autoantibodies directed against the epithelial adhesion protein integrin αVβ6 have been identified that strongly associate with ulcerative colitis (UC) and primary sclerosing cholangitis (PSC) with potential disease-driving effects. The presence of anti-αVβ6 in PSC without IBD is under debate. Moreover, data on the effect of proctocolectomy on autoantibody levels remains scarce. Methods Anti-αVβ6-directed autoantibodies were detected by a commercial enzyme-linked immunosorbent assay in patient sera, including healthy controls (N = 62), ulcerative colitis (N = 36), Crohn’s disease (CD, N = 65), PSC with concomitant IBD (78 samples from N = 41 patients), PSC without clinically manifest IBD (41 samples from N = 18 patients). Additionally, sera after proctocolectomy were studied (44 samples / N= 10 patients: 5 with UC, 5 with PSC). Immunofluorescent analyses of the target autoantigen were performed in liver, large bile ducts from surgical resections and colon tissue samples of PSC patients and single-cell RNA seq datasets were studied. Results The target autoantigen is expressed in disease-associated epithelia in the colon, in cholangiocytes in fibrotic portal fields and periductular glands of the large bile ducts as detected in immunofluorescence and single-cell RNA sequencing datasets. Anti-αVβ6 antibodies occur in 74% of patients with PSC with IBD, but also in 39% of patients with PSC without manifest IBD with lower levels. Anti-αVβ6 levels correlate with intestinal disease activity in patients with primary sclerosing cholangitis. Interestingly, proctocolectomy reduces autoantibody levels more strongly in UC than in PSC patients. Conclusion Disease-associated anti-integrin αVβ6 autoantibodies are also present in PSC without IBD with lower levels, assay sensitivity being a crucial determinant of detection. Moreover, proctocolectomy more strongly reduced autoantibody levels in UC than in PSC. Preferential autoantibody persistence in PSC after proctocolectomy might explain the increased incidence of chronic pouchitis in PSC.

P0046 Proteomic Profiles Associated with Residual Ultrastructural Anomalies in the Epithelium of IBD patients in Endoscopic Remission

Abstract Background Increased permeability, mucosal barrier and epithelial anomalies (crypt distortion, wider crypt lumen, epithelial gaps) as well as altered tight junctions have been described in active ulcerative colitis (UC) and Crohn’s Disease (CD). Probe based Confocal laser endomicroscopy (pCLE) is able to monitor epithelial anomalies in vivo1. This study aimed at comparing the proteomic profiles of the colonic epithelium associated with pCLE anomalies in UC, CD and non-IBD subjects. Methods Biopsies were collected in IBDs in endoscopic remission (n=37) and non-IBD control (CT) (n=10) undergoing colonoscopy with pCLE as previously described1. Biopsy sampled in the right colon and the rectum were analysed by mass spectrometry label free proteomics, with enrichment of epithelial cells using laser capture microdissection2. Comparisons of epithelial protein profiles, in presence (1) or absence (0) of fluorescein leaks were obtained in the colon and rectum, in CD, UC and non-IBD-CT. Significant proteins obtained were confronted and the entire protein profiles were evaluated using Gene Ontology functional annotations. Results Colon and rectum samples analyses provided 3227 and 3303 protein identifications (≥50% occurrence in minimum 1 group). Unique proteins (≥50% occurrence in only 1 group) were identified in the colon (n=40) and rectum (n=18). Differential analyses (0vs1) in the colon highlighted 165 and 152 significant proteins in CD and UC respectively, from which 13 were in common. In non-IBD CT (0vs1) 88 proteins were discriminant, with 9 and 6 being shared with CD and UC respectively. In the rectum, 74 and 172 discriminant proteins were obtained in CD and UC, from which 16 were also found significant in colon CD and UC protein lists(0vs1). DAZAP, STK4, LAMC1 and PDCD10 showed similar distribution tendencies in both segments in CD0vs1. While HNRPH1 and 2, VASP, TMX1 and PREP distributions were akin in UC0vs1 in both segments (Fig.1.A). GO analyses highlighted more numerous proteins with a decrease when leaks were observed in IBDs than in non-IBD-CT (Fig.2. Right arm of volcano plots). These proteins are associated with junction proteins, matrix and cytoskeleton, proteasome, protease, peptidase functions, or are involved in apoptosis and innate immune controls and morphogenesis. STK4 product, a key regulator of the HIPPO and MAPK pathways, showed a drop (0>1) in IBDs and non-IBD CT with leaks (Fig.1.B). Conclusion This proteomic study reveals proteins with perturbed epithelial distributions associated with pCLE fluorescein leaks. Functional analyses are required to disclose and understand our findings and might confirm very early epithelial key players involved in IBD epithelial homeostasis loss. References 1Jean-Philippe Loly, Sophie Vieujean, Catherine Reenaers, Catherine Van Kemseke, Laurence Seidel ScD, Edouard Louis, Joan Somja In-Depth Assessment of Endoscopic Remission in Inflammatory Bowel Disease Treated by Anti-TNF or Vedolizumab Inflamm Bowel Dis. 2024 Feb 1;30(2):240-246. doi: 10.1093/ibd/izad066. DOI: 10.1093/ibd/izad066 2.Angela-Maria Merli, Sophie Vieujean, Charlotte Massot, Noella Blétard, Florence Quesada Calvo, Dominique Baiwir, Gabriel Mazzucchelli, Laurence Servais, Odile Wéra, Cécile Oury, Laurence de Leval, Christine Sempoux, Roberto Manzini, Sena Bluemel, Michael Scharl, Gerhard Rogler, Edwin De Pauw, C Coimbra Marques, Arnaud Colard, Anne Vijverman, Philippe Delvenne, Edouard Louis, Marie-Alice Meuwis Solute carrier family 12 member 2 as a proteomic and histological biomarker of dysplasia and neoplasia in ulcerative colitis J Crohns Colitis 2020 Sep 13:jjaa168. doi: 10.1093/ecco-jcc/jjaa168.

