Synphilin-1 is a protein that interacts with α-synuclein and has been implicated in Parkinson’s disease. However, the cellular and molecular mechanisms underlying the effect of synphilin-1 in Parkinson’s disease remain poorly understood. This study aimed to elucidate the molecular function of synphilin-1 using integrated transcriptomic and proteomic in silico analyses, followed by in vitro validation. Synphilin-1 overexpression enhanced cell viability and attenuated pathways associated with cell death. Among the identified regulatory molecules, p53 emerged as a key mediator linking synphilin-1 to suppression of anoikis. Notably, p53 expression demonstrated predominant nuclear localisation in midbrain tissues of individuals with Parkinson’s disease. These findings suggest that synphilin-1 promotes cell survival by suppressing p53-mediated anoikis. This regulatory relationship could be crucial for understanding neuroprotective or pathological mechanisms in Parkinson’s disease.
Sodium-Glucose Transport Protein 2 inhibitors (SGLT2i), initially developed as antidiabetic agents and now established as foundational therapies for heart failure, have also shown antihypertensive effects in clinical trials involving patients with diabetes and heart failure. However, the underlying mechanisms remain incompletely understood. Given the diverse roles of arachidonic acid (AA) and its metabolites in blood pressure regulation, we investigated the antihypertensive effects of SGLT2i in hypertensive patients and an animal model, and explored whether modulation of AA metabolism contributes to these effects. We first confirmed the antihypertensive effects of SGLT2i in a retrospective cohort study and spontaneously hypertensive rats (SHRs). Targeted metabolomic analysis of plasma and tissues from SHRs identified 20-hydroxyeicosatetraenoic acid (20-HETE) originating from the renal cortex as a key metabolite modulated by SGLT2i. Among the enzymes responsible for 20-HETE production, CYP4A but not CYP4F was found to be downregulated by dapagliflozin at both mRNA and protein levels. Immunofluorescence colocalization further localized this effect to proximal tubular epithelial cells, where SGLT2i reduced CYP4A expression and subsequent 20-HETE production, leading to attenuated renal inflammation, fibrosis and blood pressure elevation. Together, these findings not only confirm the antihypertensive effects of SGLT2i but also delineate a novel antihypertensive mechanism by which lower blood pressure, demonstrating that modulation of arachidonic acid metabolism contributes partially to blood pressure-lowering effects.
Dysregulated lipid metabolism is implicated in renal injury associated with diabetic nephropathy, acute kidney injury, chronic kidney disease, nephrotic syndrome, and renal cell carcinoma. However, its causal role and mechanisms remain ambiguous. Mitochondria-associated ER membranes (MAMs) are contact sites between the endoplasmic reticulum and mitochondria that facilitate the integration of lipid trafficking, mitochondrial metabolism, calcium signaling, and redox homeostasis within cells. Recent evidence from patient biopsies and experimental renal models suggests that altered MAM integrity is linked to ectopic lipid deposition, mitochondrial dysfunction, oxidative stress, and renal injury. The present review examines evidence suggesting that MAM dysregulation may contribute to the abnormal metabolism of phospholipids (PLs), ceramides, cholesterol, fatty acids, and triglycerides in renal cells, thereby addressing a gap between previous reviews on renal lipotoxicity and those focusing on MAM-dependent calcium signaling in kidney diseases. Key mechanisms include impaired PL transfer with disrupted cardiolipin remodeling, ceramide-associated mitochondrial injury, defective fatty acid oxidation, and acyl-CoA synthetase long-chain family member 4-mediated PL peroxidation, leading to renal ferroptosis. Direct evidence for MAM-regulated lipid droplet degradation in the kidney is limited; thus, findings from non-renal cells are differentiated from kidney-specific observations. MAM-associated proteins have emerged as potential therapeutic targets in preclinical studies. However, renoprotective effects of sodium-glucose cotransporter 2 inhibitors and glucagon-like peptide-1 receptor agonists related to MAMs remain indirect and necessitate validation. Restoring the structural and functional integrity of MAMs could represent a promising strategy to mitigate lipid-induced renal injury.
