Gastroesophageal reflux disease (GERD) is a digestive disease characterized by endoplasmic reticulum stress (ERS) imbalance and oxidative stress-induced cellular injury. However, the key ERS-related regulators involved in GERD progression remain unclear. Here, we identified PCK2 as a novel GERD-associated ERS regulator and explored its role and underlying mechanisms in GERD development. Bioinformatics analysis of the GEO dataset GSE148381 identified four overlapping ERS-related candidate genes in GERD tissues. Among these candidates, PCK2 was selected based on its consistent upregulation in ABS-treated HET-1 A cells, as confirmed by RT-qPCR and western blotting, with a 1.2-fold increase in mRNA expression and a 2.67-fold increase in protein abundance compared with control cells. Functional studies demonstrated that PCK2 silencing attenuated ERS activation by reducing ATF6, PERK, p-PERK, CHOP, and XBP1 expression. PCK2 knockdown markedly alleviated oxidative stress, reducing ROS and MDA levels by approximately 91% and 34%, respectively, decreasing apoptosis by approximately 36%, and improving cell viability, whereas PCK2 overexpression exerted opposite effects. In vivo, PCK2 knockdown alleviated esophageal injury and suppressed ERS responses. Mechanistically, Gene Set Enrichment Analysis revealed enrichment of PCK2-associated genes in the MAPK pathway, and further experiments showed that PCK2 promoted p38 MAPK activation through enhanced p38 phosphorylation. Pharmacological inhibition of p38 signaling with SB203580 abolished PCK2-induced ERS, oxidative stress, and apoptosis in HET-1 A cells. Collectively, our findings indicate that PCK2 acts as a potential therapeutic target in GERD by promoting p38 MAPK activation, thereby exacerbating ERS, oxidative stress, and apoptosis.
The IQ motif and SEC7 domain-containing protein 2 (IQSEC2) is a guanine nucleotide exchange factor (GEF) for ARF family small GTPases and is encoded by the IQSEC2 gene located on chromosome Xp11.22. More than 120 pathogenic variants in IQSEC2 have been identified in patients with neurodevelopmental disorders. In this study, we aimed to clarify the role of IQSEC2 during neonatal mouse dentate gyrus development using in vivo electroporation. Electroporation of a GFP expression vector together with a short hairpin RNA (shRNA) targeting IQSEC2 caused dentate granule neurons to stop at the boundary between the granule cell layer and the hilus (GCL/hilus). IQSEC2-deficient dentate granule cells exhibited increased distal dendritic complexity compared with control cells. IQSEC2 knockdown also reduced spine head diameter without affecting spine density. In contrast, exogenous IQSEC2 expression increased proximal dendritic complexity in neonatal dentate granule cells. We examined the ARF selectivity of IQSEC2 GEF activity and found that IQSEC2 activates ARF1, ARF3, and ARF6. Functional analyses revealed that ARF1 knockdown also confined dentate granule cells to the GCL/hilus region, which was consistent with the observation following IQSEC2 knockdown. In contrast, knockdown of ARF3 or ARF6 had no significant effect. Together, these results suggest that the IQSEC2 and ARF1 play a critical role in regulating neuronal positioning and dendritic development in the neonatal dentate gyrus.
Atomic Force Microscopy (AFM) is a promising tool to measure dissociation rates and binding distances in the single-molecule regime. However, interpretation of the obtained rupture data can be challenging. The unbinding process between two complex molecules is often characterized by a spectrum of barrier heights that cannot be parametrized by a single barrier height. In standard analysis, different barriers can be discerned only by measuring over at least 4-5 orders of magnitude of the pulling force rate, which can be difficult to achieve with AFM. Here, we used fits of the full rupture force histograms to address this complexity. We found that multiple unbinding paths can be discerned from measurements at just a few force rates using this approach. We also show how multiple rupture events - situations where multiple bonds breaking simultaneously appear as single rupture events - can be addressed in the analysis.
