Search Results (2,189)

Background: Preeclampsia (PE) is a pregnancy-specific disease and hypertensive disorder with a multifactorial pathogenesis involving complex molecular regulatory networks. Recent studies highlight the critical role of non-coding RNAs, particularly miRNAs and lncRNAs, in PE development. This study investigates the molecular interaction between miR-7151-5p and the lncRNA KCNQ1OT1 and their functional contributions to PE pathogenesis. Methods: An integrative approach combining RNAhybrid-based bioinformatics, dual-luciferase reporter assays, qRT-PCR, Transwell migration and invasion assays, and RNA sequencing was employed to characterize the binding between miR-7151-5p and KCNQ1OT1 and assess their influence on trophoblast cell function and gene expression. Results: A bioinformatic analysis predicted a stable binding site between miR-7151-5p and KCNQ1OT1 (minimum free energy: –37.3 kcal/mol). The dual-luciferase reporter assay demonstrated that miR-7151-5p directly targets KCNQ1OT1, leading to suppressed transcriptional activity. In HTR8/SVneo cells, miR-7151-5p overexpression significantly downregulated both KCNQ1OT1 and Notch1 mRNA, whereas its inhibition showed no significant changes, suggesting additional regulatory mechanisms of Notch1 expression. Transwell assays indicated that miR-7151-5p overexpression suppressed trophoblast cell migration and invasion, whereas its inhibition enhanced these cellular behaviors. RNA-seq analysis further revealed that miR-7151-5p overexpression altered key signaling pathways, notably the TGF-β pathway, and significantly modulates PE-associated genes, including PLAC1, ANGPTL6, HIRA, GLA, HSF1, and BAG6. Conclusions: The regulatory effect of miR-7151-5p on KCNQ1OT1, along with its influence on trophoblast cell dynamics via Notch1 and TGF-β signaling pathways, highlights its role in PE pathogenesis and supports its potential as a biomarker in early PE screening. Full article

Background and Objectives: A thorough comprehension of the essential molecules and related processes underlying the carcinogenesis, proliferation, and recurrence of acute myeloid leukemia (AML) is crucial. This study aimed to investigate the expression levels, diagnostic and prognostic significance and biological roles of Bcl-2-associated athanogene 4 (BAG4) in AML carcinogenesis. Materials and Methods: Gene expression profiles were analyzed using publicly available datasets, particularly GSE9476 and TCGA, using tools such as GEO2R, GEPIA2, UALCAN and TIMER2.0. The immune infiltration correlation was examined using the GSCA platform, while the function of BAG4 at the single-cell level was analyzed via CancerSEA. Protein–protein and gene–gene interaction networks were constructed using STRING and GeneMANIA, and enrichment analyses were performed using GO, KEGG and DAVID. Expression validation was performed using RT-qPCR in HL-60 (AML) and HaCaT (normal) cells, and ROC curve analysis evaluated the diagnostic accuracy. Results: BAG4 was significantly overexpressed in AML tissues and cell lines compared with healthy controls. High BAG4 expression was associated with poor overall survival and strong diagnostic power (AUC = 0.944). BAG4 was positively associated with immune cell infiltration and negatively associated with CD4+/CD8+ T and NK cells. At the single-cell level, BAG4 was associated with proliferation, invasion, and DNA repair functions. Functional network analysis showed that BAG4 interacted with apoptosis and necroptosis-related genes such as BCL2, BAG3 and TNFRSF1A and was enriched in pathways such as NF-κB, TNF signaling and apoptosis. Conclusions: BAG4 is overexpressed in AML and is associated with adverse clinical outcomes and immune modulation. It may play an important role in leukemogenesis by affecting apoptotic resistance and immune evasion. BAG4 has potential as a diagnostic biomarker and treatment target in AML, but further in vivo and clinical validation is needed. Full article

