Search Results (786)

Bacterial and viral diseases significantly reduce soybean (Glycine max) yield and quality. RNA modifications, particularly N⁶-methyladenosine (m⁶A), are increasingly recognized as having a regulatory role in plant–pathogen interactions, but the m⁶A methylome of soybean during viral and bacterial infection has not yet been characterized. Here, we performed transcriptome sequencing and MeRIP-seq (methylated RNA immunoprecipitation followed by high-throughput sequencing) of soybean leaves infected with Soybean mosaic virus (SMV) and/or Pseudomonas syringae pv. glycinea (Psg). In general, m⁶A peaks were highly enriched near stop codons and in 3′-UTR regions of soybean transcripts, and m⁶A methylation was negatively correlated with transcript abundance. Multiple genes showed differential methylation between infected and control plants: 1122 in Psg-infected plants, 539 in SMV-infected plants, and 2269 in co-infected plants; 195 (Psg), 84 (SMV), and 354 (Psg + SMV) of these transcripts were both differentially methylated and differentially expressed. Interestingly, viral infection was predominantly associated with hypermethylation and downregulation, whereas bacterial infection was predominantly associated with hypomethylation and upregulation. GO and KEGG enrichment analysis revealed shared processes likely affected by changes in m⁶A methylation during bacterial and viral infection, including ATP-dependent RNA helicase activity, RNA binding, and endonuclease activity, as well as specific processes affected by only one pathogen. Our findings shed light on the role of m⁶A modifications during pathogen infection and highlight potential targets for epigenetic editing to increase the broad-spectrum disease resistance of soybean. Full article

Hepatitis E virus (HEV) is a zoonotic pathogen that can infect pregnant women and cause adverse pregnancy outcomes, including miscarriage and preterm delivery. The previous study demonstrated that HEV genotype 3 (HEV-3) inhibits complete autophagic flux in both mouse placental tissue and human trophoblast cells (JEG-3), evidenced by reduced expression of ATG proteins (including LC3, Beclin1, ATG4B, ATG5, and ATG9A) and accumulation of p62. However, the specific regulatory pathway involved remains unclear. Thus, eukaryotic expression vectors for HEV open reading frames (ORFs) were constructed, and ORF2 and ORF3 proteins were transiently overexpressed in JEG-3 cells via liposome transfection. While both ORF2 and ORF3 significantly reduced LC3B protein levels (p < 0.01), only ORF2 induced p62 accumulation (p < 0.01), indicative of autophagic inhibition, which indicates that ORF2 was the key viral protein mediating autophagy suppression in JEG-3. The results of WB and RT-qPCR showed that ORF2 suppressed the PI3K/Akt/mTOR pathway while enhancing nuclear translocation of TFEB (p < 0.01) and AMPK phosphorylation (p < 0.01), suggesting paradoxical activation of upstream autophagy regulators. Through co-transfection of mCherry-LC3 with ORF2, co-localization studies, and AlphaFold 3-based intermolecular interaction predictions, we propose that ORF2 directly binds LC3B to block autophagosome formation. Finally, co-immunoprecipitation confirmed physical interaction between HEV ORF2 and LC3B, elucidating the molecular mechanism of HEV-induced autophagy suppression in trophoblasts. These findings reveal the molecular mechanism by which HEV inhibits autophagy leading to miscarriage in mice, providing new insights into HEV-induced reproductive damage. Full article

