Search Results (10,501)

Salmonella enterica serovar Typhimurium (ST) is an intracellular pathogen that survives within macrophages and disseminates to systemic organs, thereby evading host immune defenses. Previous studies have shown that the probiotic yeast Saccharomyces boulardii CNCM I-745 improves survival in ST-infected mice, reduces bacterial translocation, and modulates cytokine expression, including the upregulation of interferon-γ and the downregulation of interleukin-10, both of which are involved in the regulation of inducible nitric oxide synthase (iNOS), a key mediator of macrophage antimicrobial activity. The present study was designed to investigate the transcriptional regulation of iNOS and associated antimicrobial responses in ST-infected RAW264.7 murine macrophages pretreated or cotreated with S. boulardii. Gene expression levels of iNOS and selected cytokines were analyzed in RT-qPCR assays. Bacterial adhesion was quantified by colony-forming unit (CFU) counting, and intracellular survival was assessed using a gentamicin protection assay. S. boulardii did not affect bacterial adhesion, but it significantly reduced intracellular ST survival, particularly under pretreatment conditions (p < 0.05). This effect was associated with increased iNOS gene expression. Interferon-γ expression was mainly induced by pretreatment, whereas tumor necrosis factor-α and interleukin-10 were modulated under cotreatment conditions. These findings indicate that S. boulardii modulates macrophage antimicrobial gene expression and suggest that probiotic pretreatment enhances innate immune responses against intracellular bacterial infections. Full article

Boule is the ancestral member of the Deleted in Azoospermia (DAZ) family and is pivotal for gametogenesis and male fertility in most animals. However, there is a dearth of information on molluscan boule. Here, we identified a counterpart (Pcbol) from the genome of Pomacea canaliculata, which has emerged as a cosmopolitan alien species and notorious pest that causes devastating damage to aquatic biodiversity, freshwater ecosystems and crop production in invaded ranges. This study aimed to investigate the biological roles of Pcbol in male reproduction and to decipher the molecular mechanisms underpinning its modulation via dsRNA-delivered RNA interference (RNAi). The bioinformatic analysis showed that the Pcbol genomic sequence is 12,934 nt in length, harboring an open reading frame of 294 nt that encodes 97 aa residues, with an RRM domain evolutionarily conserved among molluscan orthologues. Spatiotemporal expression profiling indicated the predominant abundance of Pcbol in adult males and testis tissues. dsPcbol, injected at a dose of 4 μg/per snail for 5 days, yielded optimal silencing at both transcript and translation levels of Pcbol, as revealed by qRT-PCR and Western blotting. Immunofluorescence echoed a pronounced reduction in Pcbol signal intensity following RNAi. In addition to the arrested reproductive gland phenotype, the number of sperm cells substantially dwindled upon dsPcbol treatment relative to the dsGFP control. In biochemical and fecundity assays, Pcbol depletion triggered a significant decrease in Te/SP/Arg content and suppressed the number of deposited eggs and hatchability. Furthermore, spermatogenic genes like CDC25/TSSK1/SPATA17/DDX4/Dmrt2/Sox2/Kelch10/SPO11 displayed considerable downregulation post Pcbol silencing, with molecular docking predicting a strong affinity between CDC25 and Pcbol. These molecular modules may interact with Pcbol to mediate knockdown effects on spermatogenesis dysfunction. Collectively, our findings not only confirmed that boule was indispensable for spermatogenesis and male fertility in a mollusk, but also highlighted the Pcbol-based male sterile technique (MST), which can be incorporated into precision pest management (PPM) strategies for sustainable control of P. canaliculata. Full article

