International Journal of Molecular Sciences

Brown adipose tissue (BAT) is a unique tissue with mitochondria specialized for thermogenesis via the BAT-specific uncoupling protein 1 (UCP1). Ucp1-/- mice cannot tolerate acute exposure to cold, illustrating the necessity of UCP1 for efficient mitochondrial thermogenesis. However, these mice adapt to low temperatures through a gradual acclimation process, suggesting a high degree of mitochondrial plasticity in brown and beige fat cells. This phenomenon, which remains to be fully elucidated, indicates the potential for these mitochondria to implement effective thermogenic mechanisms in the absence of uncoupling protein 1 (UCP1). Here, we investigated mitochondrial remodeling in beige and brown fat of Ucp1-/- mice to determine how they fulfill their thermogenic role. Upon gradual acclimation to a cold environment, Ucp1-/- mice exhibited body metabolic parameters and temperatures in the interscapular region similar to those of wild-type mice of BAT, highlighting effective thermogenesis. Interestingly, mitochondrial patch-clamp analysis and a mitochondrial Ca2+ swelling assay revealed a dramatic increase in Ca2+ uptake depending on the mitochondrial calcium uniporter (MCU) in BAT mitochondria from Ucp1-/- mice when robust thermogenesis was required. Mitochondrial remodeling was accompanied by markedly increased tethering between mitochondria and the endoplasmic reticulum (ER) in Ucp1-/- mice, confirming a significant restructuring of the contact sites between the ER and mitochondria, likely to adapt to a new Ca2+ homeostasis. Respiratory complexes also underwent significant reorganization, which partly led to a reduction in their assembly. Levels of ATP synthase and its F1 subcomplex increased, suggesting a major source of ATP consumption and energy expenditure. We propose a new role for MCU as a key regulator of mitochondrial plasticity, enabling efficient thermogenesis in beige and brown adipose tissues in the absence of UCP1.

Synthetic 6-Br-Q0C10 has been shown to exhibit a partial electron transfer activity of native coenzyme Q in the isolated mitochondria. It reduces energy coupling efficiency by approximately 30%, suggesting that it may be useful in modulating cell growth in tissue culture. Whether or not it behaves in the same way in the whole cells, or animal, however, has not yet been fully examined. Recently we have investigated the effect of 6-Br-Q0C10 across multiple cell lines using three detection methods. Treatment with 6-Br-Q0C10 reduces cell proliferation in all cell lines tested, with different effectiveness. Obesity-related cell lines were the most susceptible, and a pronounced inhibitory effect was also observed in cancer cell lines. These results strengthen the idea of using 6-Br-Q0C10 to manage obesity or to retard the growth of rate cancer cells and thus prolonging life.

Sepsis remains a life-threatening condition with limited therapeutic options targeting immune dysregulation. The CD47-SIRP "dont eat me" signaling axis, well characterized in tumor immune evasion, has not been systematically investigated in the context of sepsis. In this study, we performed a targeted transcriptomic analysis of phagocytosis- and "dont eat me" -related genes using the GSE228541 dataset (14 sepsis patients, 15 healthy controls). We identified 8 significantly differentially expressed genes within the curated gene panel. Key changes included downregulation of CD47 (logFC = -0.88, FDR = 5.6 x 10-4) and marked upregulation of PRTN3 (logFC = 2.68, FDR = 6.1 x 10-4). Gene Ontology (GO) enrichment demonstrated prominent alterations in pathways including negative regulation of phagocytosis (GO:0050765, FDR = 7.6 x 10-22), endocytosis, and inflammatory responses. Co-expression network analysis identified SNX3, DYSF, and PLSCR1 as hub genes within this regulatory module. Immune infiltration analysis showed increased M1 macrophage polarization and neutrophil activation in sepsis. Using LASSO regression, we constructed a 6-gene diagnostic signature (PLSCR1, SNX3, DYSF, PRTN3, CSK, CD47) that discriminated sepsis from controls with good performance (AUC = 0.933 in the test subset). Downregulation of CD47 suggests impaired "self" recognition, which may contribute to aberrant phagocytosis during sepsis. Elevated PRTN3 is consistent with neutrophil activation and extracellular trap formation, linking innate immune activation to tissue injury. This targeted transcriptomic analysis reveals coordinated transcriptional reprogramming of phagocytosis-regulatory genes in sepsis and supports the CD47-SIRP axis as a candidate therapeutic target for further investigation.