Chronic thromboembolic pulmonary hypertension (CTEPH) is a rare, progressive form of pre-capillary pulmonary hypertension characterized by persistent, organized thromboemboli in the pulmonary vasculature, leading to vascular remodeling, elevated pulmonary artery pressures, right heart failure, and significant morbidity and mortality if untreated. Despite advances, CTEPH remains underdiagnosed due to nonspecific symptoms and overlapping features with other forms of pulmonary hypertension. Basic Methodology: This review synthesizes data from large international registries, epidemiologic studies, translational research, and multicenter clinical trials. Key methodologies include analysis of registry data to assess incidence and risk factors, histopathological examination of lung specimens, and molecular studies investigating endothelial dysfunction and inflammatory pathways. Diagnostic modalities and treatment outcomes are evaluated through observational studies and randomized controlled trials. Recent Advances and Affected Population: Research has elucidated that CTEPH arises from incomplete resolution of pulmonary emboli, with subsequent fibrotic transformation mediated by dysregulated TGF-β/TGFBI signaling, endothelial dysfunction, and chronic inflammation. Affected populations are typically older adults, often with prior venous thromboembolism, splenectomy, or prothrombotic conditions, though up to 25% have no history of acute PE. The disease burden is substantial, with delayed diagnosis contributing to worse outcomes and higher societal costs. Microvascular arteriopathy and PAH-like lesions in non-occluded vessels further complicate the clinical picture. Conclusions: CTEPH is now recognized as a treatable disease, with multimodal therapies—surgical endarterectomy, balloon pulmonary angioplasty, and targeted pharmacotherapy—significantly improving survival and quality of life. Ongoing research into molecular mechanisms and biomarker-driven diagnostics promises earlier identification and more personalized management. Multidisciplinary care and continued translational investigation are essential to further reduce mortality and optimize outcomes for this complex patient population. Full article

Parkinson’s disease (PD) is a neurodegenerative disorder marked by the progressive loss of dopaminergic neurons in the nigrostriatal pathway. The dopamine active transporter (DAT), a key protein involved in dopamine reuptake, serves as a selective biomarker for dopaminergic terminals in the striatum. DAT binding has been extensively studied using in vivo imaging techniques such as Single-Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET). To support the design of new radiotracers targeting DAT, we employ Quantitative Structure–Activity Relationship (QSAR) analysis on a structurally diverse dataset composed of 57 compounds with known affinity constants for DAT. The best-performing QSAR model includes four molecular descriptors and demonstrates robust statistical performance: R² = 0.7554, Q²_LOO = 0.6800, and external R² = 0.7090. These values indicate strong predictive capability and model stability. The predicted compounds are evaluated using a docking methodology to check the correct coupling and interactions with the DAT. The proposed approach—combining QSAR modeling and docking—offers a valuable strategy for screening and optimizing potential PET/SPECT radiotracers, ultimately aiding in the neuroimaging and early diagnosis of Parkinson’s disease. Full article

Background/Objectives: In gastric cancer, only a subset of patients benefit clinically from neoadjuvant chemoimmunotherapy, underscoring the need for robust biomarkers that can predict treatment responses and guide personalized immunotherapy. This study aimed to characterize the immune microenvironment of gastric tumors and identify predictive markers associated with therapeutic efficacy. Methods: We prospectively enrolled 16 patients with histologically confirmed, PD-L1–positive (CPS ≥ 1) gastric adenocarcinoma (T_2–4N_0–1M₀). All patients received eight cycles of FLOT chemotherapy combined with pembrolizumab. Treatment response was assessed by Mandard tumor regression grading. Spatial transcriptomic profiling (10x Genomics Visium) and multiplex immunofluorescence were used to evaluate tumor-infiltrating immune cell subsets and PD-1 expression at baseline and after treatment. Results: Transcriptomic analysis differentiated the immune landscapes of responders from non-responders. Responders exhibited elevated expression of IL1B, CXCL5, HMGB1, and IFNGR2, indicative of an inflamed tumor microenvironment and type I/II interferon signaling. In contrast, non-responders demonstrated upregulation of immunosuppressive genes such as LGALS3, IDO1, and CD55, along with enrichment in oxidative phosphorylation and antigen presentation pathways. Multiplex immunofluorescence confirmed a higher density of FoxP3⁺ regulatory T cells in non-responders (median 5.36% vs. 2.41%; p = 0.0032). Notably, PD-1⁺ CD8⁺ T cell and PD-1⁺ FoxP3⁺ Treg frequencies were significantly elevated in non-responders, suggesting that PD-1 expression within cytotoxic and regulatory compartments may contribute to immune evasion. No substantial differences were observed in PD-L1 CPS or PD-1⁺ B cells and PD-1⁺ macrophages. Conclusions: Our findings identify PD-1⁺ CD8⁺ T cells and PD-1⁺ FoxP3⁺ Tregs as potential biomarkers of resistance to neoadjuvant chemoimmunotherapy in gastric cancer. Transcriptional programs centered on IL1B/CXCL5 and LGALS3/IDO1 define distinct immune phenotypes that may guide future combination strategies targeting both effector and suppressive arms of the tumor immune response. Full article

Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, comprises embryonal (ERMS) and alveolar (ARMS) subtypes with distinct histopathological features, clinical outcomes, and therapeutic responses. To better characterize their molecular distinctions, we performed untargeted plasma proteomics and metabolomics profiling in children with ERMS (n = 18), ARMS (n = 17), and matched healthy controls (n = 18). Differential expression, functional enrichment (GO, KEGG, RaMP-DB), co-expression network analysis (WGCNA/WMCNA), and multi-omics integration (DIABLO, MOFA) revealed distinct molecular signatures for each subtype. ARMS displayed elevated oncogenic and stemness-associated proteins (e.g., cyclin E1, FAP, myotrophin) and metabolites involved in lipid transport, fatty acid metabolism, and polyamine biosynthesis. In contrast, ERMS was enriched in immune-related and myogenic proteins (e.g., myosin-9, SAA2, S100A11) and metabolites linked to glutamate/glycine metabolism and redox homeostasis. Pathway analyses highlighted subtype-specific activation of PI3K-Akt and Hippo signaling in ARMS and immune and coagulation pathways in ERMS. Additionally, the proteomics and metabolomics datasets showed association with clinical parameters, including disease stage, lymph node involvement, and age, demonstrating clear molecular discrimination consistent with clinical observation. Co-expression networks and integrative analyses further reinforced these distinctions, uncovering coordinated protein–metabolite modules. Our findings reveal novel, subtype-specific molecular programs in RMS and propose candidate biomarkers and pathways that may guide precision diagnostics and therapeutic targeting in pediatric sarcomas. Full article

Background/Objectives: Chronic manganese (Mn) exposure is a recognized environmental contributor to Parkinsonian syndromes, including Mn-induced Parkinsonism (MnIP). This study aimed to evaluate whole-blood Mn levels and investigate disease/exposure-status-related alterations in metabolomic and lipidomic profiles. Methods: A case–control study (N = 97) was conducted in Brescia, Italy, stratifying participants by Parkinsonism diagnosis and residential Mn exposure. Whole-blood Mn was quantified using ICP-MS. Untargeted metabolomic and lipidomic profiling was conducted using LC-MS. Statistical analyses included Mann–Whitney U tests, conditional logistic regression, ANCOVA, and pathway analysis. Results: Whole-blood Mn levels were significantly elevated in Parkinsonism cases vs. controls (median: 1.55 µg/dL [IQR: 0.75] vs. 1.02 µg/dL [IQR: 0.37]; p = 0.001), with Mn associated with increased odds of Parkinsonism (OR = 2.42, 95% CI: 1.13–5.17; p = 0.022). The disease effect metabolites included 3-sulfoxy-L-tyrosine (β = 1.12), formiminoglutamic acid (β = 0.99), and glyoxylic acid (β = 0.83); all FDR p < 0.001. The exposure effect was associated with elevated glycocholic acid (β = 0.51; FDR p = 0.006) and disrupted butanoate (Impact = 0.03; p = 0.004) and glutamate metabolism (p = 0.03). Additionally, SLC-mediated transmembrane transport was enriched (p = 0.003). The interaction effect identified palmitelaidic acid (β = 0.30; FDR p < 0.001), vitamin B6 metabolism (Impact = 0.08; p = 0.03), and glucose homeostasis pathways. In lipidomics, triacylglycerols and phosphatidylethanolamines were associated with the disease effect (e.g., TG(16:0_10:0_18:1), β = 0.79; FDR p < 0.01). Ferroptosis and endocannabinoid signaling were enriched in both disease and interaction effects, while sphingolipid metabolism was specific to the interaction effect. Conclusions: Mn exposure and Parkinsonism are associated with distinct metabolic and lipidomic perturbations. These findings support the utility of omics in identifying environmentally linked Parkinsonism biomarkers and mechanisms. Full article

