Search Results (327)

Dextran sodium sulfate (DSS) is widely used in vivo to induce epithelial injury; however, the epithelial-specific responses to DSS and mechanisms associated with barrier restoration remain incompletely understood. Here, we establish an in vitro DSS-induced epithelial damage model using differentiated Caco-2 cell monolayers. Exposure to 2.5%(w/v) DSS for 24 h resulted in significant upregulation of the pore-forming protein Claudin-2(CLDN2) (2.677-fold) and elevated oxidative marker protein levels of NADPH oxidase 1 (NOX1), accompanied by increased reactive oxygen species (ROS) production confirmed via dihydroethidium (DHE) staining. Increased epithelial permeability was observed by transepithelial electrical resistance (TEER) analysis, without significant cytotoxicity. Surprisingly, ELISA analysis revealed significant downregulation of Interleukin-6 (IL-6) (0.364 ± 0.034 pg/mL) following DSS exposure. Treatment with 1 ng/mL recombinant IL-6 (rIL-6) improved epithelial barrier function during continued DSS exposure, whereas a higher dose of 5 ng/mL did not produce the same restorative response. Furthermore, treatment of DSS-damaged Caco-2 monolayer with 10 mM butyrate significantly improved epithelial barrier function. Collectively, this study establishes a reproducible DSS-induced Caco-2 epithelial injury model and demonstrates concentration-dependent effects of rIL-6 and barrier-restorative effects of butyrate, providing an epithelial-specific platform for investigating intestinal barrier dysfunction relevant to IBD. Full article

Streptococcus suis (S. suis) is a zoonotic pathogen capable of causing meningitis and other diseases. However, the molecular mechanisms underlying its ability to cross the blood brain barrier (BBB) and invade the central nervous system remain incompletely understood. In this study, non-targeted metabolomics was used to investigate the effects of S. suis type 2 strain HA9801 infection on the metabolic profile of human brain microvascular endothelial cells (hBMECs), and to examine associations between key metabolites and BBB permeability. The results showed that HA9801 infection induced changes in the metabolic profile of hBMECs, with significant enrichment of the histidine metabolism pathway. Notably, several histamine-related metabolites were altered. Changes in HDC and HNMT gene expression, together with alterations in histamine-related metabolites detected by non-targeted metabolomics, were consistent with changes in histamine-related metabolism. These observed metabolic alterations were accompanied by significant accumulation of endogenous histamine, which was accompanied by reduced expression of tight junction protein genes (TJP1, OCLN, and CLDN5) and increased BBB permeability. Moreover, elevated histamine levels were associated with increased invasion and translocation of S. suis across the BBB. This study suggests that S. suis impairs BBB integrity in association with alterations in the host histidine–histamine metabolic axis, which were accompanied by increased histamine levels, reduced expression of tight junction protein genes, and enhanced bacterial invasion and translocation. These findings identify histamine-associated signaling as a potential pathway for further investigation in S. suis-associated BBB dysfunction. Full article

Background/Objectives: Advanced gastroesophageal adenocarcinoma (GEA) carries a poor prognosis, with median overall survival of 13–20 months despite standard chemotherapy. Since trastuzumab’s approval, biomarker-driven precision therapies have expanded rapidly. This review comprehensively summarises current and emerging targeted agents across the key molecular targets driving advanced GEA. Methods: A comprehensive literature review was conducted using PubMed, Google Scholar and Cochrane Library in accordance with PRISMA guidelines, searching English-language human studies published between February and July 2026, supplemented by updates during editing to reflect current standards. Results: HER2-directed therapy has progressed from trastuzumab through dual blockade, immunotherapy combinations, next-generation ADCs (notably trastuzumab deruxtecan) and bispecific antibodies such as zanidatamab, which has now overtaken trastuzumab in the first-line setting. CLDN18.2-targeted zolbetuximab has demonstrated survival benefit in biomarker-selected patients, with newer ADCs, BiTEs and the first approved solid-tumour CAR-T therapy (satricabtagene autoleucel) extending this target further. VEGFR2 inhibition with ramucirumab remains a cornerstone in later lines, while novel VEGF/PD-1(L1) bispecifics are under investigation. FGFR2b-targeted bemarituzumab showed early promise that weakened on phase 3 confirmation, and MET/EGFR-directed agents, including savolitinib and amivantamab, require stringent biomarker selection to demonstrate benefit amid tumour heterogeneity. Conclusions: Novel targeted agents, particularly to HER2 and CLDN18.2, have demonstrated survival benefit despite ongoing challenges. Other lines are more investigational. Full article