Background: Sulfate transporters (SULTRs) are integral membrane proteins responsible for sulfate uptake, translocation, and plant adaptation to abiotic stresses. However, knowledge regarding the SULTR gene family in the economically important crop, Brassica rapa (Chinese cabbage), limited. The aim of this study is to conduct a genome-wide identification and functional characterization of BrSULTR genes and to explore their potential functions under abiotic stress. Methods: We identified 19 BrSULTR genes in the B. rapa genome by performing homology searches with Arabidopsis thaliana SULTR sequences as queries. Subsequent bioinformatics analysis included phylogenetic classification, chromosomal localization, gene structure, conserved motif dissection, cis-regulatory element prediction, and protein–protein interaction (PPI) network analysis. Tissue-specific expression profiles of BrSULTRs were assessed using publicly available transcriptome data. Furthermore, their expression dynamics under salt (150 mM NaCl) and low-temperature (4 °C) stress were investigated by integrating transcriptomic, proteomic, and qRT-PCR data. Results: The 19 identified BrSULTR members were phylogenetically categorized into four subfamilies and were mapped unevenly across seven chromosomes. Promoter analysis identified an array of cis-regulatory elements associated with development, hormone response, and stress response. Expression profiles revealed distinct tissue-specific patterns in roots, stems, leaves, flowers, and siliques. Under salt stress, BrSULTR13 was significantly upregulated, while BrSULTR9 and BrSULTR11 were significantly suppressed under low-temperature stress. PPI network projection indicated that the Arabidopsis homologs of BrSULTR5 may physically interact with stress-regulating enzymes such as APS and APR. Conclusions: Our work presents a comprehensive genomic and functional overview of the BrSULTR gene family in B. rapa. The results underscore the potential functions of BrSULTRs, highlighting their involvement in sulfate transport and abiotic stress responses. These insights establish valuable insights and a foundation for further research aiming at improving stress tolerance in B. rapa through the manipulation of sulfur metabolism pathways. Full article

Background: Muscle atrophy is a major feature of Limb Girdle Muscular Dystrophy R1 (LGMDR1) patients, but its underlying molecular mechanisms have not been fully explored. While the ubiquitin–proteasome system (UPS) is known to be involved in muscle protein degradation, inflammation commonly observed in LGMDR1 patients may further activate the UPS. This study aimed to explore the role of inflammation in the muscle atrophy of LGMDR1 patients. Methods: Muscle biopsies from six confirmed LGMDR1 patients (with CAPN3 variants and reduced calpain-3 protein expression) were analyzed for atrophy-related markers, MuRF1 and Atrogin-1, using qRT-PCR and Western blotting. The expression of cytokines, TNF-α, IL-1β, and IL-6 was analyzed by qRT-PCR from muscle biopsies and by ELISA from serum samples. The NFκB, FOXO1, and FOXO3 gene expression was analyzed using qRT-PCR and Western blotting from muscle biopsies. Results: Elevated TNF-α levels were associated with increased UPS activity, reflected by upregulated NFκB, FOXO1, MuRF1, and Atrogin-1 expression in LGMDR1. Conclusion: Our findings indicate that increased TNF-α expression is associated with muscle wasting in LGMDR1 patients by targeting UPS pathway mediators that activate ubiquitin ligases—MuRF1 and Atrogin-1. These findings suggest that targeting TNF-α signaling and its downstream factors may help develop therapeutic interventions to prevent muscle atrophy in LGMDR1 patients. Full article

West Nile virus (WNV) is a mosquito-borne flavivirus of growing importance for both human and equine health in Europe. Horses are highly susceptible to neurological disease and, because they share ecological exposure with humans, they represent valuable sentinels for detecting local viral circulation within a One Health framework. However, diagnosis of WNV infection in equines is complicated by the short and low-level viraemia, which limits the sensitivity of molecular assays, and by serological cross-reactivity with related flaviviruses and the confounding effects of vaccination. In this narrative review, we summarise the current diagnostic tools for WNV in horses, including direct detection methods (RT-qPCR, virus isolation, antigen detection) and indirect serological approaches (IgM and IgG ELISA, virus neutralisation tests), and discuss their practical performance and constraints in clinical and surveillance settings. We further examine equine surveillance systems, passive clinical reporting, active serosurveys and sentinel cohorts, and their integration with vector, avian and environmental monitoring. Key challenges include methodological heterogeneity, limited access to confirmatory testing and variable cross-sector data sharing. Finally, we outline future directions, highlighting the need for harmonised laboratory protocols, innovative field-deployable diagnostics, genomic surveillance and integrated, multi-source monitoring systems to strengthen early warning capacity and improve preparedness for WNV outbreaks in equine populations. Full article