Urinary extracellular vesicles (U-EVs) are gaining increasing interest as non-invasive liquid biopsy tools for clinical use. Prostate cancer (PCa) is amongst the highest cancer-related cause of death in men, and therefore, the identification of non-invasive robust biomarkers is of high importance. This study assessed U-EV profiles from individuals affected by PCa at Gleason scores 6–9, compared with healthy controls. U-EVs were characterised and assessed for proteomic cargo content by LC-MS/MS analysis. The U-EV proteomes were compared for enrichment of gene ontology (GO), KEGG, and Reactome pathways, as well as disease–gene associations. U-EVs ranged in size from 50 to 350 nm, with the majority falling within the 100–200 nm size range for all groups. U-EV protein cargoes from the PCa groups differed significantly from healthy controls, with 16 protein hits unique to the GS 6–7 and 88 hits to the GS 8–9 U-EVs. Pathway analysis showed increased enrichment in the PCa U-EVs of biological process GO (5 and 37 unique to GS 6–7 and GS 8–9, respectively), molecular function GO (3 and 6 unique to GS 6–7 and GS 8–9, respectively), and cellular component GO (10 and 22 unique to GS 6–7 and GS 8–9, respectively) pathways. A similar increase was seen for KEGG pathways (11 unique to GS 8–9) and Reactome pathways (102 unique to GS 8–9). Enrichment of disease–gene associations was also increased in the PCa U-EVs, with highest differences for the GS 8–9 U-EVs (26 unique terms). The pathway enrichment in the PCa U-EVs was related to several key inflammatory, cell differentiation, cell adhesion, oestrogen signalling, and infection pathways. Unique GO and KEGG pathways enriched for the GS 8–9 U-EVs were associated with cell–cell communication, immune and stress responses, apoptosis, peptidase activity, antioxidant activity, platelet aggregation, mitosis, proteasome, mRNA stability oxytocin signalling, cardiomyopathy, and several neurodegenerative diseases. Our findings highlight U-EVs as biomarkers to inform disease pathways in prostate cancer patients and offer a non-invasive biomarker tool for clinical use. Full article

Radon (Rn) exposure has a strong association with lung cancer risk and is influenced by epigenetic modifications. To investigate the characterization of DNA methylation (DNAm) episignatures for radon-induced lung cancer, we detected the specific changes in DNAm in blood and lung tissues using reduced representation bisulfite sequencing (RRBS). We identified the differentially methylated regions (DMRs) induced by radon exposure. The bioinformatics analysis of the DMR-mapped genes revealed that pathways in cancer were affected by radon exposure. Among them, the DNAm episignatures of MAPK10, PLCG1, PLCβ3 and PIK3R2 were repeated between lung tissue and blood, and validated by the MassArray. In addition, radon exposure promoted lung cancer development in the genetic engineering mouse model (GEMM), accompanied by decreased MAPK10 and increased PLCG1, PLCβ3, and PIK3R2 with mRNA and protein levels. Conclusively, radon exposure significantly changes the genomic DNAm patterns in lung tissue and blood. The DNAm episignatures of MAPK10, PLCG1, PLCβ3 and PIK3R2 have a significant influence on radon-induced lung cancer. This brings a new perspective to understanding the pathways involved in radon-induced lung cancer and offers potential targets for developing blood-based biomarkers and epigenetic therapeutics. Full article

Background/Objectives: The role of tertiary lymphoid structures (TLSs) in cancer prognosis is well established, yet their significance in early-stage EGFR-mutant lung adenocarcinoma remains unclear. While outcomes for early-stage lung cancer are generally better than those of late-stage disease, recurrence remains a significant challenge. This study investigates the prognostic value of TLSs and their molecular characteristics in early-stage EGFR-mutant lung adenocarcinoma. Methods: TLSs were identified in tumor samples using multiplex immunohistochemistry (IHC), and their density was quantified. The PD-L1 tumor proportion score (TPS) and TLS density were analyzed for associations with disease-free survival (DFS). Gene expression profiling was performed to compare tumor microenvironment signatures between high- and low-TLS-density groups. Results: High TLS density correlated with significantly longer DFS (43 vs. 20.5 months, p = 0.0082). No relationship was found between TLS density and PD-L1 TPS or EGFR mutation subtype. Transcriptomic analysis revealed upregulated immune response genes in the high-TLS-density group, including those involved in T and B cell activation. Low-TLS-density tumors exhibited gene signatures promoting tumor growth, such as cell cycle and WNT pathway activation. Conclusions: In summary, TLS density is a potential prognostic biomarker for DFS in early-stage EGFR-mutant lung adenocarcinoma, independent of PD-L1 TPS or EGFR mutation subtype. Enhanced immune activation in high-TLS-density tumors highlights TLSs as a potential target for improving outcomes in these patients. Full article