Ribosomal protein S3 (RPS3) is an essential structural component of the 40S ribosomal subunit, yet growing evidence highlights crucial extraribosomal roles in genome maintenance, cell-cycle control, and immune signaling. Dysregulation of RPS3 contributes to diverse human disorders, including cancer, inflammatory diseases, neurodegeneration, and resistance to antimicrobial and anticancer therapies. As a cofactor of NF-κB and a participant in DNA damage responses, RPS3 occupies a node that integrates stress signaling with transcriptional reprogramming, enabling both protective and pathological outcomes. The present review critically evaluates mechanistic insights into RPS3 biology, emphasizing recent findings that delineate its context-dependent effects, discrepancies across models, and remaining gaps that restrict translational applications. Understanding these complexities is essential to assess RPS3's potential as a biomarker and therapeutic target.
Cardiovascular diseases are the leading cause of mortality worldwide, with atherosclerosis and formation of arterial plaques being a major underlying cause. Rupture or erosion of the plaque fibrous cap can result in thrombus formation, arterial occlusion, and a stroke or myocardial infarction. Plaque changes, and endothelial cell barrier leakiness, may result in material leakage, including proteins and fragments into plasma either directly or in extracellular vesicles (EVs). Here, we report comparative LC-MS/MS analyses of plasma-derived EVs and plasma from subjects with impaired vascular status and healthy controls. Analysis of plasma-derived EVs detected 7228 peptides and 763 proteins, with 87 proteins being differentially abundant with these including arterial-cell species. Sub-group analysis based on biological sex showed no statistically significant differences for males, whereas females exhibited eight differentially expressed proteins. Subject age effects were minimal. Plasma analysis detected 4366 peptides and 497 proteins, with 188 proteins being significantly altered in abundance between the groups. Subgroup analysis by biological sex revealed 103 differentially expressed proteins in females and 84 in males. No differences were detected in specific collagen fragments. Gene Set Enrichment Analysis revealed altered biological processes related to immune regulation, humoral immune response, proteolysis, and cellular components including plasma lipoprotein particle, extracellular space, and membrane-associated structures. KEGG pathway analysis emphasized enrichment of pathways linked to complement and coagulation cascades, platelet activation, focal adhesion, endocytosis, and inflammation. Together, these data illustrate the potential of LC-MS/MS to examine the role of inflammation and arterial wall cells in shaping the proteome of EVs and plasma in health and disease.
Maintaining genome integrity is essential for survival across all forms of life. Consequently, DNA damage response (DDR) mechanisms are evolutionarily ancient and broadly conserved. Despite their importance as major pathogens of humans, plants, and animals, fungal DDR mechanisms have primarily been studied as model systems to simplify and advance our understanding of DDRs in humans. Antifungal resistance is a major contributor to mortality from human fungal infections, which are associated with an estimated 3.8 million deaths annually. The ability of fungi to balance spontaneous production of beneficial mutations with the preservation of genomic integrity has emerged as a potential mechanism underlying the acquisition of antifungal resistance. In addition, several components of DNA damage repair pathways play direct roles in the virulence of fungal pathogens. In the present review, we provide an overview of DDR pathways, their function and conservation, and highlight specific roles in contributing to genome plasticity, adaptation, virulence, and the emergence of antifungal resistance.
3β-Hydroxysteroid dehydrogenases (3βHSDs) are key enzymes in steroid metabolism, catalyzing C3 oxidation-reduction and Δ5→Δ4 isomerization reactions that govern metabolic flux across multiple steroidogenic pathways. However, the functional diversity of 3βHSDs involved in bufadienolide metabolism in amphibians remains poorly explored. Here, we systematically characterized the 3βHSD gene family in the Asian toad (Bufo bufo gargarizans) using integrated transcriptomic, biochemical, and metabolomic analyses. Seven Bg-3βHSD genes were identified from multi-tissue transcriptomes generated under control and Pb2+ exposure conditions, and six were heterologously expressed for functional evaluation. In vitro assays revealed pronounced functional divergence among Bg-3βHSD isoforms. Bg-3βHSD1 primarily catalyzed bidirectional C3 redox reactions of C21 steroids and bile acid-related substrates, consistent with canonical steroidogenic roles. In contrast, Bg-3βHSD2 enzyme exhibited broad substrate specificity and high catalytic efficiency toward hormones, bile acids, and bufadienolides. In addition to canonical C3 redox reactions and Δ5→Δ4 isomerization, Bg-3βHSD2 also displayed additional oxidation activity at the C17 position for several steroid substrates. A third homolog, Bg-HSD3B7 (GenBank accession no. XM 044303756.1), selectively converted 7α-hydroxylated sterols, suggesting a potential role in classical bile acid metabolism. Integration of tissue-specific expression profiles with bufadienolide distribution patterns suggests that Bg-3βHSD2 may contribute to connecting classical steroid metabolism with bufadienolide biosynthesis in adrenal tissue. Together, the present study identifies Bg-3βHSD2 as an efficient and versatile steroid-transforming enzyme, expands our knowledge of functional diversity within the amphibian 3βHSD family, and provides insights into the enzymatic basis of steroid and bufadienolide metabolism in B. bufo gargarizans.