Although calpain-5/CAPN5 is widely expressed in mammals, little is known regarding its functions. Pathogenic mutations of CAPN5 are causal for a devastating autoimmune eye disease, neovascular inflammatory vitreoretinopathy (NIV). To provide insight into both the physiological and pathological roles of CAPN5, it is essential to identify candidate interaction partners and possible substrates. Human SH-SY5Y neuroblastoma cells, transfected with full-length catalytically dead (Cys81Ala) CAPN5-3×FLAG, were used for anti-FLAG co-immunoprecipitation (co-IP) and quantitative proteomics using Sequential Window Acquisition of all THeoretical mass spectra (SWATH-MS). Fifty-one proteins were enriched at least four-fold, p < 0.01, relative to cells transfected with an empty FLAG vector. A high proportion (24/51) of candidate CAPN5 interaction partners are associated with protein quality control, including components of the chaperonin, chaperone, and ubiquitin–proteasome systems. Additional candidate interactors include tubulins, kinases, phosphatases, G proteins, and mitochondrial proteins. CAPN5 interactions for 14 of the candidate proteins were confirmed by co-IP and immunoblotting. Of these 14 proteins, 11 exhibited in vitro calcium-induced proteolysis following co-IP with WT CAPN5-3×FLAG. Impaired calcium-induced proteolysis of co-IP proteins was observed for the pathogenic CAPN5 variants R243L and R289W. Further studies are needed to validate the association of candidate CAPN5 interactors with proteins and complexes suggested by the SWATH-MS and co-IP results, and the possible role of CAPN5 within such complexes. The possible involvement of CAPN5 in protein quality control is relevant to NIV, as defects in protein quality control have been implicated in inherited retinal disorders. Proteomic data are available via ProteomeXchange with identifier PXD068008. Full article

Cancer remains one of the most common causes of death worldwide and imposes enormous social and economic burdens. Human tumor-associated NADH oxidase (ENOX2, also known as tNOX) is a cancer cell-specialized NADH oxidase that is expressed on the membranes of cancer cells. In this study, we investigated the potential role of ENOX2 in regulating stemness properties in oral cancer through a combination of in vitro, in vivo, and bioinformatics approaches. We found that ENOX2 physically interacted with the stem cell transcription factor, SOX2, in co-immunoprecipitation experiments. The expression and activity of ENOX2 were elevated in p53-functional SAS and p53-mutated HSC-3 oral cancer cell spheroids compared with their monolayer counterparts. Consistently, SIRT1, a downstream effector modulated by ENOX2 through NAD⁺ generation, was also upregulated in spheroid cultures. Functional studies further established that ENOX2 overexpression significantly enhanced spheroid formation, self-renewal properties, stem cell marker expression, and PKCδ expression, whereas ENOX2 knockdown produced the opposite effects. In xenograft models, ENOX2-overexpressing oral cancer cell spheroids exhibited enhanced tumorigenicity, while ENOX2-silenced spheroids formed significantly smaller tumors. Complementary analyses of public transcriptomic and proteomic datasets revealed elevated ENOX2 expression in human head and neck tumor tissues compared with adjacent normal tissues. Based on these findings and literature-supported correlations, we propose a putative ENOX2-SIRT1-SOX2 regulatory framework that may contribute to the acquisition and maintenance of stem-like properties of oral cancer cells. While the ENOX2–SOX2 interaction was experimentally validated, the roles of SIRT1 and other downstream components are inferred from bioinformatic analyses and prior studies; thus, this axis represents a hypothetical model that warrants further mechanistic investigation. Collectively, our results identify ENOX2 as a potential regulator of oral cancer stemness and provide a conceptual foundation for future studies aimed at elucidating its downstream pathways and clinical relevance in head and neck tumors. Full article

Protein complexes, assembled by scaffold proteins, act as molecular machines driving development. The mechanosensitive adapter protein Zyxin is a key example, integrating actin cytoskeleton dynamics with gene expression. However, the developmental regulation of its interactions and post-translational modifications remains poorly understood. Here, we characterize the dynamic Zyxin interactome across three early developmental stages of Xenopus laevis (from gastrulation to neurulation) using co-immunoprecipitation coupled with quantitative mass spectrometry (DDA and DIA). We identify stage-specific changes in Zyxin’s association with core focal adhesion components, transcriptional regulators and kinases. Furthermore, we uncover developmentally regulated phosphorylation events on isoforms, suggesting dynamic post-translational control of its interactions. Our work provides a comprehensive resource that positions Zyxin as a central orchestrator of cell adhesion, survival, and gene regulatory programs during morphogenesis. These findings underscore the role of Zyxin as a multifaceted regulatory hub, with important implications for understanding tissue homeostasis and related pathologies. Full article