ESRP2-microRNA-122 axis promotes the postnatal onset of liver polyploidization and maturation.

Hepatocyte polyploidy and maturity are critical to acquiring specialized liver functions. Multiple intracellular and extracellular factors influence ploidy, but how they cooperate temporally to steer liver polyploidization and maturation or how post-transcriptional mechanisms integrate into these paradigms is unknown. Here, we identified an important regulatory hierarchy in which postnatal activation of epithelial splicing regulatory protein 2 (ESRP2) stimulates processing of liver-specific microRNA (miR-122) to facilitate polyploidization, maturation, and functional competence of hepatocytes. By determining transcriptome-wide protein-RNA interactions in vivo and integrating them with single-cell and bulk hepatocyte RNA-seq data sets, we delineated an ESRP2-driven RNA processing program that drives sequential replacement of fetal-to-adult transcript isoforms. Specifically, ESRP2 binds the primary miR-122 host gene transcript to promote its processing/biogenesis. Combining constitutive and inducible ESRP2 gain- and loss-of-function mouse models with miR-122 rescue experiments, we demonstrated that timed activation of ESRP2 augments the miR-122-driven program of cytokinesis failure, ensuring the proper onset and extent of hepatocyte polyploidization.

Source of dietary protein alters the abundance of proteases, intestinal epithelial and immune proteins both directly and via interactions with the gut microbiota.

Dietary protein has been shown to impact long-term health outcomes differentially depending on its amount and source. It has been suggested that interactions of the gut microbiota with dietary proteins mediate some of the effects of dietary protein on health outcomes. However, it remains unclear what specific host responses drive the health effects of dietary proteins from different plant and animal sources. Additionally, which specific host responses are mediated by interactions of dietary protein source with the gut microbiota and which host responses are caused by dietary proteins directly is not well understood. We used metaproteomics to quantify dietary, host, and microbial proteins in fecal samples of conventional and germ-free mice fed purified dietary protein from six different plant and animal sources, including casein, egg-white, soy, brown rice, pea, and yeast. We characterized differences in the host fecal proteome across the six dietary protein sources as well as between the conventional and germ-free mice for each source to determine how the host responds to the different dietary protein sources and the role of the gut microbiota in mediating these responses. We found that both the source of dietary protein and the presence or absence of the gut microbiota drive the host response to dietary protein source in the fecal host proteome. Host proteins pertaining to immune response, digestion, and barrier function were differentially abundant in different protein sources with and without the gut microbiota. These changes in the host response correlated with changes in microbial composition and differences in protein digestibility. Our results show how dietary protein sources, through their interactions with the gut microbiota, impact several aspects of host physiology.