OBJECTIVE:To investigate the effect and mechanism of histone methylation on galactose-induced lens opacity. METHODS:Lenses from SD rats were cultured in 30 mM galactose medium to induce opacification, with intervention using four histone methyltransferase inhibitors (GSK126, Pinometostat, GSK3326595, BIX-01294). Lens opacity, structural changes (HE staining/transmission electron microscopy (TEM)), differentially expressed genes (high-throughput sequencing), and validated targets (qPCR) were analyzed. A diabetic cataract (DC) rat model was established via intraperitoneal galactose injection, with BIX-01294 intervention. Ocular lesions (fundus/slit-lamp examination), lens structure (HE staining), AR/TNF-α levels (ELISA), and Pikfyve mRNA expression (qPCR) were assessed. Ex vivo, YM201636 (a Pikfyve inhibitor) was used to intervene in galactose-induced opacity, with opacity observation and structural evaluation (HE staining/TEM). RESULTS:The four histone methyltransferase inhibitors ameliorated lens opacity to varying degrees. HE staining and TEM results showed that histone methyltransferase inhibitors alleviated lens injury, with BIX-01294 exhibiting the most pronounced effect. High-throughput sequencing of the model group and the BIX-01294 intervention group revealed that after BIX-01294 treatment, 113 genes were upregulated and 60 genes were downregulated. qPCR validation showed that mRNA expression levels of Pth1r, Fam111a, Pikfyve, and H19 were significantly downregulated after BIX-01294 intervention. Furthermore, BIX-01294 intervention reduced lens injury, decreased AR activity and TNF-α content, and lowered Pikfyve mRNA expression in DC rats. Intervention with YM201636 (a Pikfyve inhibitor) alleviated galactose-induced lens opacity and improved lens damage in vitro. CONCLUSION:Histone methyltransferases are involved in lens opacity in DC rats by regulating Pikfyve gene expression.
BACKGROUND:Osteosarcoma is a common malignant bone tumor in children and adolescents. Conventional chemotherapy has drug resistance and side effects, and new treatments are needed. Astragaloside IV (AS-IV) has anti-tumor effects, but its mechanism in osteosarcoma is unclear. METHODS:MG63/Saos2 osteosarcoma cells were divided into control, AS-IV (20/40 μM), Erastin (ferroptosis inducer), AS-IV + Lip-1 (ferroptosis inhibitor), AS-IV + short hairpin RNA negative control (shNC), and AS-IV + short hairpin RNA Transferrin Receptor 1 (shTFRC) (transfection of shTFRC lentiviral vector) groups. Cell counting kit-8, flow cytometry, immunofluorescence, kit detection, quantitative real-time polymerase chain reaction, and Western blot were used to detect cell viability, apoptosis, reactive oxygen species (ROS), iron metabolism, oxidative stress, and Janus Kinase 2/Signal Transducers and Activators of Transcription 3 (JAK2/STAT3) pathway-related indicators. RESULTS:AS-IV could concentration-dependently inhibit the viability of MG63 and Saos2 cells, promote cell apoptosis and ROS production, upregulate the levels of Fe2+, labile iron pool (LIP) and malondialdehyde (MDA), downregulate the expression of Ferritin Heavy Chain 1 (FTH1) and Glutathione Peroxidase 4 (GPX4) mRNA and the activities of superoxide dismutase (SOD) and catalase (CAT), and significantly inhibit the phosphorylation of JAK2 and STAT3. Lip-1 could reverse these effects. Moreover, after TFRC knockdown, the regulatory effects of AS-IV on the viability, apoptosis, iron metabolism, oxidative stress and JAK2/STAT3 pathway of osteosarcoma cells were significantly weakened. CONCLUSION:AS-IV is associated with ferroptosis induction and JAK2/STAT3 pathway inhibition in osteosarcoma cells, and these effects are attenuated by TFRC knockdown, providing new targets and theoretical basis for the treatment of osteosarcoma.