Obesity and related metabolic disorders are closely linked to dysregulated lipid metabolism, where the metabolic balance of diacylglycerol (DAG) played a pivotal role. Although cis-palmitoleic acid (cPOA) exhibits anti-obesity effects, its efficacy varies across dietary conditions, and its molecular mechanisms remains unclear. In this study, we investigated the dose-dependent regulatory effects of cPOA on DAG metabolic shunting in db/db mice, employing lipidomics, pathway analysis, and gene/protein expression assays. Under a basal diet, low-dose cPOA (75 mg/kg) inhibited DAG-to-triglyceride (TAG) conversion, reducing hepatic lipid accumulation, while medium-to-high doses (150–300 mg/kg) redirected DAG flux toward phospholipid metabolism pathways (e.g., phosphatidylcholine [PC] and phosphatidylethanolamine [PE]), significantly lowering body weight and adiposity index. In high-fat diet (HFD)-fed mice, cPOA failed to reduce body weight but alleviated HFD-induced hepatic pathological damage by suppressing DAG-to-TAG conversion and remodeling phospholipid metabolism (e.g., inhibiting PE-to-PC conversion). Genetic and protein analyses revealed that cPOA downregulated lipogenic genes (SREBP-1c, SCD-1, FAS) and upregulated fatty acid β-oxidation enzymes (CPT1A, ACOX1), while dose-dependently modulating DGAT1, CHPT1, and PEMT expression to drive DAG metabolic shunting. Notably, DAG(36:3, 18:1–18:2) emerged as a potential biomarker for HFD-aggravated metabolic dysregulation. This study elucidated cPOA as a bidirectional regulator of lipid synthesis and oxidation, improving lipid homeostasis through dose-dependent DAG metabolic reprogramming. These findings provide novel insights and strategies for precision intervention in obesity and related metabolic diseases. Full article

Background: Necroptosis, a regulated form of inflammatory cell death, is increasingly recognized as a key driver of sepsis and critical illness. The balance between necroptosis and apoptosis may influence immune responses and outcomes in ICU patients. Objective: To evaluate necroptosis- and apoptosis-related protein expression in critically ill pediatric and adult patients with sepsis/septic shock, trauma/SIRS, or cardiac conditions, and assess their association with clinical outcomes. Methods: In this prospective, observational study, 88 patients admitted to a tertiary ICU were categorized into four groups: sepsis/septic shock, trauma/SIRS, cardiac disease, and healthy controls. Serum levels of RIPK1, RIPK3, MLKL, A20, caspase-8, IL-1β, and IL-18 were measured within 24 h of admission using ELISA. Biomarkers were analyzed by disease group, age, and severity indices. Results: Patients with sepsis—both adults and children—exhibited significantly elevated levels of RIPK1, IL-1β, and IL-18 (p < 0.001) and reduced levels of caspase-8 (p = 0.015), indicating activation of the necroptosis pathway. A20 was significantly upregulated (p < 0.001) and independently associated with lactate levels. RIPK1, IL-1β, and IL-18 were positively correlated with ICU length of stay and illness severity, whereas caspase-8 showed an inverse correlation. ROC analysis demonstrated strong predictive performance for sepsis/septic shock using RIPK1 (AUC = 0.81), IL-18 (AUC = 0.71), and A20 (AUC = 0.71); conversely, caspase-8 was inversely associated with sepsis (AUC = 0.32). Conclusions: Necroptosis appears to play a central role in the pathophysiology of sepsis across age groups. Elevated levels of RIPK1, IL-1β, IL-18, and A20 may serve as biomarkers of disease severity, while reduced caspase-8 supports a shift away from apoptosis toward necroptotic cell death. These findings highlight the potential of necroptosis-related pathways as targets for risk stratification and therapeutic intervention in critically ill patients of all ages. Full article