Role of TGF-β1/Smad3 signalling pathway in renal tubulointerstitial fibrosis and renal damage in elderly rats with isolated systolic hypertension induced by increased pulse pressure

Objective Elevated systolic blood pressure and increased pulse pressure are closely associated with renal damage; however, the exact mechanism remains unclear. Therefore, we investigated the effects of increased pulse pressure on tubulointerstitial fibrosis and renal damage in elderly rats with isolated systolic hypertension (ISH). Additionally, the role of renal tubular epithelial-mesenchymal transition (EMT) and its upstream signalling pathways were elucidated. Methods Ten-month-old male rats were randomly divided into control and ISH groups, with seven rats in each group administered warfarin and vitamin K1 for 6 weeks. Blood pressure, renal function, mean blood flow in the common iliac artery, and diastolic vessel diameter were assessed, and the rat kidney medulla was collected for histological, genetic, and protein level analysis. Results Increased pulse pressure, abnormal renal function, and increased shear stress were detected in rats with ISH. Histology assessments revealed fibrosis in the interstitium of ISH rats. Epithelial marker E-cadherin protein expression was decreased, while the protein expression of interstitial markers α-SMA and Vimentin was increased, and transforming growth factor (TGF)-β1/Smad3 signalling was upregulated in the kidney tissue of ISH rats. Conclusions Increased pulse pressure in elderly rats with ISH caused an increase in shear stress. These effects led to the development of EMT and the activation of its upstream TGF-β1/Smad3 signalling pathway, ultimately leading to renal tubular interstitial fibrosis causing renal injury.

Fluorinated chitosan mediated transepithelial delivery of sanguinarine-loaded platinum (IV) prodrug for intravesical instillation therapy of muscle-invasive bladder cancer.

Cisplatin-based neoadjuvant chemotherapy is first-line strategy to inhibit progression and metastasis of muscle-invasive bladder cancer (MIBC). However, its clinical efficacy is often limited by drug resistance and severe systemic side effects, highlighting the urgent need for innovative therapeutic approaches. Despite advancements in cisplatin-based regimens, research on intravesical cisplatin delivery systems remains scarce. In this study, we developed an amphiphilic platinum(IV) prodrug micellar platform (Pt (IV)-DI-PEG) capable of efficiently encapsulating sanguinarine (San), which was further coated with fluorinated chitosan (FCS) to construct San@Pt(IV)-DI-PEG@FCS nanoparticles (SPFNPs) for intravesical instillation even targeting MIBC. The resulting SPFNPs demonstrated several advantages: the FCS coating facilitated enhanced trans-epithelial drug delivery by regulating bladder epithelial tight junction proteins, enabling efficient intravesical administration; Second, the glutathione (GSH)-responsive reduction of the Pt(IV) prodrug promoted tumor-targeted release of San and localized accumulation of Pt(II), while simultaneously depleting intracellular GSH. Furthermore, the released San induced reactive oxygen species (ROS) production, oxidative cleavage and inhibit the activation and function of poly (ADP-ribose) polymerase, collectively impairing nucleotide-excision repair and preventing the elimination of Pt-DNA adducts, resulting in persistent DNA damage, cell cycle arrest, and apoptosis in tumor cells. The synergistic effects of San and cisplatin were validated in both orthotopic mouse models and patient-derived orthotopic xenograft, demonstrating robust anti-tumor efficacy. This study underscores the potential of intravesical cisplatin formulations as a promising strategy for MIBC treatment, offering a shift from traditional systemic chemotherapy towards localized, targeted drug delivery systems.

In Silico Analysis of Curcumin's Targeted Cancer Therapy: Folate Receptor Pathways and Molecular Interaction Insights.