Caffeine, the most widely consumed stimulant worldwide and primarily sourced from coffee, is well known for its central nervous system effects. Emerging evidence indicates that caffeine also modulates key cellular processes, including DNA repair. It inhibits the kinase activity of ATM and ATR-essential DNA damage response proteins, and impairs homologous recombination (HR)-mediated repair through multiple mechanisms. However, its effects on nonhomologous end joining (NHEJ), a major double-strand break (DSB) repair pathway, have been underexplored. In a recent study, we reported that caffeine inhibits NHEJ primarily by interfering with Ligase IV/XRCC4 complex, using in vitro and ex vivo model systems. Given coffees role as a primary dietary caffeine source, this study investigates the impact of Coffea arabica decoction on NHEJ-mediated DSB repair. High-performance liquid chromatography (HPLC) quantified caffeine levels in the decoction, followed by in vitro and ex vivo assays to evaluate NHEJ efficiency. Results demonstrate that coffee decoction inhibits end joining of both compatible and noncompatible DNA ends in cell-free systems derived from normal and cancer cells. Extrachromosomal repair assays confirmed impaired intracellular NHEJ, leading to accumulation of unrepaired DSBs in human cells. Kinetic analysis of {gamma}-H2AX foci formation and resolution revealed persistent DNA breaks and reduced repair kinetics. Reconstitution experiments verified that the decoction specifically targets the Ligase IV/XRCC4 complex. These findings, building on our previous work, establish coffee decoction as a potent NHEJ inhibitor, mirroring purified caffeines effects. This underscores caffeines interference with endogenous DNA repair, with profound implications for cancer therapy by sensitizing tumors to genotoxic treatments.

Sugar-induced cell death (SICD) remains an intriguing but poorly studied phenomenon in the physiology of Saccharomyces cerevisiae. Recently, it was shown that SICD development largely depends on the redirection of glucose fluxes between glycolysis and the pentose phosphate pathway (PPP). In particular, inhibition of glycolysis by iodoacetamide (IAA) was observed to reduce SICD levels. This study is devoted to further investigation of the relationship between SICD and the functionality of the two PPP branches. It was shown that deletion of the ZWF1 gene does not affect the decrease in SICD levels in IAA-treated cells. This allows us to conclude that the oxidative branch of the PPP is not involved in the suppression of SICD/ROS. Deletion of the GLR1 gene and attenuation of the TRR1 gene also did not restore SICD levels in cells after IAA treatment. The obtained results indicate that the level of reduced glutathione or thioredoxin does not affect SICD genesis. The addition of 5 mM ribose-5-phosphate (R5P) to the incubation medium led to suppression of SICD by 79%. At the same time, the addition of 5 mM ribose + 5 mM Pi suppressed SICD by only 20%. Suppression of SICD by 5 mM R5P in the{Delta} pho3 strain (83%) excludes the mechanism of extracellular dephosphorylation of R5P to ribose, its subsequent transport into the cell, and re-phosphorylation inside the cell. Furthermore, more than 70% suppression of SICD in the{Delta} end3 strain with 5 mM R5P excludes endocytosis as a mechanism of R5P import into the cell. The observed effect of R5P can be explained by the moonlighting function of some unknown protein. Thus, SICD development in S. cerevisiae cells depends on the final product of the non-oxidative PPP--R5P.