Neuroinflammation has emerged as a convergent pathological signature of Parkinson's disease (PD), shaping both its onset and progression. Persistent activation of glial cells, driven by elevated proinflammatory cytokines, creates a self-reinforcing inflammatory milieu that accelerates dopaminergic neurodegeneration. Microglia, astrocytes, and Toll-like receptor (TLR)-mediated pathways converge to amplify this response, linking innate immune activation to chronic neurotoxicity. At the transcriptional level, the JAK/STAT signaling cascade governs cytokine-driven inflammation by promoting the expression of proinflammatory mediators, thereby sustaining immune activation. In contrast, the TGF-β/SMAD signaling functions as a critical counter-regulatory mechanism, maintaining immune quiescence and neural homeostasis through canonical SMAD-dependent signaling. However, in PD, this anti-inflammatory pathway becomes impaired, diminishing its neuroprotective influence. Emerging evidence suggests that hyperactivated JAK/STAT signaling can suppress TGF-β/SMAD activity, establishing a pathological dominance of proinflammatory signaling over neuroprotective restraint. This review synthesizes current understanding of the mechanistic interplay between these critical pathways, proposing that the imbalance between JAK/STAT activation and TGF-β/SMAD suppression represents a key candidate regulatory interface governing the neuroinflammatory trajectory of PD. Unraveling this crosstalk provides new insight into how signaling hierarchies shape glial phenotypes and neurodegeneration, while highlighting potential therapeutic strategies aimed at restoring immune equilibrium to mitigate PD progression.
BACKGROUND:Heart failure (HF) remains a leading cause of death worldwide. Stem cells represent a promising strategy for myocardial regeneration due to their differentiation potential and paracrine effects. This study investigated the effect of human pericardial fluid Sca-1+/Nanog+ cells Conditioned Medium (hPFCs-CM) on doxorubicin (DOX)-induced damage to AC16 cardiomyocytes using transcriptomic analysis. METHODS:Human AC16 cardiomyocytes were divided into Control (CON), DOX, DOXh, and hPFCs groups. Cell viability, cytotoxicity (LDH release), reactive oxygen species (ROS) levels, and apoptosis were assessed. Transcriptomic sequencing was performed on CON, DOX, and DOXh groups. MYH3 and IFI16 genes were knocked down using siRNA, followed by Western blot analysis of apoptosis. RESULTS:DOX significantly increased ROS and apoptosis compared with CON group (P < 0.05), while hPFCs-CM attenuated these effects (P < 0.05). RNA-seq identified 52 differentially expressed genes between DOX and DOXh groups (adjP < 0.05). Three target genes (MYH3, IFI16, and KCTD14) were selected for RT-qPCR validation. Further functional assays revealed that MYH3 knockdown significantly potentiated the anti-apoptotic activity of hPFCs-CM (P < 0.05), whereas IFI16 knockdown showed no significant effect. CONCLUSION:hPFCs-CM effectively attenuated DOX-induced AC16 cardiomyocyte damage through MYH3, providing potential molecular targets for future mechanistic studies in myocardial injury treatment.
BACKGROUND:Chronic kidney disease (CKD) is accompanied by renal injury, gut-barrier disruption, dysbiosis, inflammation, and disordered iron regulation. We examined whether a clinical multi-strain probiotic produces coordinated renal, intestinal, and metabolic effects in experimental CKD. METHODS:Male Sprague-Dawley rats underwent 5/6 nephrectomy (5/6 Nx). Dose-response phenotyping compared sham, untreated 5/6 Nx, three Bifid Triple Viable Tablets (BTV) doses, and valsartan (n = 3/group). Independent cohorts provided targeted validation (untreated versus high-dose BTV; n = 6/group) and paired fecal 16S rRNA/serum metabolomics (n = 4/group). Technical replicates were averaged within animals. RESULTS:High-dose BTV was associated with lower blood urea nitrogen (BUN), serum creatinine (Scr), inflammatory cytokines, and hepcidin, plus qualitatively less renal and gastrointestinal injury. Occludin and zonula occludens-1 (ZO-1) integrated fluorescence did not differ from untreated 5/6 Nx rats (all adjusted P > 0.05; n = 3/group). Validation confirmed lower BUN (27.87 ± 2.09 vs 16.64 ± 3.82 mmol/L; P = 0.000264), Scr (235.75 ± 27.41 vs 132.05 ± 19.50 μmol/L; P = 0.0000343), and interleukin-6 (IL-6; 188.11 ± 8.67 vs 92.21 ± 14.51 pg/mL; P = 5.72 × 10-7) with high-dose BTV. Omics detected 1966 metabolites, 393 differential metabolites, and 160 nominal genus-metabolite associations. CONCLUSIONS:BTV was associated with reproducible renal biochemical and inflammatory improvement, qualitative gastrointestinal differences, and exploratory microbial-metabolic changes. Barrier-protein findings were nonsignificant, and small, separate cohorts preclude causal inference.