This study explores the therapeutic potential of curcumin (CUR) in cancer therapy, specifically examining its targeted transport through folate receptors and its interaction with certain proteins in breast cancer cell lines. We employed molecular docking technique to assess the binding affinities of CUR with proteins 1H1Q, 1UOM, 4JDD, 5U2D and MCF10A normal breast epithelial cell line protein 5UGB. Out of these, the CUR-1H1Q complex exhibited the greatest binding affinity. To assess the stability of this complex in a biological setting, we conducted molecular dynamics simulations of the 1H1Q-CUR complex for a duration of 100ns. The simulations demonstrated an extremely stable Cα-backbone, exhibiting a consistently low root mean square deviation. The radius of gyration measurements suggested a condensed structure with specific areas of flexibility. The simulation revealed a consistent hydrogen bond between CUR and 1H1Q, indicating a robust and long-lasting interaction between the two molecules. The results indicate that the cytotoxicity of curcumin on MCF7 cancer cell lines is mainly affected by its interactions with several proteins found in these cancer cells. Among the four proteins tested, 1H1Q has the greatest influence. The high affinity of these proteins for curcumin, which results in the creation of stable complexes, seems to trigger cell death. Curcumin's biocompatibility and toxicological effects were investigated in both normal and cancerous cell lines. The study revealed enhanced biocompatibility and potential toxicity in cancerous cell lines, while demonstrating reduced toxicity in normal cell lines.

The Cancer Chimera: Impact of Vimentin and Cytokeratin Co-Expression in Hybrid Epithelial/Mesenchymal Cancer Cells on Tumor Plasticity and Metastasis.

The epithelial-mesenchymal transition (EMT) program is critical to metastatic cancer progression. EMT results in the expression of mesenchymal proteins and enhances migratory and invasive capabilities. In a small percentage of cells, EMT results in the expression of stemness-associated genes that provide a metastatic advantage. Although EMT had been viewed as a binary event, it has recently become clear that the program leads to a spectrum of phenotypes, including hybrid epithelial/mesenchymal (E/M) cells that have significantly greater metastatic capability than cells on the epithelial or mesenchymal ends of the spectrum. As hybrid E/M cells are rarely observed in physiological, non-diseased states in the adult human body, these cells are potential biomarkers and drug targets. Hybrid E/M cells are distinguished by the co-expression of epithelial and mesenchymal proteins, such as the intermediate filament proteins cytokeratin (CK; epithelial) and vimentin (VIM; mesenchymal). Although these intermediate filaments have been extensively used for pathological characterization and detection of aggressive carcinomas, little is known regarding the interactions between CK and VIM when co-expressed in hybrid E/M cells. This review describes the characteristics of hybrid E/M cells with a focus on the unique co-expression of VIM and CK. We will discuss the structures and functions of these two intermediate filament proteins and how they may interact when co-expressed in hybrid E/M cells. Additionally, we review what is known about cell-surface expression of these intermediate filament proteins and discuss their potential as predictive biomarkers and therapeutic targets.

Peptide Mimicking Loop II of the Human Epithelial Protein SLURP-2 Enhances the Viability and Migration of Skin Keratinocytes.

The secreted human protein SLURP-2 is a regulator of epithelial homeostasis, which enhances the viability and migration of keratinocytes. The targets of SLURP-2 in keratinocytes are nicotinic and muscarinic acetylcholine receptors. This work is devoted to the search for the SLURP-2 functional regions responsible for enhancing keratinocyte viability and migration. We produced synthetic peptides corresponding to the SLURP-2 loop regions and studied their effect on the viability and migration of HaCaT skin keratinocytes using the WST-8 test and scratch-test, respectively. The highest activity was exhibited by a loop II-mimicking peptide that enhanced the viability of keratinocytes and stimulated their migration. The peptide activity was mediated by interactions with α7- and α3β2-nAChRs and suppression of the p38 MAPK intracellular signaling pathway. Thus, we obtained new data that explain the mechanisms underlying SLURP-2 regulatory activity and indicate the promise of further research into loop II-mimicking peptides as prototypes of wound healing drugs.

Circadian Rhythm Enhances mTORC1/AMPK Pathway-Mediated Milk Fat Synthesis in Dairy Cows via the Microbial Metabolite Acetic Acid.