Cellular processes are controlled by interconnected networks of protein-protein interactions that can be dynamically regulated by post-translational modifications such as phosphorylation. Dysregulation of signaling pathways can drive cellular transformation and contribute to cancer treatment resistance. Mass spectrometry (MS)-based approaches have emerged as key technologies to study both protein function and their dynamic regulation at a network level. Modern proteomics allows investigators to study how signaling networks are rewired in response to genetic lesions, external cues, and targeted therapies, enabling the comparison of baseline (steady-state) networks to perturbed states. Here, we briefly describe key advancements in proteomics to study signaling dynamics, including affinity-purification combined with MS, proximity proteomics (e.g., BioID, APEX), and phosphoproteomics. We highlight how proteomics has led to the identification of comprehensive protein-protein interaction networks, to the delineation of protein subcellular localization maps and to discoveries regarding their dynamics and rewiring in disease. Finally, we comment on the future directions of proteomics to study signaling dynamics, enabled by next-generation MS instruments and AI-driven data analysis, and discuss how these developments are paving the way for clinical translation by bringing quantitative network biology into patient-relevant contexts.
Blastocyst culture is an essential part of IVF/ICSI treatments, however, it has been shown to increase the risk of preterm deliveries as well as large for gestational age infants. The effect of the extended culture on offspring phenotype is thought to be mediated by epigenetic mechanisms, since this developmental period coincides with extensive epigenetic remodelling. Here, we compare genome-wide DNA methylation of cord blood and umbilical cord artery samples collected from newborns resulting from cleavage-stage transfer (n = 25), blastocyst-stage transfer (n = 25), and spontaneous conception (n = 30). Epigenome-wide association studies, global methylation analyses, and epigenetic age comparison did not reveal statistically significant differences between the cleavage-stage and blastocyst-stage groups. However, we identified some loci with a >10% difference in median methylation level between the groups, which should be studied further with a larger sample size. The genome-wide DNA methylation of spontaneously conceived and cleavage-stage newborns were comparable while in the spontaneous to blastocyst-stage comparison, cg25263722 was differentially methylated (P = 5 × 10-8) in umbilical cord artery. Our study did not identify major effects of extended culture on DNA methylation, which is reassuring with respect to the future health of newborns conceived via assisted reproductive technologies.
Pemphigus vulgaris (PV) is a life-threatening autoimmune bullous disease with an unclear pathogenesis. Our previous study indicated that the expression of miR-125b-5p was significantly elevated in PV patients. MiR-125b-5p has been reported to target P63 mRNA and inhibit the expression of P63, while p53-apoptosis-effector-related-to-PMP-22 (PERP) is a downstream protein regulated by P63. However, the role and underlying molecular mechanisms of miR-125b-5p in PV remain unclear. Here, we explored the roles of miR-125b-5p, P63, and PERP in regulating desmosome integrity and their contribution to PV pathogenesis. The dual-luciferase reporter assay showed that miR-125b-5p inhibited P63 expression by directly binding to the 3' UTR of P63 mRNA. Mouse tongue tissues were cultured in vitro and treated with desmoglein (Dsg) 3-monoclonal antibody (mAb) or with miR-125b-5p overexpression before Dsg3-mAb treatment. Overexpression of miR-125b-5p significantly reduced the mRNA and protein levels of P63 and PERP in Dsg3-mAb-treated tongue tissue. Transmission electron microscopy analysis of the structure of desmosomes revealed that, in tongue tissue with miR-125b-5p overexpression and Dsg3-mAb treatment, the interdesmosal width was significantly increased, the thickness of keratin insertion into the membrane was markedly reduced, and the desmosome structures exhibited evident damage. Together, these findings indicate that miR-125b-5p reduced the expression of both P63 and PERP by targeting P63, thereby contributing to the disruption of desmosome integrity.