Juvenile colorectal polyps (JP) are the most common polypoid lesions of the colon in children and the leading cause of lower gastrointestinal bleeding in pediatric patients. Although histologically classified as benign hamartomatous lesions, they arise in a context of allergic sensitization and IgE-mediated inflammation, suggesting that the epithelial compartment may play an active role in their pathogenesis. To date, most studies have focused on the immune cell infiltrate, leaving the molecular programs operating within the epithelium largely unexplored. Whole-transcriptome RNA sequencing (RNA-seq) was performed on epithelial cells isolated from pediatric JP (n = 8) and paired tissue circumjacent to polyp (TCP) (n = 7) obtained from children with a history of rectal bleeding and IgE sensitization to food allergens. Differential expression, functional enrichment (GO, KEGG, GSEA), and cross-dataset comparison with TCGA colorectal adenocarcinoma (CRC) was conducted. Candidate genes were validated by RT-qPCR in independent samples, including colorectal cancer (CRC) and inflammatory bowel disease (IBD) tissue. Differential expression analysis identified 3273 differentially expressed genes (2342 upregulated, 931 downregulated in JP vs. TCP), with 220 genes exceeding 32-fold change, including SERPINB3 (log2FC = 12.16), MMP1 (log2FC = 10.19), NMUR2 (log2FC = 10.08) and CHI3L1 (log2FC = 8.87). JP epithelium displayed a coherent type 2 inflammatory signature, encompassing upregulation of the alarmin IL33, eosinophil-attracting chemokines (CCL11, CCL24), IgE receptor subunits (FCER1A, FCER1G), and a coordinately activated leukotriene and prostaglandin biosynthetic program (ALOX5, ALOX5AP, PTGS2). Concurrent alterations in epithelial identity were observed, including downregulation of absorptive enterocyte and intestinal stem cell markers (CDX2, LGR5, ASCL2) alongside upregulation of secretory and regenerative programs, including the ectopic gastric-type mucin MUC5AC. Tight junction dysregulation—notably upregulation of the pore-forming claudin CLDN2 and loss of barrier-sealing claudins (CLDN3, CLDN4, CLDN23)—was consistent with impaired epithelial permeability. Pathway analyses confirmed activation of type 2 immune and extracellular matrix remodeling programs, with concurrent suppression of mitochondrial oxidative phosphorylation. Cross-dataset comparison with TCGA CRC data identified 381 co-upregulated and 87 co-downregulated genes shared between JP and CRC, including SERPINE1, CXCL8, ICAM1, and MMP3, several of which were associated with poorer disease-specific survival in CRC patients. RT-qPCR validation confirmed elevation of CHI3L1 and SERPINE1 in both JP and CRC, while SERPINB4 appeared JP-specific. Epithelial cells from pediatric juvenile colorectal polyps associated with allergic sensitization display a comprehensive type 2 inflammatory transcriptome alongside profound alterations in lineage identity, barrier integrity, and metabolic programming. Partial convergence with CRC-associated gene expression programs—in the absence of histological dysplasia—suggests that chronic allergic inflammation activates conserved epithelial remodeling pathways shared across mucosal tissues. CHI3L1, SERPINE1, and SERPINB4 are candidate biomarkers warranting validation in larger cohorts. Full article