Salmonella typhimurium (S.T) infection of the central nervous system (CNS) induces severe inflammation, leading to elevated expression of inducible nitric oxide synthase (iNOS) in microglia. This process catalyzes excessive production of nitric oxide (NO), resulting in irreversible damage to neuronal mitochondria. Asiatic acid (AA) is a small molecule with neuroprotective potential; however, its ability to counteract nerve injury induced by S.T and the underlying mechanisms remain unclear. In this study, we established an S.T-infected mouse model (in vivo) and an S.T-stimulated microglial model using BV-2 cells (in vitro) and employed techniques including immunofluorescence (IF), Western blot, co-immunoprecipitation (Co-IP), and RNA extraction and quantitative reverse transcription PCR (RT-qPCR) to systematically evaluate the protective effects and mechanisms of AA. The results showed that pre-treatment with AA significantly reduced the expression of iNOS and the production of NO caused by S.T infection in mouse hippocampal tissue and BV-2 cells. Mechanistically, AA exerts its effects by inhibiting the upstream Toll-like receptor 2 (TLR2)/Notch and nuclear factor-κB (NF-κB) signaling axis. It interferes with the nuclear translocation of Notch and p65 proteins and their complex formation under S.T stimulation, thereby blocking downstream expression of iNOS and production of NO. This study reveals a novel mechanism by which AA alleviates infection-related neuroinflammation through targeting Notch-p65 interactions, providing a new theoretical basis for its clinical application. Full article

Foot-and-mouth disease virus (FMDV) infection elicits sustained, high-level interleukin-10 (IL-10) secretion in cattle and pigs, which correlates with lymphopenia and immunosuppression. We previously showed that macrophages are the principal source of IL-10 during FMDV infection in mice, but the viral trigger and host pathways remained unknown. In the present study, we examined whether the FMDV structural protein VP3 regulates IL-10 expression. To this end, a eukaryotic VP3 expression vector was transfected into porcine alveolar macrophages (3D4/21 cells), and IL-10 expression together with related signaling pathways was interrogated by qRT-PCR, ELISA, Western blot, co-immunoprecipitation (Co-IP), confocal microscopy, and luciferase reporter assays. The results showed that VP3 significantly increased IL-10 mRNA and protein levels (p < 0.001) in a time-dependent manner. Mechanistically, VP3 promoted phosphorylation of PI3K, AKT, and mTOR; this effect was abolished by the PI3K inhibitor LY294002, which also abrogated VP3-induced IL-10 secretion (p < 0.05). Furthermore, VP3 upregulated mRNA expression of STAT3, ATF1, and CREB (p < 0.05) and enhanced IL-10 promoter activity. The STAT3 inhibitor Stattic reduced IL-10 secretion by 22% (p < 0.05). Co-IP and confocal microscopy confirmed direct binding of VP3 to PI3K in the cytoplasm. In conclusion, FMDV VP3 induces IL-10 overexpression by directly activating the PI3K/AKT-mTOR signaling pathway, thereby elucidating a key mechanism of FMDV-induced immunosuppression. Full article