Purpose: To evaluate the efficacy and safety of oral L-ergothioneine (EGT) in alleviating pain and associated symptoms in women with primary dysmenorrhea (PD). Methods: In this randomized, double-blind, placebo-controlled trial, 40 women with PD (aged 18-30 years) were randomized (1:1) to receive EGT capsules (120 mg/day) or a matching placebo for 3 consecutive menstrual cycles. Outcomes evaluated at baseline and post-cycle included peak pain (Visual Analog Scale, VAS), Dysmenorrhea Symptom Score, and the COX Menstrual Symptom Scale (CMSS). Results: EGT significantly improved PD symptoms over 3 cycles. Mean VAS for peak pain decreased from 4.80 {+/-} 1.12 to 2.32 {+/-} 1.59 in the EGT group (p < 0.001), compared to a non-significant reduction (4.10 {+/-} 1.30 to 3.45 {+/-} 1.69) in the placebo group. The between-group difference at cycle 3 was significant (p < 0.01). A linear mixed-model confirmed a significant Time x Group interaction (p < 0.001), showing an accelerated decline in symptom severity for EGT. Furthermore, 84% of EGT-treated patients achieved [&ge;]50% VAS reduction versus 35% in the placebo group (p = 0.003). Serum inflammatory biomarkers showed no significant between-group differences or correlation with VAS improvements, suggesting EGT's analgesic effects likely operate via cytoprotective pathways independent of classical inflammatory cascades. No adverse events were reported. Conclusion: Oral EGT supplementation (120 mg/day) effectively and progressively mitigates menstrual pain and systemic symptoms in PD, offering a well-tolerated, non-pharmacological intervention. Trial Registration: ChiCTR2500112557; Retrospectively registered on 2025-11-17.

This study aimed to investigate the dynamics of gene expression in pigs during heat stress (HS), focusing on both short-term (STHA) and long-term (LTHA) heat acclimation phases. A total of 12 castrated males were exposed to thermoneutral temperatures (24{degrees}C) for 14 days (TN) and then to a constant temperature of 30{degrees}C for 21 days. Rectal temperature measurements indicated a biphasic thermoregulatory response, with an initial peak followed by acclimation. Using whole blood transcriptome analysis at seven time points between day 5 before the initiation of HS challenge and day 13 post HS. A total of 525 genes were differentially expressed during the STHA (day 0-day 2) phase. A switch in the expression of most genes was observed around 20 hours after HS. Functional pathway enrichment analysis identified through shape-based clustering revealed the activation of the immune system, especially mediated through toll-like receptor signaling pathways. The LTHA phase (day 2-day 13) revealed 985 differentially expressed genes, with pathways associated with various metabolisms, including mitochondrial fatty acid beta-oxidation, and electron transport, ATP synthesis, and heat production by uncoupling proteins. Interestingly, oxidative phosphorylation was predicted to be activated during the LTHA, particularly in Complex V, whereas other complexes showed mixed regulation. Comparative pathway analysis indicated distinct metabolic adaptations between STHA and LTHA, with up-regulation of glucose and lipid metabolism in late STHA and down-regulation of lipid metabolism during LTHA. This study contributes to a better understanding of the time course of adaptation mechanisms in pigs to HS, underlying a coordinated regulation during STHA involving several stress-specific mechanisms (via the HSP) and metabolic variation to help pigs achieve homeothermy.