Conventional methods for isolating extracellular vesicles (EVs) are often limited by long processing times, a low purity, and a reliance on specialized equipment. To overcome these challenges, we developed the DeSEI (amine-functionalized Diatomaceous earth-based Syringe platform for EV Isolation), a novel platform employing low-cost, amine-functionalized diatomaceous earth (ADe) within a simple syringe–filter system. The capture mechanism leverages the electrostatic interaction between the positively charged ADe and the negatively charged EV surface, enabling a rapid and efficient isolation. The optimized 30 min protocol yields intact EVs with morphology, size, and protein markers comparable to those from ultracentrifugation, ensuring minimal cellular contamination. Notably, DeSEI exhibited a nearly 60-fold higher recovery efficiency of EV-derived miRNA compared to ultracentrifugation. The platform further proved its versatility with a rapid one-step miRNA extraction protocol and a user-friendly cartridge format. The direct miRNA extraction capability is particularly advantageous for a streamlined biomarker analysis, while the cartridge design illustrates a clear pathway toward developing point-of-care diagnostic tools. The DeSEI offers a promising alternative to existing methods for EV-based research by providing a combination of speed, simplicity, and procedural flexibility that does not require specialized equipment. Full article

Background/Objectives: Gastric cancer (GC) remains a significant global health burden due to its high mortality rate and frequent diagnosis at advanced stages. This study aimed to identify reliable diagnostic biomarkers and elucidate molecular mechanisms underlying GC by integrating transcriptomic data from independent platforms and applying machine learning techniques. Methods: Two transcriptomic datasets from the Gene Expression Omnibus were analyzed: GSE26899 (microarray, n = 108) as the discovery dataset and GSE248612 (RNA-seq, n = 12) for validation. Differential expression analysis was conducted using limma and DESeq2, selecting genes with |log2FC| > 1 and adjusted p < 0.05. The top 200 differentially expressed genes (DEGs) were used to develop machine learning models (random forest, logistic regression, neural networks). Functional enrichment analyses (GO, KEGG, Hallmark) were applied to explore relevant biological pathways. Results: In GSE26899, 627 DEGs were identified (201 upregulated, 426 downregulated), with key genes including CST1, KIAA1199, TIMP1, MSLN, and ATP4A. The random forest model demonstrated excellent classification performance (AUC = 0.952). GSE248612 validation yielded 738 DEGs. Cross-platform comparison confirmed 55.6% concordance among core genes, highlighting CST1, TIMP1, KRT17, ATP4A, CHIA, KRT16, and CRABP2. Enrichment analyses revealed involvement in ECM–receptor interaction, PI3K-Akt signaling, EMT, and cell cycle. Conclusions: This integrative transcriptomic and machine learning framework effectively identified high-confidence biomarkers for GC. Notably, CST1, TIMP1, KRT16, and ATP4A emerged as consistent, biologically relevant candidates with strong diagnostic performance and potential clinical utility. These findings may aid early detection strategies and guide future therapeutic developments in gastric cancer. Full article

Background: Constipation is a common gastrointestinal disorder with a significant impact on quality of life. Methods: Constipation was induced in male ICR mice via 25% cotrimoxazole gavage (20 mL/kg/day for 7 days). Mice were divided into prevention (pre-MBSFL), treatment (MBSFL), and control groups. MBSFL was prepared by fermenting mung bean starch with Lactobacillus plantarum (1:3 w/v ratio, 37 °C for 48 h), and administered via daily oral gavage (250 mg/kg bw) for 14 days. Fecal parameters (water content and first black stool latency), gastrointestinal motility (gastric emptying and small intestinal propulsion), serum biomarkers (NO, VIP, SP, and 5-HT), and intestinal gene expression (5HTR₄, SERT, and MAO_A) were analyzed. Results: MBSFL intervention restored fecal water content by 38%, reduced first black stool latency from 6.2 h to 3.1 h, and improved small intestinal propulsion by 64%. Additionally, it downregulated serum NO (25%) and VIP (32%) while upregulating SP (49%) and 5-HT (78%) levels. Intestinal 5HTR4 and SERT expression increased by 78% and 71%, respectively, with MAOA suppression (25%). Microbial analysis revealed a 140% increase in Dubosiella and 49% in Lactobacillus abundance, alongside a 62% reduction in Mucispirillum. MBSFL contained polysaccharides (12.3% w/w) and organic acids, including hydroxy butyric acid (4.2 mg/mL). Conclusions: MBSFL alleviates constipation through dual mechanisms: modulating 5-HT pathway activity and restoring gut microbiota homeostasis. Full article