Background/Objectives: Claudin 18.2 (CLDN18.2) is expressed in a substantial proportion of pancreatic ductal adenocarcinoma (PDAC) and has emerged as a promising therapeutic target. However, the positivity threshold for enrollment in CLDN18.2-directed trials remains undefined, and ongoing studies use different criteria. We quantified how different CLDN18.2 thresholds influence the size of the candidate population within the same PDAC cohort. Methods: In this retrospective single-center study, CLDN18.2 was assessed by immunohistochemistry (43-14A clone, Ventana BenchMark) in 99 PDAC patients (metastatic, n = 71; non-metastatic, n = 28). The candidate population was evaluated at three thresholds: two used in current CLDN18.2 trials (any-intensity [1+/2+/3+] staining in ≥40% and moderate-to-strong [2+/3+] staining in ≥75% of tumor cells) and an exploratory lower bound of any detectable expression (≥5%). Between-threshold reclassification was analyzed as paired data; clinicopathological and survival analyses were exploratory. Results: The candidate proportion was 40.4% (40/99) at ≥75%, 43.4% (43/99) at ≥40%, and 64.6% (64/99) at ≥5%; among metastatic patients, it was 36.6%, 38.0%, and 59.2%, respectively. Lowering the threshold from ≥75% to ≥40% caused virtually no reclassification (1/71 metastatic, 1.4%; McNemar p = 1.0), whereas lowering it to ≥5% reclassified 16 metastatic patients (22.5%; 95% CI, 14.4–33.5; p < 0.001) as candidates. Among the 64 tumors with detectable expression, median staining was 80% (IQR, 20–90%). Survival analyses were exploratory and underpowered (52 deaths; 80% power for HR ≥ 2.24); no association between CLDN18.2 and overall or progression-free survival was detected at any threshold. Conclusions: The stringent thresholds used in current drug development identified nearly identical candidate populations, whereas relaxation to any detectable expression substantially enlarged the pool; CLDN18.2 behaved as a targetable rather than a prognostic biomarker. Our findings highlight the need for a PDAC-specific, response-based CLDN18.2 threshold. Full article

Background/Objectives: Claudin 18.2 (CLDN18.2) has emerged as a clinically actionable therapeutic target in gastrointestinal malignancies; however, its expression profile and prognostic significance in cholangiocarcinoma (CCA) remain incompletely characterized. This study investigated CLDN18.2 expression in CCA and evaluated its associations with clinicopathological characteristics and overall survival (OS). Methods: This retrospective study included 124 patients with histologically confirmed CCA who were diagnosed between 2010 and 2025. CLDN18.2 expression was assessed by immunohistochemistry using two predefined positivity thresholds (≥10% and ≥75% of tumor cells showing moderate-to-strong membranous staining). Associations with clinicopathological variables were analyzed, and prognostic significance was evaluated using Kaplan–Meier survival analysis and Firth’s penalized Cox regression. Results: Using a 10% cutoff, CLDN18.2 positivity was detected in 10.5% (13/124) of cases; using a 75% cutoff, it was detected in 4.8% (6/124) of cases. At the 75% threshold, CLDN18.2 expression was significantly associated with anatomical location (p = 0.013) and histological subtype (p = 0.002), with the highest positivity observed in perihilar CCA (30.0%) and large-duct intrahepatic CCA (21.7%). The median OS was 9.5 months. CLDN18.2 positivity was not significantly associated with mortality in a univariable Firth’s penalized Cox regression at either the 10% cutoff (hazard ratio (HR) = 1.641, 95% confidence interval (CI): 0.883–2.816; p = 0.112) or the 75% cutoff (HR = 1.884, 95% CI: 0.762–3.877; p = 0.155). In multivariable Firth’s penalized Cox regression, CLDN18.2 positivity at the 10% cutoff was independently associated with an approximately two-fold increased risk of mortality (HR = 2.072, 95% CI: 1.090–3.666; p = 0.028), whereas no independent prognostic significance was observed at the 75% cutoff (HR = 1.906, 95% CI: 0.768–3.951; p = 0.150). The absence of adjuvant therapy was independently associated with increased mortality in both models (10% cutoff: HR = 4.870, 95% CI: 1.671–13.784; p = 0.004; 75% cutoff: HR = 4.296, 95% CI: 1.521–11.899; p = 0.006). Conclusions: CLDN18.2 expression in CCA varies according to anatomical location, histological subtype, and positivity threshold. The relatively high expression observed in perihilar CCA, and large-duct intrahepatic CCA, and the independent prognostic significance identified at the 10% cutoff support the potential role of CLDN18.2 as a prognostic biomarker and therapeutic target in selected patients with CCA. Given the limited sample sizes in some anatomical and histological subgroups, these findings should be interpreted cautiously and validated in larger prospective multicenter studies. Full article