Lipopolysaccharide (LPS), a ubiquitous bacterial component of food, is neutralized by a variety of mechanisms that help to establish a threshold, which when exceeded results in an inflammatory TLR4 mediated response. Notably both CEACAM1 and CD36 affect downstream signaling of TLR4 to LPS. Furthermore, CEACAM1 associates with CD36 in hepatocytes, regulating lipid storage and bile acid (BA) secretion that includes reverse transport of LPS to the intestine. Direct binding of LPS-Ra micelles to soluble CEACAM1 or soluble CD36 was analyzed by surface plasmon resonance (SPR), size exclusion chromatography (SEC) and transmission electron microscopy (TEM). Direct binding of CEACAM1 to CD36 was analyzed by SPR and proximity ligation assays. Molecular models were generated by Alpha Fold and Molecular Dynamics. LPS Binding: SPR binding constants of KD= 1.04 x 10-10 M and KD= 3.38 x 10-10 M were obtained for LPS-Ra micelle binding to sCEACAM1 and sCD36, respectively. On SEC, the molecular sizes of LPS-Ra micelles bound to sCEACAM1 and sCD36 were approximately 500 and 800 kDa, respectively. In addition, LPS binding to both was reduced by sodium cholate and sodium deoxycholate. Alpha Fold predicted a binding site of LPS-Ra to CD36, while Molecular Dynamic studies of an N-domain mutant of CEACAM1, that breaks a conserved salt bridge, revealed the presence of an open form that is predicted to bind LPS. sCEACAM1 to sCD36 Binding: A KD of 5.28 x 10-8 M was obtained for sCEACAM1 binding to immobilized sCD36 by SPR. Antibody-based-proximity ligation demonstrated the association of the ectodomains of CEACAM1 and CD36 on hepatic cells and when co-expressed in HEK cells. In addition, biotin-based proximity ligation demonstrated association of the cytoplasmic domains of CEACAM1 and a CD36-BioID2 fusion protein when co-expressed in HEK cells. Alpha Fold predicted both head-to-head (trans) and side-to-side (cis) binding of the N-domain of CEACAM1 to CD36, from which a membrane model of their cis-interaction could account for the proximity ligation results. Both CEACAM1 and CD36 share a common LPS micelle binding function, as well as binding to each other, and together, may regulate uptake and excretion of micellar LPS.

Purified proteins are routinely flash frozen for use in functional and structural studies, providing a convenient way to reproduce results across complex experiments. Rho guanine-nucleotide exchange factors (RhoGEFs) are no exception to this practice, yet the effects of freezing on their activity and stability remain largely uncharacterized. This gap potentially affects the characterization of these important enzymes and how results are interpreted with respect to their prospective use as therapeutic targets. Here, we tested the isolated DH/PH tandems of P-Rex1, P-Rex2, and PRG under different cryoprotectant conditions and monitored activity and thermostability over time after flash freezing. Our results show a clear divergence between the activity of fresh and frozen purified RhoGEF protein samples in as little as one week for some conditions. Specifically, the variability in data collected on frozen samples was greatly increased. Despite these differences, thermostability seems to be preserved for much longer timepoints across RhoGEFs. Moreover, despite eventual changes in both activity and thermostability with respect to freezing, there are no obvious changes in global conformation between fresh and frozen samples of the isolated P-Rex2 DH/PH tandem. From our data, there are few generalizable trends between the different RhoGEFs and no single cryoprotective agent tested was a silver bullet to preserve both activity and thermostability across RhoGEFs. Overall, our findings emphasize the unpredictable effects of freezing RhoGEFs. As such, RhoGEF freezing should be carefully characterized for each protein and critically viewed when comparing analyses between different studies.

The enzyme nitric oxide synthase (NOS) breaks down the semi-essential amino acid L-arginine (L-Arg) in the cell to produce citrulline and nitric oxide (NO). NO is a crucial signalling molecule in cells that controls the metabolism of fats and carbohydrates. The aim of this study was to investigate two important genes in the L-Arg-NOS-NO signalling pathway, AMPK and ACC-1, as markers of the molecular mechanisms that are triggered when liver cells sense elevated L-Arg. Mouse liver epithelial insulin-sensitive BNL CL2 cells were used as a model system and cultured with 0, 400 or 800 {micro}M L-Arg. Cell growth parameters were analysed alongside qRT-PCR based analysis of target transcripts involved in lipid and glucose metabolic pathways. In a further experiment, NOS inhibitor; L-NAME (40 mM) and external NO donor; SNAP (100 {micro}M) were added and the effect on target gene expression analysed. L-Arg addition impacted culture viability and cell growth. AMP-activated protein kinase (AMPK) was regulated in response to L-Arg addition with increasing extracellular concentrations elevating AMPK mRNA and protein expressions. L-NAME decreased target gene expression in an L-Arg addition dependent manner. SNAP (100 {micro}M) addition increased target gene expression after 6 and 24 h. NO, produced as a result of L-Arg addition and the factors L-NAME and SNAP, that regulate NO bioavailability, impacted BNL CL2 cell NO/AMPK/ACC-1 signalling pathways via regulating mRNA expression and subsequently protein expression.