Acne vulgaris is a common chronic inflammatory skin disorder with a multifactorial pathogenesis involving genetic predisposition, hormonal regulation, microbial factors, and the cutaneous immune microenvironment. In recent years, psychosocial influences and their association with acne onset and progression have drawn increasing attention. Chronic stress may disturb immune homeostasis via neuroendocrine regulatory pathways and thereby contribute to acne pathophysiology. However, the molecular mechanisms by which chronic stress modulates acne development and exacerbation remain unclear. In the present study, we established a chronic stress-induced murine model of acne to characterize stress-related behavioral alterations, skin histopathological changes, serum corticosterone levels, and inflammatory mediators profiles in both serum and skin tissue. We further performed transcriptomic profiling to identify differentially expressed genes and to elucidate candidate regulatory pathways and molecular targets. Our findings indicate that chronic stress indeed contributes to the pathological progression of acne. However, stress hormones do not appear to directly drive inflammatory worsening. Instead, transcriptomic analysis indicated that chronic stress may play an important role in complex neuroendocrine-metabolic-immune interactions involved in acne exacerbation. These preliminary results provide experimental evidence exploring the role of chronic stress in acne pathogenesis and suggest potential targets for acne prevention and treatment.
The multidrug-resistance 2 (Mdr2-/-) mouse is an animal model of biliary liver injury and fibrosis. This genetic equivalent of the human disorder progressive familial intrahepatic cholestasis type 3 histologically resembles primary sclerosing cholangitis. Bile acid accumulation during cholestasis is linked to the down-regulation of the iron regulatory hormone hepcidin, thus suggesting a link between liver disease and iron homeostasis. In the present study, we investigated iron homeostasis in the Mdr2-/- mouse model of cholestasis. Iron levels and expression of iron-related genes were analysed by real-time PCR and western blotting. Accumulation of iron and the hepcidin response were analysed in wild-type and Mdr2-/- mice challenged with either an iron-deficient or a 1% carbonyl iron diet. Mdr2-/- mice on a control diet had reduced hepatic iron stores when compared with age-matched controls, despite lower hepatic hepcidin expression and a corresponding elevation in hepatic transferrin receptor 1 expression. Mdr2-/- mice fed a 1% carbonyl iron diet were resistant to hepatic iron accumulation, despite increased serum iron, suggesting impaired hepatocyte iron uptake in this model. In conclusion, Mdr2-/- have abnormal hepatic iron homeostasis, potentially resulting from cholestasis. Impaired hepatic iron uptake may explain the relative paucity of liver iron in cholestatic compared with hepatocellular conditions.
Connexin 36 (Cx36)-deficient mice exhibit deregulation of insulin secretion and glucose homeostasis. Mouse islet beta cells express Cx36 and connexin 30.2 (Cx30.2). Human beta cells express Cx36; however, it remains unknown whether they express Cx31.9 protein, the ortholog of Cx30.2. To address this question, sections of 23 human pancreatic autopsy samples from both sexes were analyzed by real-time qRT-PCR with oligonucleotides against Cx31.9 and by immunohistochemistry (IHC) with a specific antibody against this connexin. Mean levels of Cx31.9 mRNA were similar to those of Cx36 mRNA, used as a control gene, in all human samples analyzed (n = 7). IHC studies showed that Cx31.9-positive staining was localized to most islet cells in donor samples (15 of 16). The density of Cx31.9 staining per μm2 was calculated from 31 islet images from 10 donor samples. Pancreatic serial sections revealed that the spatial distribution of anti-Cx31.9-labeled cells is similar to that of anti-Cx36- and anti-insulin-positive cells. Moreover, anti-Cx31.9 staining was also localized in anti-CK19-positive epithelial ductal cells and in the endothelial cells of small, medium, and large-diameter vessels. Cx31.9 protein is expressed in endocrine islet beta cells as well as in exocrine ductal epithelial and vascular endothelial cells. In addition, there is a balance of Cx31.9 and Cx36 mRNA levels in the human pancreas.