Antiretroviral therapy (ART) is essential for controlling human immunodeficiency virus (HIV) infection, requiring strict daily adherence for lifelong viral suppression. However, this continuous oral dosing results in persistent exposure of the gastrointestinal tract (GIT), raising the need to investigate the effects of TC-ART (Tri-combinationL: Abacavir, Dolutegravir, Lamivudine–ART) on epithelial integrity, barrier recovery mechanisms, and surface barrier architecture. TC-ART exposure (125 µM to 4000 µM) showed marked alterations in transepithelial resistance, permeability, and wound-healing abilities even at sub-cytotoxic doses. The dose exposure range at the mid-dose level showed the highest transcriptional activity, characterized by a downregulation of junctional genes [claudins (CLDNs), desmogleins (DSGs), and junctional plakoglobin (JUP)] and signaling mediators [the signal transducer and activator of transcription 3 (STAT3), mitogen-activated protein kinase 1 and 3 (MAPK1/3), and catenin beta 1 (CTNNB1)], along with reduced IL-9 expression that is linked to mucin loss. These transcriptional changes were consistent with structural findings, including partial transepithelial electrical resistance (TEER) recovery followed by a decline, delayed wound closure, and waning of the apical mucin layer in a dose-dependent manner. However, several cytokines, like IL-2 and IL-6, showed increased secretion despite lower transcriptional levels, suggesting alternative regulatory control during early stress responses. Together, these results support that TC-ART exposure alters epithelial responses in a way that may transition from early adaptation to signs of impaired recovery, leading to a gradual decline in mucosal barrier function. Such concentration- and time-dependent epithelial stress may contribute to gastrointestinal disturbances observed in treated HIV populations, emphasizing the need for incorporating intestinal epithelial health endpoints in drug safety evaluations. Full article

Background: The early-life gut microbiota plays a critical role in programming intestinal barrier function and immune homeostasis, with profound implications for long-term host health. However, the effects of early-life compound probiotic intervention on the maturation of intestinal barrier function and the underlying molecular mechanisms remain incompletely understood, particularly in large-animal models relevant to human physiology. Methods: In this preclinical study, 3627 neonatal piglets—a well-established translational model for human infant gut development—were orally administered a novel compound probiotic formulation comprising Bifidobacterium longum subsp. infantis BZ, Lactobacillus plantarum LZ, and Pediococcus acidilactici PZ during early life. A total of 1512 fecal samples collected at seven time points from birth to day 180 were analyzed by 16S rRNA sequencing, and 525 samples from three developmental windows (days 10, 25, and 70) were subjected to LC-MS-based metabolomics. The candidate metabolite indole-3-lactic acid (ILA) was further mechanistically validated in a DSS-induced colitis mouse model and in IPEC-J2 cells. Results: Early-life probiotic intervention significantly enhanced intestinal barrier integrity, as evidenced by improved intestinal morphology and upregulated expression of tight junction proteins—zonula occludens-1 (ZO-1), occludin (OCLN), and Claudin-1 (CLDN1)—in the jejunum (p < 0.05). Notably, intervention at birth was more effective than post-weaning administration, and two administrations (birth + weaning) yielded superior outcomes compared with a single administration (p < 0.05). Microbiome analysis revealed enhanced microbial diversity and enrichment of beneficial genera during the juvenile-to-adult transition (p < 0.05). Metabolomic profiling identified ILA as a signature metabolite consistently elevated by probiotic supplementation. Conclusions: These findings provide evidence that early-life compound probiotic intervention is associated with improved intestinal barrier maturation, with ILA identified as a key candidate metabolite that may mediate this effect, as supported by functional validation in murine and cellular models. This provides a mechanistic rationale for probiotic-based strategies to support intestinal health in human infants during critical developmental windows. Full article