NLRP3 inflammasome is a cytosolic multi-protein complex that plays a crucial role in the immune system, responding to various exogenous and endogenous stimuli by triggering protective inflammatory responses. However, aberrant NLRP3 inflammasome activation is implicated in numerous inflammatory diseases. Therefore, the NLRP3 inflammasome is an important pharmacological target for the treatment of multiple diseases. In this context, we screened various US-FDA-approved drugs for NLRP3 inflammasome inhibition. We found that among various drugs, minoxidil hydrochloride (MXL) effectively inhibits NLRP3 inflammasome, evidenced by reduced secretion of IL-1{beta} and IL-18 in J774A.1 cells treated with MXL. The IC50 values of MXL for inhibition of IL-1{beta} and IL-18 were calculated to be 1.2 and 1.06 {micro}M, respectively. MXL was found to prevent ASC oligomerization, thereby inhibiting the NLRP3 inflammasome and leading to CASP1 cleavage. Further investigation revealed that MXL also utilizes AMPK-mediated autophagy to modulate NLRP3 inflammasome activity. Using siAMPK and bafilomycin A1, an end-stage autophagy inhibitor, we elucidated crosstalk between the NLRP3 inflammasome and autophagic pathways, which was modulated by MXL. Furthermore, we demonstrated the efficacy of MXL in two different mouse models of inflammation, involving the NLRP3 inflammasome. MXL at doses of 10 and 20 mg/kg effectively inhibited the activation of NLRP3 inflammasome by monosodium urate in the air pouch model and by ATP in the peritoneal inflammation model, as evidenced by reduced secretion of 1{beta} and IL-18 in the lavage. Our study identifies MXL as a potent NLRP3 inflammasome inhibitor, warranting further investigation as a potential therapeutic agent for inflammatory diseases.

This report identifies a bidirectional signaling axis connecting lipid metabolism to nuclear mechanotransduction, with the potential to control fatty acid/triglyceride metabolism. The sterol regulatory element-binding (SREBP) family of transcription factors control fatty acid, triglyceride and cholesterol synthesis and metabolism. The family consists of three members: SREBP1a, SREBP1c, and SREBP2, that are regulated by intracellular cholesterol levels and insulin signaling. The SREBP2-dependent control of the LDL receptor gene is a well-established target for cholesterol-lowering therapeutics and the activity of SREBP1c is an attractive target in metabolic disease. In the current report, we identify SYNE4 (nesprin-4), a component of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, as a direct target of the SREBP family of transcription factors, and show that nesprin-4 in turn supports SREBP1c function. We identify functional SREBP binding sites in the human SYNE4 promoter and demonstrate that these are required for the sterol- and SREBP-dependent regulation of the promoter. Furthermore, we show that the endogenous SYNE4 gene is also regulated by SREBP1/2 and intracellular sterol levels. Interestingly, SREBP2 is responsible for the sterol regulation of the SYNE4 gene in HepG2 cells, while SREBP1 is the major regulator in MCF7 cells, demonstrating that diberent cell types use diberent SREBP paralogs to regulate the same promoter/gene. Importantly, we find that nesprin-4 is a positive regulator of SREBP1c expression and function in HepG2 cells and during the diberentiation of human adipose-derived stem cells. In summary, the current report identifies a novel regulatory interaction between lipid metabolism and the LINC complex. Importantly, we demonstrate that this signaling axis is bidirectional, forming a closed loop that has the potential to control SREBP1c activity and thereby fatty acid and triglyceride synthesis/metabolism. Based on our data, we propose that the nesprin-4-dependent regulation of SREBP1c could represent a novel therapeutic target in metabolic disease.