Metacaspases are Ca2+-dependent cysteine proteases essential for programmed cell death. This study elucidates the regulatory mechanisms of Saccharomyces cerevisiae type I metacaspase (ScMCA-IA), focusing on Ca2+-dependent autoprocessing and the function of its N-terminal hydrophobic prodomain. Through cloning, heterologous expression, and site-directed mutagenesis, the roles of Asp236, Asp252, Asp253, and Asp283 in ScMCA-IA autoprocessing were systematically investigated. A truncated variant (ScMCA-IA-ΔN86) was engineered to assess prodomain contribution. Results demonstrate that ΔN86 exhibits gradual autoprocessing and processing patterns consistent with intermolecular cleavage. The N-terminal region critically modulates Ca2+ affinity and processing kinetics. Mutagenesis revealed distinct aspartate contributions to maturation efficiency: D236A compromised truncated variant stability; D252A attenuated processing efficiency; D253A yielded predominantly unprocessed enzyme; while D283A profoundly impaired autoprocessing, establishing Asp283 as a critical residue for calcium-dependent autoprocessing and acquisition of the processed conformation. These findings provide mechanistic insights into metacaspase regulation, with Asp283 playing a key role in the Ca2+-dependent proteolytic maturation. However, direct catalytic activity was not measured; thus, the relationship between processing and enzymatic activation remains to be determined.
The development of targeted therapies has revolutionized cancer treatment by improving the specificity and efficacy of therapeutic options. Aptamers, short single-stranded nucleic acids, have emerged as a promising class of molecules for targeted therapy. In this manuscript, we explore the therapeutic application of the epithelial cell adhesion molecule (EpCAM) aptamer in cancer therapy. EpCAM is overexpressed in various types of cancer and has a vital role in tumor progression and metastasis. These aptamers selectively bind to the EpCAM glycoprotein on the surface of cancer cells, facilitating the targeted cellular internalization of therapeutic payloads and modulating tumor-associated signaling pathways. This review outlines recent advances in EpCAM aptamer-based strategies, focusing on their evaluation in drug-aptamer conjugates, diverse nanoparticle platforms (including lipid, polymeric, dendritic, silica, serum albumin, iron oxide, and carbon nanotube nanoparticles) for targeted delivery, clinical imaging, and cancer diagnosis. In addition, advanced therapeutic modalities such as DNA nanostructures, immunotherapy, and photothermal therapy employing EpCAM aptamers are critically discussed. Finally, we address the technical challenges, current limitations, and the clinical translation bottlenecks of this innovative platform to provide a comprehensive roadmap from bench to bedside.
Diabetic wounds are a serious healthcare concern, affecting approximately 25% of individuals with diabetes globally. Their healing process is often delayed by chronic hyperglycemia, oxidative stress, and persistent inflammation, all of which disrupt normal tissue repair. This review explores the molecular signaling pathways involved in diabetic wound healing and discusses emerging therapeutic strategies targeting these pathways. Key signaling routes such as Wnt/β-catenin, MAPK/ERK, NF-κB/NLRP3, Hippo-YAP, HIF-1α/VEGF-SDF-1α, AKT/eNOS, PI3K/Akt, Nrf2, TGF-β/Smad, and Notch are analyzed for their roles in cell growth, new blood vessel formation, inflammation reduction, and extracellular matrix remodeling. Diabetes disrupts these pathways through advanced glycation end-products, reactive oxygen species, and persistent inflammation, which impair keratinocyte migration, decrease angiogenesis, and prolong inflammation. Recently, promising therapeutic strategies have included nanoparticle-based pathway activation, exosome therapies, and bioactive compounds. Given the extensive interactions among these signaling pathways, therapeutic approaches should target multiple pathways rather than individual signaling components. Future therapeutic strategies may increasingly emphasize personalized medicine, leveraging AI-assisted diagnostics and customised combination therapies to improve management of chronic diabetic wounds.