Gastrointestinal (GI) cancers represent a diverse group of malignancies that remain a major cause of cancer-related morbidity and mortality worldwide. Their management is increasingly complex, reflecting differences in tumor biology, anatomical location, stage, and molecular profile. In recent years, advances in molecular diagnostics, immunotherapy, and targeted treatment have moved clinical decision-making beyond a purely organ- and stage-based approach toward more individualized, biomarker-guided care. This narrative review summarizes established and emerging biological and targeted therapies used in esophageal, gastric and gastroesophageal junction, colorectal, pancreatic, hepatocellular, and biliary tract cancers. It focuses on immune checkpoint inhibitors targeting PD-1, PD-L1, and CTLA-4; HER2-directed monoclonal antibodies and antibody–drug conjugates; antiangiogenic and anti-EGFR therapies; and newer strategies involving CLDN18.2, FGFR2b, and tumor-agnostic alterations such as NTRK fusions. The review also considers the predictive biomarkers used to guide treatment selection and the growing integration of systemic therapy with surgery in neoadjuvant, perioperative, adjuvant, and conversion settings. However, clinical efficacy alone does not determine whether new treatments become part of routine practice. Regulatory approval, reimbursement, access to molecular testing, and the availability of specialized multidisciplinary care are equally important. The rapidly evolving treatment landscape for GI cancers therefore requires clinical decisions that account for tumor biology, anatomical resectability, molecular eligibility, expected benefit, treatment-related toxicity, and local access to therapy. Expanding access to comprehensive biomarker testing and effective molecularly guided treatments will be essential to translate progress in precision oncology into more personalized and equitable care for patients with GI cancers. Full article

Background/Objectives: Extracellular matrix remodeling, cellular stress responses, and blood–brain/blood–tumor barrier-related alterations are important processes involved in brain tumor biology. This exploratory cross-sectional pilot study evaluated circulating circulating protein disulfide isomerase A4 (PDIA4), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and claudin-2 (CLDN2) concentrations in vitamin D-deficient patients with different brain tumor entities and aimed to provide preliminary effect-size estimates for future studies. Methods: A total of 62 vitamin D-deficient participants were included, comprising patients with glioblastoma (GBM; n = 15), brain metastases (n = 20), and meningioma (n = 8) and surgical controls with degenerative spine disease (n = 19). Serum biomarker concentrations were measured using enzyme-linked immunosorbent assays. Global between-group effect sizes were estimated using η², while exploratory pairwise comparisons and associations with tumor size were assessed using non-parametric methods. Effect-size estimates were prioritized, with p-values reported to provide complementary inferential context. Results: The largest global between-group effect-sizes were observed for CLDN2 (η² = 0.35) and MMP-2 (η² = 0.22), whereas the estimated effects were small for MMP-9 (η² = 0.05) and negligible for PDIA4 (η² = 0.00). Exploratory pairwise comparisons indicated lower serum MMP-2 concentrations in patients with GBM than in surgical controls and lower CLDN2 concentrations in patients with GBM and brain metastases than in controls. Positive exploratory associations were observed between tumor size and PDIA4 concentration in the GBM group (Spearman’s rho = 0.56; 95% CI: 0.02–0.90) and between tumor size and MMP-2 concentration in patients with brain metastases (rho = 0.46; 95% CI: 0.01–0.90). Conclusions: These preliminary effect-size estimates, together with the exploratory correlations with tumor size, may inform biomarker selection and sample-size planning in future prospective studies. The findings should not be interpreted as robust diagnostic or prognostic evidence and require validation in larger, independent, and well-controlled cohorts. Full article