Equine asthma (EA) is a highly prevalent, chronic, inflammatory disease of the lower airways ranging from mild-to-moderate to severe clinical presentations. Diagnosis currently relies on bronchoalveolar lavage fluid (BALF) cytology, an invasive method associated with interobserver variability, which highlights the need for more reproducible approaches. MicroRNAs (miRNAs) are small noncoding RNAs involved in post-transcriptional gene regulation. They are stable and readily detectable in body fluids and have shown promising results as biomarkers in human asthma. The aim of this study was to characterize miRNA abundance profiles in BALF and serum from horses with distinct EA endotypes to evaluate their biomarker potential and explore their involvement in disease pathogenesis. A total of 43 horses were included and classified as either EA (n=32) or controls (n=11), based on clinical examination and BALF cytology. The EA horses were further divided into three endotypes based on BALF inflammatory cell composition: neutrophilic asthma (n=10), mastocytic asthma (n=15), and mixed asthma (n=7). RNA was isolated from both serum and BALF samples and analyzed by quantitative real-time PCR (qPCR) targeting 103 miRNAs linked to asthma and pulmonary inflammation in humans. Differential miRNA abundance was analyzed across EA endotypes. The most significantly differentially abundant miRNAs were used for in silico target prediction and pathway enrichment analyses. Horses with mixed EA had significantly lower levels of eca-miR-125a-3p and eca-miR-125b-5p in BALF compared to controls. Additionally, eca-miR-146a-5p abundance was significantly increased in BALF from horses with neutrophilic EA compared to mastocytic EA. Target and pathway enrichment analyses for eca-miR-146a-5p identified immune-relevant pathways, such as MAPK and T-cell receptor signaling, supporting its involvement in inflammatory processes associated with asthma. This study identified three promising candidates, eca-miR-125a-3p, eca-miR-125b-5p, and eca-miR-146a-5p, as potential biomarkers associated with different EA endotypes. These miRNAs are interesting candidates for further investigation in an independent cohort.

In our society, ageing, longevity, and neurodegenerative diseases are major concerns of public health. Recently, in Drosophila, we have identified a new cluster of three snoRNAs, including jouvence, and showed that each of them affect longevity and neurodegeneration. As these snoRNAs are required in the epithelium of the gut, these results point-out a causal relationship between the epithelium of the gut and the neurodegenerative lesions through the metabolic parameters, indicating a gut-brain axis. Here, we demonstrate that each snoRNA pseudouridylates a specific site on ribosomal-RNA, which consequently affects the amount of ribosomes as well as the translational efficacy. Moreover, using TRAP experiment assay, we also show that these lacks of pseudouridylations modify the translation of specific genes involved in lipid metabolism. Consequently, these lead to a chronic deregulation of trigycerides and sterols levels, whose correlate to an increase of neurogenerative lesions in old flies, as well as to a modification of longevity.

The RNA exosome is an essential and ubiquitous RNase with exonucleolytic activity, involved in ribosome biogenesis and RNA quality control in eukaryotes. It is present both in nucleus and cytoplasm, and interacts with specific cofactors in each cell compartment, which are essential for recruitment and activity control of the exosome. Posttranslational modifications are known to regulate enzyme activity and protein interaction, although their precise roles are individually specific. In this study, we investigated the phosphorylation status of proteins associated with the nuclear (Rrp6) and core (Rrp46) subunits of the RNA exosome in Saccharomyces cerevisiae. Using co-immunoprecipitation followed by phosphopeptide enrichment and high-resolution mass spectrometry, we identified 121 phosphorylation sites on proteins functionally related to rRNA processing. Differential phosphorylation patterns between Rrp6 and Rrp46 co-immunoprecipitations are consistent with distinct exosome assemblies and suggest potential regulatory roles for phosphorylation. The results shown here highlight the role of phosphorylation in the recruitment and control of the exosome in RNA processing and degradation, offering new insights into the posttranscriptional control of gene expression.