Bone remodeling is maintained through the coordinated actions of osteoblasts, osteoclasts, and osteocytes, among which osteocytes serve as major regulators of osteoclast-mediated bone resorption through the receptor activator of the nuclear factor-κB ligand (RANKL)–osteoprotegerin (OPG) signaling axis. While molecular signals regulating osteocytic RANKL-OPG expression are fairly understood, how post-transcriptional mechanisms impact osteocyte function remains poorly defined. HuR (human antigen R) encoded by Elavl1 (embryonic lethal abnormal vision-like 1), a ubiquitously expressed RNA-binding protein, is known for stabilizing AU-rich element-containing transcripts involved in inflammatory and stress responses; however, its role in osteocyte-derived bone resorption is unknown. In this study, we examined the effect of HuR loss on osteocyte–osteoclastogenesis. Short hairpin RNA (shRNA)-mediated HuR knockdown in MLO-Y4 osteocyte-like cells resulted in a significant increase in OPG mRNA and its protein expression, whereas RANKL levels remained unchanged, leading to a significantly reduced RANKL/OPG ratio. Both co-culture and conditioned-medium assays demonstrated that HuR-deficient osteocytes produced a markedly diminished osteoclastogenic environment. Actinomycin D chase experiments showed no alteration in OPG mRNA decay kinetics, and RNA immunoprecipitation (RIP)-PCR failed to detect HuR–OPG interactions, indicating that HuR regulates OPG expression through indirect mechanisms rather than mRNA binding. These findings identify HuR as an indirect regulator of osteocyte-derived OPG expression that impacts osteoclast differentiation and reveal a previously unrecognized mechanism by which HuR contributes to bone remodeling. Full article

Cerebral ischemia–reperfusion injury triggers mitochondrial dysfunction and oxidative stress, exacerbating neuronal apoptosis. Emerging evidence highlights hydrogen sulfide (H₂S) as a gasotransmitter modulating redox balance, autophagy, and apoptosis. This study investigates the neuroprotective mechanisms of Enriched Environment (EE) against ischemic injury, focusing on mitochondrial dynamics and H₂S-mediated pathways. Using MCAO mice and OGD/R-treated SH-SY5Y neurons, interventions targeting H₂S synthesis, hypoxia-inducible factor 1-alpha (HIF-1α), and mitophagy were implemented. Behavioral, histological, and molecular analyses demonstrated EE significantly improved neurological outcomes, suppressed apoptosis, and attenuated oxidative damage (reduced MDA, elevated MnSOD/glutathione). Mechanistically, EE enhanced mitophagy via dual pathways: canonical PINK1/parkin-mediated mitochondrial clearance, corroborated by transmission electron microscope and LC3B/parkin colocalization, and non-canonical HIF-1α/BNIP3L axis activation. Transcriptomic and Co-immunoprecipitation (Co-IP) data revealed EE upregulated endogenous H₂S biosynthesis post-injury by promoting dopamine-induced calcium influx, which activated calmodulin-dependent signaling to stimulate cystathionine β-synthase/γ-lyase expression. Pharmacological blockade of H₂S synthesis or HIF-1α abolished mitochondrial protection, confirming H₂S as a central mediator. Notably, H₂S exerted antiapoptotic effects by restoring mitochondrial integrity through synergistic mitophagy activation and oxidative stress mitigation. These findings propose a novel neuroprotective cascade: EE-induced dopaminergic signaling potentiates H₂S production, which coordinates PINK1/parkin and HIF-1α/BNIP3L pathways to eliminate dysfunctional mitochondria, thereby preserving neuronal homeostasis. This study elucidates therapeutic potential of EE via H₂S-driven mitochondrial quality control, offering insights for ischemic brain injury intervention. Full article

Myopathy encompasses a group of diseases characterized by abnormalities in both muscle function and structure. However, the underlying regulatory mechanisms of newly formed myofiber development remain poorly defined. No promising therapeutic approach has been developed, but numerous medication options are available to alleviate symptoms. Our previous studies demonstrated that adenosine kinase (ADK) is critical in regulating adenosine metabolism, pathological angiogenesis, pathological vascular remodeling, and vascular inflammatory diseases. Adenosine dynamically distributes between extracellular and intracellular, and adenosine concentration regulates ADK expression. However, the mechanism by which adenosine triggers an ADK-dependent intracellular signaling pathway to regulate skeletal muscle regeneration is not well defined. This study aimed to evaluate whether the adenosine-induced intracellular signaling pathway is involved in regulating myopathy, and how it regulates the development of newly formed myofibers. In this study, an intramuscular injection of cardiotoxin was used to induce a skeletal muscle injury model; satellite cells and C2C12 cells were employed. Whether adenosine regulates satellite cell activity, new myofiber formation and differentiation, as well as fusion of myofibers, were determined by H&E staining, BrdU incorporation assay, and spheroid sprouting assay. Interaction between ADK and PFKFB3 was evaluated by IF staining, PPI network analysis, molecular docking simulation, and CO-immunoprecipitation assay. The results demonstrated that adenosine dynamically distributes between extracellular and intracellular through concentrative nucleoside transports or equilibrative nucleoside transporters, and it rapidly induces an ADK-dependent intracellular signaling pathway, which interacts with PFKFB3-mediated glycolytic metabolism to promote satellite cell activity, new myofiber formation, differentiation, and fusion, and eventually enhances skeletal muscle regeneration after injury stress. The remarkable endogenous regeneration capacity of skeletal muscle, which is regulated by adenosine-triggered intracellular signaling, presents a promising therapeutic strategy for treating muscle trauma and muscular dystrophies. Full article