The chemical composition of donkey and sheep milk showed significant differences, with donkey milk containing less fat and protein and a higher level of lactose, while sheep milk had more dry matter and a higher proportion of casein and saturated, unsaturated, monounsaturated, and polyunsaturated fatty acids. This study investigated the biological effects of in vitro-digested donkey and sheep milk, and their blends at different ratios, on differentiated human intestinal Caco-2 cells. Gene expression analysis showed that treatment with digested donkey milk (100%) significantly altered the expression of the autophagy-related gene (SQSTM1), tight junction (CLDN4), and innate defense genes (DEFB1, MUC2, and MUC5), showing a non-cytotoxic response and a profile consistent with a barrier-related transcriptional response. In contrast, sheep milk (100%) and certain milk blends (70% and 50% sheep’s milk) down-regulated genes involved in the autophagy process (ULK1, AMBRA, BECN1, ATG5, GABARAP, and SQSTM1), tight junction genes (OCLN and CDH1), and innate defense genes (DEFB1 and MUC2), suggesting a distinct epithelial response that warrants further confirmation. Metabolomic profiling revealed clear compositional and functional distinctions among the digested milks, identifying 166 metabolites that were differentially regulated (136 compounds were down-regulated and 30 up-regulated). Donkey milk digestion yielded a metabolite profile enriched in polar peptides and compounds with reported antioxidant associations, whereas sheep milk digestion showed a different pattern dominated by hydrophobic peptides and lipid-related metabolites. These findings indicate that, under the tested in vitro conditions, donkey milk digestion is associated with gene expression and metabolomic patterns consistent with epithelial barrier support. Full article

Chronic kidney disease is associated with neurovascular complications including cognitive impairment, potentially involving blood–brain barrier (BBB) impairment. The protein-bound uremic toxin indoxyl sulfate (IS) promotes endothelial injury, but its effects on human brain microvascular endothelial cells remain incompletely understood. Human cerebral microvascular endothelial cells (hCMEC/D3) were exposed to IS 200 and 900 μM. BBB integrity was assessed by the FITC-dextran (4 kDa) transwell permeability assay and claudin-5 immunofluorescence. Transcriptional responses were quantified by qPCR for aryl hydrocarbon (AhR) target genes, oxidative stress-associated and inflammatory markers, senescence, and junction-associated genes. Senescence-associated phenotypic changes were evaluated by SA-β-galactosidase staining and cytokine array profiling. IS increased endothelial permeability at 24 and 48 h (~1.5-fold relative to control) without evidence of cytotoxicity and reduced claudin-5 staining intensity. IS strongly upregulated AhR target genes, including CYP1A1, CYP1B1, and CYP1A2. NFE2L2 and IDO1 increased, while NFKB1 remained unchanged. SA-β-gal positivity increased, accompanied by elevated GM-CSF and G-CSF secretion, while CDKN1A decreased at IS 900 µM and CDKN2A remained unchanged. CDH5 was downregulated, TJP1 increased at 900 µM, and CLDN5 remained unchanged. These findings indicate that IS exposure is associated with impaired BBB integrity, AhR-related transcriptional responses, oxidative stress-associated transcriptional changes, junctional remodeling, and senescence-like endothelial features. However, causal attribution to individual pathways requires inhibition or knockdown studies. Full article

This study aimed to characterize claudin 18.2 (CLDN18.2) expression in HER2-negative gastric or gastroesophageal junction cancer (GC/GEJC) and to evaluate whether CLDN18.2 status is associated with clinicopathological features and outcomes after first line chemoimmunotherapy. We retrospectively analyzed 189 patients with HER2-negative GC/GEJC treated at our institution from October 2019 to September 2024. CLDN18.2 expression was assessed by immunohistochemistry using two prespecified positivity thresholds: moderate to strong membranous staining (2+) in ≥40% or ≥75% of tumor cells. CLDN18.2 positivity was observed in 92/189 patients (48.7%) using the ≥40% threshold and 69/189 (36.5%) using the ≥75% threshold. PD L1 CPS ≥ 5 was less frequent in CLDN18.2 positive than in CLDN18.2 negative tumors at both thresholds (≥40%: 8.7% vs. 23.7%, p = 0.003; ≥75%: 8.7% vs. 20.8%, p = 0.019). Among 87 patients receiving first line chemoimmunotherapy, CLDN18.2 status was not associated with significant differences in objective response rate, progression free survival, or overall survival. CLDN18.2 positive tumors showed lower PD L1 expression, but CLDN18.2 status did not identify a subgroup with differential benefit from first line chemoimmunotherapy. These findings suggest that CLDN18.2 status does not appear to serve as a predictive biomarker for immune checkpoint inhibitor-based treatment. Full article