IP3R-Grp75-VDAC1 protein complex at the mitochondria-ER contact sites (MERCS) is involved in response to nutrients and control of glucose and energy metabolism, however, early alterations of the complex and MERCS in response to increased fat intake remain inconclusive. We investigated early effects of high-fat diet (HFD) on IP3R-Grp75-VDAC1 protein expression in correlation with ER-mitochondrial interaction in the liver of mice. Five-week-old mice were fed an HFD or a standard diet (SD) for 2 weeks (2W) or 8 weeks (8W). MERCS fractionation by a gradient ultracentrifugation, Western blot, transmission electron microscopy (TEM), Oroboros high-resolution respirometry were used to analyse liver tissues, while real-time PCR was used to profile genes responsive to HFD. No macroscopic morphological or functional alterations were observed in mice at 2W, while, expectedly, at 8W of HFD mice gained weight and glucose intolerance. Total IP3R protein was reduced at both 2W and 8W points by a post-transcriptional mechanism, while in MERCS, IP3R, VDAC1 and Grp75 were reduced at 8W time-point. TEM analysis revealed a significant reduction of mitochondrial coverage by MERCS, mitochondrial fragmentation and shortening of ER-mitochondria distance already at 2W time-point. Mitochondrial function and metabolism were largely spared. Markers of altered protein homeostasis such as Lmp2, Mecl-1 and Lmp7 showed an early upregulation. In conclusion, HFD induces early alterations in liver MERCS that precede gain of weight and glucose intolerance, suggesting their primary role in obesity and metabolic diseases and as potential therapeutic target.

In mice, oral epithelial stem cells (OESCs) are essential for oral mucosal homeostasis and repair. Less is known regarding the role of OESCs in the human oral mucosa. Here, we studied the behaviour of OESCs and their contribution to tissue maintenance and repair in oral lichen planus (OLP). OLP is a chronic T cell-mediated disease characterized by basal keratinocyte degeneration, epithelial atrophy, acanthosis, and hyperkeratosis. Using immunohistological techniques and semi-automated image analysis, we observed that in OLP proliferative activity was increased in the normally largely quiescent basal cell compartment. In areas of OLP mucosa with intact basal cell layer, expression of NGFR, KRT15, and KRT19-markers of slowly cycling reserve OESCs, was strongly reduced or absent. In contrast, expression of CSPG4, a marker for actively cycling stem cells, was increased in OLP basal cells. Tissue compartmentalization, as evaluated by keratin expression, was strongly disturbed. Taken together, our findings indicate that the inflammation in OLP leads to activation and proliferation of OESCs that give rise to a population of cells with an aberrant differentiation programme. Along with the well-documented epithelial up-regulation of anti-apoptotic proteins in OLP, this likely reflects an attempt by the epithelium to avoid overt ulceration.

Proteins can be actively packaged into extracellular vesicles (EVs) through mechanisms dependent on the stimulus that activated the cells. Identifying proteins released in endothelial EVs in response to stimuli relevant to cardiovascular disease (CVD) may therefore reveal potential biomarkers that provide information about the vascular endothelium. This study aimed to identify differentially expressed proteins in EVs released from human umbilical vein endothelial cells (HUVEC) in response to stimuli relevant to vascular endothelium activation. HUVEC were stimulated with TNF (10 ng/mL) or oxLDL (10 {micro}g/mL). Apoptosis was assessed using a flow cytometric DNA fragmentation protocol and caspase-3/7 activity assay. Size distributions of EVs were examined by nanoparticle tracking analysis. Isolated EVs were examined using tandem liquid-chromatography-mass spectrometry (LC-MS/MS). While treatment of HUVECs with TNF or oxLDL resulted in non-significant elevations in levels of EVs, only TNF increased apoptosis. Mass spectrometry quantified 1355 proteins and revealed significant differences in the proteome of EVs from TNF-treated HUVEC compared to EVs from oxLDL-treated or untreated cells. Several candidate biomarkers were significantly and differentially expressed in response to TNF, including E-selectin and dual specificity phosphatase 7. This study further associated E-selectin on endothelial-derived EVs with endothelial apoptosis and may offer a biomarker of endothelial damage in patients with CVD.