The Mre11/Rad50 (MR) complex uses adenosine triphosphate (ATP) binding and hydrolysis to coordinate the recognition and processing of DNA double-strand breaks. Although Mre11 and DNA stimulate the relatively slow ATPase activity of Rad50, the mechanism by which this occurs remains incompletely understood. In the present study, we investigated a basic patch on bacteriophage T4 Rad50, consisting of Arg154, Arg155, and Lys156, that was predicted to contribute to DNA binding. Mutation of these residues caused only modest changes in DNA affinity, indicating that this region is unlikely to function primarily as a direct DNA-contact surface. In contrast, the effects on ATP hydrolysis were pronounced. R154A and the TripleA mutant displayed strong ATPase activation in the presence of Mre11 alone, approaching the activity of the wild-type MR complex bound to DNA. DNA titrations further showed that these mutants were relatively insensitive to increasing double-stranded DNA concentrations, consistent with a shift in the conformational equilibrium toward an ATPase-active-like state. However, ATP-dependent stimulation of repetitive nucleotide excision was reduced for all mutants, with the strongest defect observed for TripleA, indicating that enhanced ATP hydrolysis alone is not sufficient to support processive nuclease activity. A mutation at Asp479 had a related but distinct effect, supporting long-range coupling within the T4 MR complex. Overall, the results support a model in which a basic patch near the base of the Rad50 coiled-coils contributes to an allosteric pathway linking Mre11 and DNA engagement with productive ATP hydrolysis and its coupling to nuclease output.
Antibiotic heteroresistance is a form of within-isolate susceptibility heterogeneity in which an apparently susceptible bacterial population contains rare subpopulations capable of growth at substantially higher antibiotic concentrations. It is commonly defined as resistant minorities occurring at frequencies ≥10-7 and growing at concentrations at least eight-fold above those that inhibit the dominant population, as demonstrated by population analysis profiling or related assays. This phenomenon has been described in diverse Gram-negative and Gram-positive pathogens and across multiple drug classes and is frequently characterized by instability, with resistant subpopulations expanding under treatment and contracting once drug pressure is relieved. This dynamic behavior contributes to systematic under-detection by routine antimicrobial susceptibility testing, which is optimized for population-average endpoints and may miss clinically relevant resistant tails, helping explain treatment failure despite 'susceptible' results. Mechanistically, heteroresistance spans a continuum from genotypic processes, including gene amplification, plasmid copy-number variation, transposition, and point mutations, to phenotypic mechanisms driven by reversible regulatory and physiological states such as transcriptional reprogramming, stochastic expression variability, and intergenerational phenotypic memory. These mechanisms can coexist and shift under antibiotic selection, supporting the view that heteroresistance may act as an evolutionary intermediate linking susceptibility and stable resistance. Recent advances in single-cell phenotyping, copy-number-aware genomics, transcriptomics, pharmacodynamic and population modeling, and machine-learning-assisted diagnostics offer new opportunities to detect and interpret susceptibility distributions. Integrating these approaches into clinical microbiology and stewardship will be essential to improve risk stratification, guide therapy, and predict resistance evolution.
Vitamin D (VD) is involved in bone health, immunity, cardiovascular function, and cancer prevention. Recent studies suggested the potential of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] and the VD metabolite ratio (VMR = [24,25(OH)2D3/25(OH)D] × 100) as indicators of VD levels. An assessment of VD levels relies on serum measurements. The present study investigated the utility of urinary VD metabolites as a non-invasive alternative and their potential as surrogate markers for serum VD metabolites. We used residual serum and urine from the annual health check-ups of employees at Shinshu University Hospital, Japan (n = 506). Urine samples were mixed with β-glucuronidase. Serum and urine were processed via solid-phase extraction, followed by DAP-PA derivatization. LC-MS/MS was used to quantify serum and urinary 25(OH)D3, 25(OH)D2, 3-epi-25(OH)D3, and 24,25(OH)2D3 and urinary 23,25(OH)2D3. Serum whole parathyroid hormone (PTH) levels were measured, and urinary VD metabolites were adjusted for urinary creatinine. We also investigated whether serum and urinary VD metabolites correlated with PTH. The results showed that urinary 24,25(OH)2D3 and 23,25(OH)2D3 strongly correlated with serum 25(OH)D [25(OH)D3 + 25(OH)D2] (all ρ >0.7, P<0.001). Urinary 24,25(OH)2D3, 23,25(OH)2D3, and VMR had stronger inverse correlations with whole PTH (ρ = -0.297, -0.274, -0.308, respectively; all P<0.001), whereas serum 25(OH)D showed a weak correlation (ρ = -0.174, P<0.001). In conclusion, urinary VD metabolites, particularly 24,25(OH)2D3, 23,25(OH)2D3 and the VMR, correlated with serum values and PTH, suggesting their potential as non-invasive markers for the VD status.