To date, the S46 peptidase family is limited to two members, dipeptidyl-peptidase (DPP)7 and DPP11, which are widely distributed among Gram-negative bacteria and present in both oral and intestinal microbiota. Both peptidases are indispensable for the growth of the periodontopathic bacterium Porphyromonas gingivalis. They have a 40.0% amino acid identity, and exhibit specificity for hydrophobic and acidic P1 residues, respectively. This specificity is associated with S1 Gly666 in DPP7 and the equivalent residue, Arg673, in DPP11. Recent studies have revealed a relaxed P1 specificity of DPP7 even for neutral amino acids and hydrophilic Asn. Thus, the broad utility of DPP7 suggests that the ancestral enzyme of the S46 family resembled DPP7. The aim of the present study was to reconstruct the evolutionary divergence of these two DPPs. DPP7 hydrolyzed the tetrapeptidyl substrate LE-|-MP- and LD-|-MP-4-methylcoumaryl-7-amide (MCA) at 4.0% and 7.3%, respectively, of the efficiency of DPP11, whereas DPP11 did not hydrolyze the DPP7 substrate LN-|-MP-MCA, indicating that DPP7 partially complements the role of DPP11. A single amino acid substitution at the S1 site in DPP7 (Gly666Arg) enhanced hydrolysis toward Asp and Glu to a level higher than that of DPP7 wild-type, while a reverse substitution in DPP11 (Arg673Gly) abolished hydrolytic activities toward all substrates examined. Taken together, DPP7 might represent the ancestral form of S46-family peptidases, from which the dpp11 gene arose through gene duplication and subsequent mutations initiated by Gly666Arg substitution.
Altered protein glycosylation is a hallmark of cancer and has been implicated in the regulation of tumor cell behavior and tissue organization. N-Acetylgalactosaminyltransferase 6 (GALNT6), an initiating enzyme of mucin-type O-glycosylation, has been associated with breast cancer progression. However, whether GALNT6 exhibits subtype-specific expression and distinct histopathological localization patterns in breast cancer tissues has remained unclear. In this study, we performed subtype-stratified analyses of TCGA-BRCA RNA-seq data. We identified preferential GALNT6 upregulation in luminal A breast cancer, whereas expression was low in basal-like tumors and normal breast tissue. Gene co-expression network analysis further demonstrated that GALNT6-associated modules in luminal A breast cancer were enriched for estrogen receptor (ER) signaling, mammary epithelial development, and apical membrane-associated pathways, indicating close association with luminal epithelial transcriptional programs. To further characterize GALNT6 localization patterns in clinical tissues, we performed region-based histopathological and immunofluorescence analyses of luminal A breast cancer specimens. These analyses revealed architecture-dependent subcellular localization of GALNT6 associated with distinct Golgi organization patterns, showing apical/luminal-biased distribution in gland-forming regions and less polarized and more broadly distributed cytoplasmic localization in non-gland-forming tumor regions. E-cadherin was enriched at basolateral intercellular junctions, particularly in GALNT6-high tumors. Together, these findings identify GALNT6 as a luminal A-associated glycosylation-related marker linked to luminal epithelial transcriptional programs and architecture-dependent subcellular localization patterns in breast cancer.
Alternative splicing dysregulation contributes to colorectal cancer (CRC) progression, yet the roles of specific splicing factors, such as SF3B3, remain poorly defined. To identify key dysregulated splicing factors, RNA sequencing was performed comparing high-metastatic (SW620) and low-metastatic (SW480) CRC cell lines. Notably, SW480 and SW620 are isogenic cell lines derived from the primary tumor and lymph node metastasis of the same CRC patient, respectively. Their consistent genetic background minimizes confounding factors, enabling reliable detection of metastasis-associated molecular alterations. Functional assays-including MTT, wound healing, and transwell experiments-combined with molecular analyses confirmed the critical role of the SF3B3-STOX1 axis. SF3B3 was markedly upregulated in metastatic CRC cells and patient tissues. Mechanistically, SF3B3 promoted CRC cell proliferation, migration, and invasion by modulating STOX1 alternative splicing, specifically enhancing exon 3 inclusion to produce the oncogenic isoform STOX1-L while suppressing STOX1-S. Notably, STOX1-L, but not STOX1-S, robustly activated the PI3K-AKT and MAPK/ERK signaling pathways. Importantly, knockdown of STOX1-L reversed the malignant phenotypes driven by SF3B3. Collectively, these results identify SF3B3 as a pivotal regulator of CRC cell metastasis in the isogenic SW480/SW620 cell model that acts through the production of STOX1-L, establishing the SF3B3-STOX1-L axis as a promising candidate therapeutic target for advanced colorectal cancer worthy of further validation in diverse CRC models.