Bisphenol A (BPA) and Bisphenol S (BPS) exposure disrupt ovarian function and granulosa cell (GC) steroidogenesis. Extracellular vesicles (EVs) and their miRNA cargo, as mediators of cellular response to environmental stimuli, might be involved in fertility and folliculogenesis. This study explored modulation of microRNA expression after 48h BPA or BPS exposure (10 {micro}M) in ovine primary GC and EVs from corresponding conditioned medium (CM EVs). Small RNA sequencing of control (0h) and 48h treated GC, CM EVs as well as follicular fluid EVs allowed identification of 533 ovine miRNAs, including 129 new sequences. BPA did not alter miRNA expression in GC, while BPS decreased cellular oar-24b miR. In contrast, BPA modified expression of 4 miRNAs in CM-EVs, including 3 new sequences, and two miRNAs were modified by BPS. Both compounds reduced expression of sequence homologous to miR-1306. Further studies are required to decipher their roles in bisphenol toxicity in GC.

BackgroundChronic inflammation and oxidative stress are central drivers of cardiovascular disease progression and remain incompletely addressed by existing pharmacological strategies. Traditional medicinal plants provide a valuable source of multi-target bioactive compounds that may modulate these interconnected pathways. Rauwolfia serpentina, a classical antihypertensive plant in Ayurveda, has been historically valued for cardiovascular indications. Yet, its antioxidant and anti-inflammatory actions beyond blood pressure regulation remain insufficiently characterised in immune-driven inflammatory models. MethodsRoot extracts of R. serpentina prepared using hot and cold ethanol and water were evaluated for antioxidant capacity using DPPH radical scavenging and phosphomolybdenum assays, along with phenolic, flavonoid, and terpenoid quantification. Protective effects against lipid peroxidation were assessed in rat liver and heart homogenates. Anti-inflammatory activity was examined in THP-1 human monocytic cells exposed to lipopolysaccharide (LPS), arachidonic acid (AA), and oxidative stress. Cytokine secretion and gene expression of TNF-, MCP-1, IL-6, and IL-8 were measured by ELISA and qRT-PCR. Intracellular reactive oxygen species and catalase activity were quantified to assess oxidative regulation. LC-MS-based metabolomic profiling was performed to characterise chemical diversity. The principal alkaloid, reserpine, was evaluated separately, and molecular docking was performed to examine its interaction with IKK. ResultsEthanolic extracts of R. serpentinas root, particularly the cold ethanolic fraction, showed superior antioxidant capacity, higher phenolic and flavonoid content, and potent inhibition of lipid peroxidation. These extracts markedly suppressed LPS-induced cytokine release and gene expression in THP-1 cells, with pronounced effects on MCP-1 and IL-6. Oxidative stress induced by arachidonic acid was attenuated through reduced intracellular ROS and preservation of catalase activity. Reserpine reproduced key features of the extract response, demonstrating strong suppression of IL-6 and MCP-1 at both transcriptional and secretory levels. Docking analysis indicated stable binding of reserpine within the IKK catalytic pocket, supporting a plausible mechanism for modulation of the NF-{kappa}B pathway. ConclusionR. serpentina root extracts exhibit coordinated antioxidant and anti-inflammatory activity in immune cell models relevant to cardiovascular inflammation. These effects are extraction-dependent and are partially mediated by reserpine through modulation of oxidative stress and inflammatory signalling pathways. The findings support the translational relevance of R. serpentina as a traditional medicine with mechanistic activity extending beyond antihypertensive action.