Tick-borne viruses (TBVs) pose significant public health and economic threats. Pakistan has endemic Crimean-Congo hemorrhagic fever virus (CCHFV), but evidence suggests broader TBV circulation. This study assessed the seroprevalence of thirteen TBVs (seven are members of the genus Orthonairovirus and six are members of the genus Bandavirus) in humans, livestock, and rats in Punjab, Pakistan. Serum samples (n = 794: 321 livestock, 253 human, and 220 rat) were collected from the Narowal, Lahore, and Faisalabad districts. Antibodies to viral NPs were detected using the luciferase immunoprecipitation system (LIPS). The overall seroprevalence was 19.14% (152/794); it was highest in livestock (27.10%), then humans (20.55%), and then rats (5.91%). The highest seroprevalence rates were 3.12% for CCHFV in livestock, 3.56% for Yezo virus (YEZV) in humans, and 0.91% for Tamdy virus (TAMV) and Tacheng tick virus 1 (TcTV-1) in rats. Neutralizing antibodies were detected against CCHFV (1 cattle, 4 humans), Bhanja virus (BHAV) (3 livestock, 1 rat), TAMV (1 cattle), Guertu virus (GTV) (1 cattle), and Dabie bandavirus (2 cattle). Sixteen samples showed antibodies to both orthonairoviruses and bandaviruses, indicating co-exposure. Further analysis showed that seropositivity was not randomly distributed. Livestock kept in commercial farming systems and people working mainly outdoors had distinctly higher exposure to TBVs than subsistence livestock and indoor workers. The results supported the circulation of TBVs among hosts within the close socio-economic/ecological integration area of Pakistan. These findings confirm the circulation of CCHFV, SFTSV, GTV, and TAMV; provide the first serologic evidence of BHAV in Pakistan; and underscore the need for further investigation into the potential circulation of additional TBVs. All results demonstrated that multiple TBVs have been circulating among humans, livestock, and rodents in Pakistan. Full article

Background: Glioblastoma (GBM) is an aggressive primary brain tumor characterized by limited therapeutic options and poor prognosis. Temozolomide (TMZ) remains the standard chemotherapy; however, its effectiveness is often hindered by the development of acquired resistance. Cuproptosis, a recently identified copper-dependent form of regulated cell death, has emerged as a potential therapeutic target. The synergistic effects of TMZ and copper, as well as the molecular mechanisms underlying their combined action, remain unclear. This study aimed to investigate the role of tripartite motif-containing protein 14 (TRIM14) and its downstream effector ATP7A in mediating TMZ- and copper-induced cuproptosis in glioma. Methods: We employed in vitro cellular assays, in vivo xenograft models, bioinformatic analysis, immunofluorescence staining, Western blotting, and co-immunoprecipitation experiments to examine the functional involvement of TRIM14 and ATP7A during combined TMZ and copper chloride (CuCl₂) treatment. Intracellular copper levels and cuproptosis markers, including Dihydrolipoamide S-acetyltransferase (DLAT), were assessed to evaluate copper-dependent cytotoxicity. Results: TMZ combined with CuCl₂ markedly enhanced cuproptosis in glioma cells, as evidenced by increased DLAT expression and elevated intracellular copper accumulation. This combination treatment significantly suppressed TRIM14 expression, downregulated the TRIM14–ATP7A axis, and inhibited non-canonical NF-κB signaling. Co-immunoprecipitation assays further revealed a potential interaction between TRIM14 and ATP7A, suggesting that TRIM14 may modulate ATP7A stability or activity. Conclusions: Our findings indicate that TMZ and copper synergistically induce cuproptosis in GBM by disrupting the TRIM14–ATP7A regulatory axis and promoting intracellular copper accumulation. Targeting TRIM14 or ATP7A to enhance cuproptosis may represent a promising therapeutic strategy to overcome TMZ resistance and improve clinical outcomes in GBM patients. Full article