Magnesium is an essential mineral involved in numerous physiological and neurobiological processes. However, the biological effects of distinct Mg forms remain insufficiently characterized. This study compared the systemic and neurobiological effects of a multi-form Mg supplement (Magnesium Breakthrough™, Mg BT™, BIOptimizers, Reno, NV, USA) with commonly used single-form Mg compounds in a rat model. Sixty-day-old male Wistar rats were assigned to a control group or to groups receiving Mg BT™ or individual Mg compounds (oxide, citrate or glycinate). Treatments were administered by gastric gavage for 30 days at 50 mg/kg/day of elemental Mg. A comprehensive panel of endpoints was evaluated, including Mg distribution in biological fluids and tissues, metabolic markers, glucose tolerance, synaptic protein expression (synaptophysin, PSD95, phospho-PSD95, drebrin), cortical gene expression (NR2B, BDNF), behavioral outcomes, and liver and kidney histology. Magnesium from the multi-form supplement increased serum Mg without affecting glucose homeostasis and modulated proteins involved in synaptic plasticity, accompanied by mild anxiolytic-like effects without changes in locomotion. No adverse histological alterations were observed, while preserved renal CLDN-19 expression indicated maintained tubular integrity. These findings suggest that supplementation with the multi-form Mg BT™ supplement influences multiple evaluated biological domains, including systemic, behavioral and molecular parameters, with effects comparable to those observed with individual magnesium compounds under the experimental conditions applied. Full article

Brown mushroom stem (BMS), a nutrient-rich by-product of the mushroom processing industry, represents a potential sustainable feed ingredient for poultry. However, its effects on intestinal morphology and barrier-related gene expression in layer chicks are not well characterized. This study evaluated the influence of dietary BMS on ileal morphology and intestinal barrier-related gene expression in layer chicks. A total of 160 Lohmann LSL Lite chicks were randomly allocated to 4 dietary treatments: a control diet and control diets containing 2%, 4%, or 6% BMS as partial replacements for soybean meal on an equivalent basis, respectively. Each treatment had 5 replicates with 8 birds per replicate, and the trial lasted 36 d. Ileal samples were collected for histomorphological evaluation and quantitative real-time PCR of barrier-associated genes, including claudin-1 (CLDN1), occludin (OCLN), tight junction protein 1 (TJP1), tight junction protein 2 (TJP2), and mucin-2 (MUC2). Data were analyzed using one-way ANOVA, and orthogonal polynomial contrasts were applied to determine linear and quadratic responses to increasing BMS inclusion levels. Villus height decreased in BMS-fed groups compared with the control (linear and quadratic effects, p < 0.001), whereas crypt depth showed a quadratic response with the lowest value at 4% BMS (p < 0.001). Expression of CLDN1 mRNA was significantly elevated at 2% BMS but declined at higher inclusion levels (quadratic effect, p = 0.001). In contrast, TJP2 mRNA expression decreased with increasing BMS inclusion (linear effect, p = 0.009). No significant differences were observed for OCLN, TJP1, or MUC2 mRNA expression (p > 0.05). A composite index of barrier-related gene expression was numerically the highest at 2% BMS supplementation but did not differ significantly among treatments. These findings suggest that low dietary inclusion of BMS may support intestinal barrier-related gene expression in layer chicks, although higher levels may alter intestinal morphology without corresponding changes in barrier-related gene expression. Optimizing BMS inclusion could support sustainable poultry production by utilizing mushroom processing by-products as an alternative feed ingredient. Full article