Interactions between volatile anesthetics and bovine serum albumin (BSA) were thermodynamically characterized using isothermal titration calorimetry (ITC). Because weak ligand binding requires appropriately matched protein and ligand concentrations for reliable thermodynamic analysis, measurements were performed at several protein concentrations to characterize the concentration-dependent titration behavior. Binding constants and thermodynamic parameters were evaluated by enthalpy-based analysis combined with Scatchard analysis of the resulting ITC data. For comparison, the binding of medium-chain fatty acids to BSA was analyzed using the same approach to contrast the thermodynamic behavior of weak- and strong-binding ligands. Fatty acid binding exhibited clear saturation behavior and could be described as finite-site binding with a well-defined number of binding sites (approximately 7-9) and relatively high binding constants (on the order of 105-106). In contrast, anesthetic binding showed pronounced protein concentration dependence, and the resulting Scatchard plots were nonlinear, indicating that the interaction cannot be explained by a simple finite-site binding model. The apparent binding constants and binding numbers were broadly consistent with trends in anesthetic hydrophobicity and potency. Although the binding enthalpy changed sign depending on the anesthetic species, this sign did not determine the essential binding mode and likely reflects solvent reorganization and hydrophobic interactions. These results indicate that fatty acids bind to serum albumin through site-specific interactions at a finite number of binding sites, whereas anesthetic interactions are better described as hydrophobic, partition-like multi-site associations on the protein surface. The present study provides a thermodynamic framework for distinguishing site-specific binding from hydrophobic association in protein-ligand interactions.
Non-Small Cell Lung Cancer (NSCLC) remains a leading cause of cancer-related deaths worldwide, creating an urgent need to explore new drugs and clarify their mechanisms of action. In recent years, lactic acid bacteria-derived extracellular vesicles (EVs) have shown great potential in anti-tumor therapy; however, research on their role in lung cancer remains limited. In this study, EVs from three common Lactobacillus species were isolated to verify their anti-tumor effects on lung cancer. Among these, the extracellular vesicles derived from Lactobacillus rhamnosus GG (LGG-EVs) exhibited the most significant anti-cancer activity. A549 and H1299 cells were treated with LGG-EVs, and transcriptome sequencing analysis was performed. The results revealed that Alcohol Dehydrogenase Iron-Containing 1 (ADHFE1)- a differentially expressed gene with notable changes before and after treatment- displayed the same expression pattern in both cell lines. Further verification was conducted using methods including Western Blotting, Transwell, wound healing, and flow cytometry. Compared with the control group, treatment with LGG-EVs led to downregulated ADHFE1 expression in both cell lines, along with inhibited malignant cellular behaviors and glycolysis capacity. Notably, overexpression of ADHFE1 significantly reversed these effects. These findings indicate that the anti-tumor effect of LGG-EVs on lung cancer may be achieved through ADHFE1-mediated metabolic reprogramming. This study is the first to identify that ADHFE1-mediated metabolic reprogramming could be a key mechanism by which LGG-EVs inhibit lung cancer, providing a new target and strategy for lung cancer treatment.
Triple-negative breast cancer (TNBC) has a poor prognosis due to the lack of effective therapeutic targets, highlighting the urgent need for new intervention strategies. This study focuses on an engineered artificial nuclease, L29E myoglobin, which forms a heterodinuclear Mg2+-H2O-heme center upon Mg2+ binding and exhibits DNA cleavage activity. We overexpressed L29E Mb in the TNBC cell line MDAMB231 and systematically evaluated its effects on cellular physiology. The results showed that, in the presence of Mg2+, the overexpression of L29E Mb significantly induced DNA damage and loss of mitochondrial membrane potential, leading to G1-phase cell cycle arrest and activation of the caspase-9/caspase-3 cascade, and ultimately promoting apoptosis. These findings establish a proof-of-concept that the complex of Mg2+-L29E Mb may function as an artificial nuclease and trigger the mitochondrial apoptotic pathway, which provides a foundation for protein-based anticancer strategies.