Livestock may respond differently to circadian rhythms, leading to differences in the composition of the animal products. Nevertheless, the circadian effects on rumen microorganisms and animal products are poorly understood. In the study, it was found that dairy cows exhibited increased milk fat levels, decreased acetic acid concentrations in the rumen fluid, and elevated acetic acid levels in the blood during the night compared to those of the day. Correlational analyses suggested a high association between Succiniclasticum, Lactobacillus, Prevotellacene NK3B31_group, Muribaculaceae_unclassified, etc., which were significantly enriched in rumen fluid at night, and milk fat levels. The differential metabolite Vitamin B6, significantly elevated at night, promoted the translocation of acetic acid into the circulation by increasing the level of rumen epithelial MCT1 protein expression. In addition, we found that both acetic acid treatment time and dose modulated the expression of lipid metabolism transcription factors (PPARγ, PPARα, and SREBP1c) and downstream genes (FASN, SCD1, ACCα, and CPT1A). Additionally, the mTORC1 and AMPK pathways were responsible for the effects of acetic acid on transcription factors and genes involved in lipid metabolism. Differences in rumen microbial taxa were observed between the day and night. Microbial metabolite (acetic acid) was found to be absorbed into the bloodstream and entered the mammary gland at night at a significantly elevated level. This regulation impacted the expression of lipid metabolism-related transcription factors (PPARγ, PPARα, and SREBP1c), as well as downstream genes through the mTORC1 and AMPK signaling pathways, ultimately affecting milk fat synthesis. These findings provide a new perspective for the microbial regulation of milk synthesis.

The power of trans-sodium crocetinate: exploring its renoprotective effects in a rat model of colistin-induced nephrotoxicity.

Colistin, a multidrug-resistant gram-negative bacterial infection medication, has been associated with renal impairment and failure. Trans-sodium crocetinate (TSC), a saffron-derived chemical recognized for its antioxidant and nephroprotective properties, was studied in this study to determine its potential to alleviate the nephrotoxic effects of colistin. Forty-two male Wistar rats were randomly classified into seven groups (n = 6): (1) control (normal saline, 12 days, i.p.), (2) colistin (22 mg/kg, 7 days, i.p.), (3-5) colistin + TSC (25, 50, and 100 mg/kg, 12 days, i.p., starting from 5 days before colistin), (6) TSC (100 mg/kg, 12 days, i.p.), (7) colistin + vitamin E (100 IU/kg, 12 days, i.p). On day 13, the rats were euthanized and the serum content of creatinine, BUN, Na+, and K+, as well as oxidative stress (GSH, MDA, SOD, CAT), inflammatory (IL-1β), apoptotic (Bax, Bcl-2, caspase-3, 8, 9), and autophagy (Beclin-1, LC3) markers, NGAL, and histopathological changes in the kidney were measured. Colistin significantly increased serum creatinine, BUN, MDA, IL-1β, caspase-3,8,9, Bax, Beclin-1, LC3, and NGAL levels in kidney tissue. It also caused inflammation, focal necrosis of tubular epithelial cells, protein cast, and acute tubular necrosis. Furthermore, colistin decreased SOD, CAT, GSH, and Bcl-2 levels. TSC and vitamin E administration along with colistin restored most of the alterations induced by colistin. Overall, it could be concluded that colistin induces oxidative stress, inflammation, autophagy, and apoptosis, which can cause kidney injury. However, TSC can also be used as a therapeutic agent to reduce injuries caused by colistin.

Potential of epithelial membrane protein 3 as a novel therapeutic target for human breast cancer.

Amplification of human epidermal growth factor 2 receptor (HER2) and overexpression of estrogen receptor (ER) and/or progesterone receptor (PR) are key determinants in the treatment planning for human breast cancer (BC). Currently, targeted therapies for BC are focused mainly on these biomarkers. However, development of resistance to targeted drugs is almost unavoidable, emphasizing the importance of biochemical and pharmaceutical advances to improve treatment outcomes. To the best of our knowledge, the present study is the first to show functional crosstalk in vitro between HER2 and epithelial membrane protein 3 (EMP3), a tetraspan membrane protein, in human BC. EMP3 overexpression significantly promoted BC cell proliferation, invasion and migration by Transwell assays via epithelial-mesenchymal transition and transactivated the HER family, resulting in increased ER and PR expression in vitro. Knocking down EMP3 notably suppressed cell proliferation and migration and was accompanied by decreased expression of HER1‑HER3 and p‑SRC proteins. Suppression of EMP3 expression enhanced sensitivity of BC cells to trastuzumab in vitro. Xenograft experiments revealed decreased expression of HER1 and HER2 in stable EMP3‑knockdown cells, resulting in decreased tumor weight and size. In patients with BC, EMP3 overexpression was detected in 72 of 166 cases (43.4%), with 18 of 43 (41.9%) HER2‑amplified BC samples co‑expressing EMP3. Co‑expression of EMP3 and HER2 was positively associated with ER expression (P=0.028) and tended to be associated with nodal metastasis (P=0.085), however this was not significant. Taken together, the present results supported the potential of targeting EMP3 as a novel therapeutic strategy for human BC via co‑expression of HER2 and EMP3.