The pharmacotherapeutic landscape for the clinical management of type-2 diabetes (T2D), obesity, metabolic dysfunction-associated steatotic liver disease, and steatohepatitis is evolving swiftly in response to the escalating global prevalence and incidence of these interrelated metabolic disorders. Although insulin and metformin formulations have long constituted the foundation of diabetes care, a paradigm shift in T2D management has been observed with the advent of novel pharmacotherapies. Gut peptide analogues are at the forefront of this transformation. The emergence of glucagon-like peptide-1 (GLP-1) receptor agonists represents a watershed moment, fundamentally reshaping the therapeutic landscape for both T2D and obesity due to multifaceted metabolic benefits. The clinical success of GLP-1-based therapies has stimulated pharmaceutical interest in other metabolic peptides. Gut-pancreatic peptides such as glucose-dependent insulinotropic polypeptide, glucagon, amylin, and peptide YY are of particular interest due to their distinct pharmacological benefits and therapeutic promise in metabolic disorders. The present review aims to provide a comprehensive and current overview of non-insulin gut-pancreatic peptide signalling-based therapies that are either clinically approved or under clinical investigation, with a focus on the emerging therapeutic convergence between T2D, obesity, and associated liver disease. The review critically narrates their mechanisms of action, therapeutic efficacy, limitations, current development status, and positioning in the treatment landscape. Furthermore, the review delineates the emerging avenues in the development of novel peptide-based pharmacotherapies, offering insights into their future potential and acquainting the reader with developments in non-insulin gut-pancreatic peptide signalling-based therapies for metabolic disorders.
A major impediment in the therapeutic success of breast cancer (BC) arises from the persistence of clinically undetectable breast cancer stem cells (BCSCs) that need addressal by targeting translationally relevant markers to restrain relapse. Aurora kinase A (AURKA), due to its negative prognostic effect, was considered in clinical trials yet showed an unappreciable response, highlighting the need for additional markers. The present study tried exploring AURKA as a tool of relevance to detect the abundance of Oct4/Sox2(octamer-binding transcription factor 4/sex-determining region Y-box 2)-expressing BCSCs. The purpose was to understand the underlying intricacies governing the limited success of AURKA inhibition and to seek an improved relevant marker profile for detecting disseminated BCSCs. Flow cytometry and chromatin immunoprecipitation assay findings correlated Oct4/Sox2/AURKA expression, proposing an Oct4/Sox2 threshold-dependent AURKA induction. Surprisingly, in BC patient blood, Oct4/Sox2+ve cells were apparently lacking AURKA, emphasizing marker profile dynamicity in disseminating BCSCs with transient cell fate alterations. Immunoprecipitation/immunofluorescence results highlighted an interaction of pAURKA with fate-determinant pNUMB, hinting toward the existence of an AURKA/pNUMB axis for mesenchymal fate induction in breast cancer cells. Oct4/Sox2/AURKA+ve cells further expressed vimentin, highlighting a mesenchymal fate in BCSCs as evident from correlated Oct4/Sox2 and vimentin expression in patient blood. Hence, our study indicated an existing Oct4/Sox2/AURKA/pNUMB axis during transient mesenchymal differentiation of BCSCs and subsequently advocated for a peripheral blood-based approach using AURKA and vimentin for tracking BCSCs, thus supporting a co-targeting strategy. Preliminary in vitro intervention using combinatorial targeting of AURKA and vimentin reduced stemness propensities. Based on these interesting observations, further in-depth studies are warranted for clinical validation.