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is caused by mutations in PKD1 or PKD2 genes, encoding polycystin-1 (PC1) or polycystin-2 (PC2), respectively, characterized by excessive cell proliferation and fluid secretion, resulting in renal cyst formation and growth. PC1 and PC2 form a complex localized on the plasma membrane, endoplasmic reticulum, and primary cilia. PC2 is a non-selective cation channel which, in renal epithelial cells, contributes to calcium transport and signaling. It has been previously shown in renal cells that high external calcium increases whole-cell currents likely mediated by PC2. In this study, we explored the possibility that the Calcium Sensing Receptor (CaSR) is involved in the functional regulation of PC2. To test this hypothesis, human conditionally immortalized Proximal Tubular Epithelial cells, isolated from urine sediments, wt or with stably downregulated PKD1 (PC1KD) or PKD2 (PC2KD) were used. Interestingly, CaSR and PC2 co-immunoprecipitated and Proximity Ligation Assay demonstrated a direct physical interaction at endogenous protein levels. Membrane potential measurements demonstrated that selective CaSR activation, elicited by the calcimimetic R568, caused plasma membrane depolarization, consistent with the modulation of PC2-mediated cation currents, which was significantly lower in PC2KD with respect to wt and PC1KD cells. To conclude, this study provides evidence for a functional coupling of CaSR and PC2, which might be relevant for therapeutic strategies to correct dysregulations occurring in ADPKD. Full article

Influenza A virus (IAV) poses a significant threat due to its rapid evolution through gene mutations and reassortments. Understanding host–virus protein interactions is vital for developing countermeasures. In this study, we developed a live-cell screening platform using the NanoBiT system for rapid discovery of host–virus protein–protein interactions (PPIs). Novel interactions between the host factor SNAPIN and the viral M1, M2 and NS2 were identified using this system. We confirmed the platform’s reliability by validating the SNAPIN-M1 interaction using independent methods including co-immunoprecipitation (Co-IP) and glutathione S-transferase (GST) pull-down assays. These results demonstrate the robustness of the PPI screening system and provide a basis for studying the role of SNAPIN in regulating IAV replication. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 9 (NSP9), the viral RNA-dependent RNA polymerase (RdRp), is essential for viral replication but its comprehensive host interactome remains uncharacterized. This study employed co-immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to systematically identify NSP9-associated host proteins. We identified 222 high-confidence host interactors, with Gene Ontology and KEGG pathway analyses revealing significant enrichment in RNA/DNA-binding proteins, ubiquitin-proteasome pathways, metabolic regulators (amino acid/lipid biosynthesis), endoplasmic reticulum processing, and cell cycle components. Protein-protein interaction network analysis further delineated six functional modules involved in RNA processing, vesicular transport, and innate immunity. Crucially, validation studies confirmed direct binding between NSP9 and key candidates (CAPZ1, PSMA3, CDK1, USP48). Functional assessment demonstrated that CDK1 overexpression significantly inhibited PRRSV replication, implicating CDK1 as a host restriction factor. These findings collectively unveil the multifaceted role of NSP9 in subverting host machinery while identifying novel host defense mechanisms and potential targets for antiviral development against PRRSV. Full article