Sustained immune activation and impaired epithelial barrier integrity in the ectocervix of women with chronic HIV infection.

Chronic systemic immune activation significantly influences human immunodeficiency virus (HIV) disease progression. Despite evidence of a pro-inflammatory environment in the genital tract of HIV-infected women, comprehensive investigations into cervical tissue from this region remain limited. Similarly, the consequences of chronic HIV infection on the integrity of the female genital epithelium are poorly understood, despite its importance in HIV transmission and replication. Ectocervical biopsies were obtained from HIV-seropositive (n = 14) and HIV-seronegative (n = 47) female Kenyan sex workers. RNA sequencing and bioimage analysis of epithelial junction proteins (E-cadherin, desmoglein-1, claudin-1, and zonula occludens-1) were conducted, along with CD4 staining. RNA sequencing revealed upregulation of immunoregulatory genes in HIV-seropositive women, primarily associated with heightened T cell activity and interferon signaling, which further correlated with plasma viral load. Transcription factor analysis confirmed the upregulation of pro-inflammatory transcription factors, such as RELA, NFKB1, and IKZF3, which facilitates HIV persistence in T cells. Conversely, genes and pathways associated with epithelial barrier function and structure were downregulated in the context of HIV. Digital bioimage analysis corroborated these findings, revealing significant disruption of various epithelial junction proteins in ectocervical tissues of the HIV-seropositive women. Thus, chronic HIV infection associated with ectocervical inflammation, characterized by induced T cell responses and interferon signaling, coupled with epithelial disruption. These alterations may influence HIV transmission and heighten susceptibility to other sexually transmitted infections. These findings prompt exploration of therapeutic interventions to address HIV-related complications and mitigate the risk of sexually transmitted infection transmission.

Impact of Circadian Clock PER2 Gene Overexpression on Rumen Epithelial Cell Dynamics and VFA Transport Protein Expression.

The circadian gene PER2 is recognized for its regulatory effects on cell proliferation and lipid metabolism across various non-ruminant cells. This study investigates the influence of PER2 gene overexpression on goat rumen epithelial cells using a constructed pcDNA3.1-PER2 plasmid, assessing its impact on circadian gene expression, cell proliferation, and mRNA levels of short-chain fatty acid (SCFA) transporters, alongside genes related to lipid metabolism, cell proliferation, and apoptosis. Rumen epithelial cells were obtained every four hours from healthy dairy goats (n = 3; aged 1.5 years; average weight 45.34 ± 4.28 kg), cultured for 48 h in vitro, and segregated into control (pcDNA3.1) and overexpressed (pcDNA3.1-PER2) groups, each with four biological replicates. The study examined the potential connection between circadian rhythms and nutrient assimilation in ruminant, including cell proliferation, apoptosis, cell cycle dynamics, and antioxidant activity and the expression of circadian-related genes, VFA transporter genes and regulatory factors. The introduction of the pcDNA3.1-PER2 plasmid drastically elevated PER2 expression levels by 3471.48-fold compared to controls (p < 0.01), confirming effective overexpression. PER2 overexpression resulted in a significant increase in apoptosis rates (p < 0.05) and a notable reduction in cell proliferation at 24 and 48 h post-transfection (p < 0.05), illustrating an inhibitory effect on rumen epithelial cell growth. PER2 elevation significantly boosted the expression of CCND1, WEE1, p21, and p16 (p < 0.05) while diminishing CDK4 expression (p < 0.05). While the general expression of intracellular inflammation genes remained stable, TNF-α expression notably increased. Antioxidant marker levels (SOD, MDA, GSH-Px, CAT, and T-AOC) exhibited no significant change, suggesting no oxidative damage due to PER2 overexpression. Furthermore, PER2 overexpression significantly downregulated AE2, NHE1, MCT1, and MCT4 mRNA expressions while upregulating PAT1 and VH+ ATPase. These results suggest that PER2 overexpression impairs cell proliferation, enhances apoptosis, and modulates VFA transporter-related factors in the rumen epithelium. This study implies that the PER2 gene may regulate VFA absorption through modulation of VFA transporters in rumen epithelial cells, necessitating further research into its specific regulatory mechanisms.

Down-regulation of ESRP2 inhibits breast cancer cell proliferation via inhibiting cyclinD1

Epithelial splicing regulatory protein 2 (ESRP2),an important alternative splicing protein of mRNA, is reported to have a dual role in tumors, which can promote or inhibit the occurrence and development of tumors. However, the function and mechanism of ESRP2 in breast cancer (BC) remain unclear. The distribution of ESRP2 expression in breast cancer and the correlation between ESRP2 expression and the overall survival rate were detected by The Cancer Genome Atlas (TCGA) database. Gene Ontology(GO)analysis, containing biological process, cellular components, and molecular function, was utilized to evaluate the potential mechanism of ESRP2 in breast cancer. The ESRP2 expression in breast cancer cell lines was detected by real-time quantitative PCR analysis (RT-qPCR) and western blotting. Cell clone was performed to examine the proliferation of ESRP2 knockdown in MCF-7 cells. The cell cycle was measured by flow cytometry assays. The role of ESRP2 knockdown in synergistic effect with chemotherapeutic agents was also determined by MTT assay. Bioinformatics analysis demonstrated that the ESRP2 gene was elevated in breast cancer cells and its overexpression was strongly correlated with shorter overall survival. GO analysis revealed that ESRP2 expression was related to cell proliferation. ESRP2 mRNA and protein expression were elevated in breast cancer cell lines, compared to the normal human breast cell line MCF-10 A. Dwon-regulation of ESRP2 inhibited cell proliferation and promoted the sensitivity of chemotherapy drug, Cisplatin(DDP) and Paclitaxel (TAXOL), in MCF-7 cells.Additionally, ESRP2 knockdown obstructed the cell cycle at the G1 phase and caused a decrease in cyclinD1 protein expression. These findings reveal that ESRP2 is highly expressed in breast cancer and is correlated with poor prognosis in breast cancer patients. ESRP2 knockdown can inhibit MCF-7 cell proliferation by arresting the cell cycle at the G1 phase and promoting the sensitivity of chemotherapy drugs (DDP and TAXOL)in MCF-7 cells. ESRP2 may be required for the regulation of breast cancer progression, as well as a critical target for the clinical treatment of breast cancer.

Longitudinal analysis of urinary I-FABP in extremely preterm infants that develop necrotizing enterocolitis.

Intestinal fatty acid binding protein (I-FABP) is an intestinal epithelial protein detectable in infants with necrotizing enterocolitis (NEC). The longitudinal behavior of I-FABP following NEC or its association with gastrointestinal or neurodevelopmental outcomes is unknown. In this secondary analysis of the Preterm Erythropoietin Neuroprotection Trial, we compared infants with and without NEC. Urine I-FABP concentrations in matched infants (n = 70) were measured serially using ELISA and compared using paired analysis. In infants with NEC, the associations of I-FABP levels with short-term outcomes and neurodevelopmental outcomes at 22-26 months corrected age were determined using non-parametric analysis. Infants with NEC were more likely to have cholestasis, death or severe neurodevelopmental impairment, cerebral palsy, and lower Bayley-III motor scores. Baseline urinary I-FABP levels were similar between groups. When compared to controls, infants with NEC had urinary I-FABP concentrations that were higher at diagnosis (median 11 vs 2.6 ng/ml, p = 0.006) and lower post-NEC (median 1 vs 5 ng/ml, p = 0.002). Diagnosis I-FABP levels were not associated with gastrointestinal or neurodevelopmental outcomes at 22-26 months corrected age. In extremely preterm infants, urinary I-FABP was elevated at NEC diagnosis and lower post-NEC compared to matched controls. I-FABP levels were not associated with adverse gastrointestinal or neurodevelopmental outcomes. Urinary intestinal fatty acid binding protein (I-FABP) levels are increased at diagnosis of NEC and fall to below baseline after NEC in extremely preterm infants. Urine I-FABP levels at NEC diagnosis are not associated with cholestasis, intestinal stricture or obstruction, need for additional intestinal surgery after NEC, or neurodevelopmental outcomes at 22-26 months corrected age. Urine I-FABP levels may be useful in the diagnosis of NEC. Diagnostic I-FABP levels do not predict short-term gastrointestinal or neurodevelopmental outcomes after NEC.

Peripheral immune signatures associated with the risk of hepatocarcinogenesis in cirrhotic Egyptian HCV patients before and after treatment with direct-acting antivirals

BackgroundAlthough direct-acting antivirals (DAAs) have revolutionized the management of chronic HCV, the debatable association with hepatocellular carcinoma (HCC) occurrence/recurrence has raised major concerns about their long-term use, especially in cirrhotic cases. The role of epithelial tight junction proteins (TJPs) in hepatocarcinogenesis has been highlighted; however, the association of their expression in peripheral blood mononuclear cells (PBMCs) with HCC has rarely been reported. This study aimed to explore the role of peripheral claudin (Cldn)1 in liver pathogenesis and its crosstalk with soluble immune mediators in HCC prognosis.MethodsThe study population included six independent subgroups: healthy controls, cirrhotic/non-cirrhotic treatment-naïve HCV patients, DAA-SVR patients, and anticancer treatment-naïve de novo HCC patients. The laboratory tests included serum levels of alpha-fetoprotein (AFP), albumin, liver transaminases, total bilirubin, and CBC profiling. The serum levels of soluble cluster of differentiation (sCD)163, IL-10, and IL-12 were estimated by corresponding ELISA kits, whereas the levels of Cldn1 and transforming growth factor (TGF)-β in PBMCs were quantified using quantitative PCR (qPCR).ResultsSerum sCD163, IL-10, and IL-12 levels were significantly higher in the HCC patient group than in the control and non-malignant patient groups (P < 0.0001). No significant difference was detected in the serum levels of the three markers between cirrhotic and non-cirrhotic patients of chronic HCV, whereas their levels were significantly different between cirrhotic and non-cirrhotic SVRs (P < 0.0001). Similarly, the transcriptional levels of peripheral Cldn1 and TGF-β were significantly higher in patients with HCC and non-malignant cirrhosis than in patients without cirrhosis (P = 0.0185–<0.0001 and 0.0089–<0.0001, respectively). Logistic regression analysis revealed a significant association between all the abovementioned markers and HCC (P = 0.0303 to < 0.0001), which was further confirmed by the results of receiver operating characteristic (ROC) analysis, which revealed an area under the curve (AUC) value ranging from 0.883 to 0.996. The calculated cutoff values demonstrated remarkable prognostic capacity, with ranges of 88–99.41% and 82.14–97.92% and positive/negative predictive values ranging from 84.62 to 98.3% and 92–98%, respectively.ConclusionSerum sCD163, IL-10, IL-12 and peripheral Cldn1 and TGF-β expression levels represent novel non-invasive HCC biomarkers that maintain their predictive power under different pathological conditions and circumvent the drawbacks of conventional prognostic markers in patients with mild cirrhosis and/or normal AFP, albumin, and/or platelet counts.

Exercise rescues cognitive impairment through inhibiting the fibrinogen neuroinflammative pathway in diabetes.

Fibrinogen is a pivotal factor in the activation of neuroinflammation and cognitive impairment. While exercise, especifically swimming, has demonstrated cognitive benefits, the molecular protective mechanisms orchestrated by exercise in response to blood-brain barrier (BBB) leakage in diabetes remain elusive. This study systematically investigates the impact of fibrinogen on neuroinflammation and the role of exercise in diabetic rats. Diabetic rats underwent an 8-week swimming exercise regimen, and subsequent assessments included changes in interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), astroglia activation, BBB permeability, and key epithelial tight junction proteins such as zona occludins (ZO)-1, Claudin-5, and matrix metalloproteinase-9 (MMP-9). Spatial learning and memory were evaluated using the Morris water maze test and the novel object recognition test. The study revealed that exercise significantly improved cognitive function, potentially by suppressing fibrinogen levels and astroglia activation. Intriguingly, heightened fibrinogen expression markedly attenuated the protective effects of exercise on BBB integrity. Fibrinogen emerged as a potential compromise to exercise protective effect by increasing expression levels of inflammatory factors IL-1β and TNF-α. In summary, our findings elucidate that fibrinogen may contribute to the deterioration of cognition and diminish the protective effects of exercise by amplifying the neuroinflammatory process through damaged BBB in diabetes.