Chronic diabetic wound remain difficult to heal because persistent inflammation, oxidative stress, and impaired regeneration delay repair, while effective noninvasive options are limited. In this study, graphene-based far-infrared radiation (FIR) therapy was evaluated in a streptozotocin (STZ)-induced diabetic rat full-thickness wound model, and mechanisms were examined in vivo and in vitro. Wound closure was quantified by serial imaging, whereas tissue remodeling and angiogenesis were assessed by H&E and Masson’s trichrome staining and CD34-based analyses. Transcriptomic responses were profiled by RNA sequencing with qRT-PCR validation, immune phenotypes were characterized by immunofluorescence, and high-glucose cell assays were performed. Re-epithelialization, collagen deposition, and neovascularization were quantified histologically. These datasets enabled integrated evaluation of inflammation, oxidative stress, and repair programs over time. Graphene FIR accelerated closure, reaching 83.9% healing by day 14 vs. 66.8% in untreated controls. Treatment was associated with downregulation of Cxcl2/Cxcl3, suppression of M1 polarization with enhanced M2 polarization, and reduced ROS accumulation. Consistently, NF-κB signaling was inhibited, supporting restoration of a pro-regenerative microenvironment. Collectively, graphene FIR represents a promising noninvasive strategy for diabetic wound repair via coordinated immunomodulatory and antioxidant actions. Full article

The yellowfin seabream (Acanthopagrus latus) is an important aquaculture species, yet endocrine gene regulation during practical fasting and feeding schedules remains poorly understood. Here, we identified and characterized two duplicated proglucagon genes (Gcga and Gcgb) and examined tissue distribution of expression and transcriptional responses to feeding-related challenges. Sequence and phylogenetic analyses confirmed that Gcga and Gcgb cluster with teleost proglucagon paralogs and contain conserved peptide domains. Both genes were broadly expressed, with the strongest relative qRT-PCR signal detected in brain and fin, while other tissues (including intestine, gill, stomach, and liver) showed comparatively low but detectable expression. Because the liver is a central metabolic organ and displayed reproducible feeding-dependent regulation, we further quantified hepatic transcription under two paradigms. In a short-term starvation–refeeding trial, hepatic Gcga was significantly suppressed during fasting and rebounded after refeeding, whereas Gcgb showed a distinct, weaker response. In an acute peri-feeding assay, hepatic Gcga and Gcgb displayed rapid but differential regulation around meal time, and Gcgb expression differed between feeding and non-feeding groups. Together, these results support transcriptional divergence between the two proglucagon paralogs in nutritional regulation within a liver-focused metabolic-response model. Our findings provide baseline molecular information for A. latus and offer endocrine insights relevant to evaluating feeding strategies in aquaculture. Full article

Diagnostic testing of foot-and-mouth disease virus (FMDV) currently utilizes reverse transcription quantitative PCR (RT-qPCR) to detect the presence of viral RNA and double antibody sandwich ELISAs (DAS-ELISAs) to determine viral serotype. Serotype identification is critical to support informed vaccine selection to combat outbreaks. While DAS-ELISAs are capable of serotype identification, the test suffers from low sensitivity and requires a viral isolate for successful detection. In this study, we developed FMDV-ONTAPS: an Oxford Nanopore Technologies Amplicon P1 Sequencing protocol involving reverse transcription-PCR to amplify P1 of the FMDV genome, and Nanopore sequencing of the amplicons to provide genetic data for serotype and subtype/topotype identification. FMDV isolates representing all seven serotypes were successfully sequenced with this method. Additionally, the protocol successfully provided serotype identification from a variety of specimen matrices obtained from experimentally infected animals that included milk, serum, oral and nasal swabs, tissue suspensions, vesicular fluid, and oral fluid. The limit of detection for FMDV cell culture isolates was comparable for both sequencing and RT-qPCR detection. RT-qPCR Cq values for clinical samples evaluated ranged from 8 to 28.21. Sequencing was successful for all samples except for a single tissue suspension sample (Cq of 28.21). Identification of FMDV serotype in clinical samples is critical for effective outbreak response, and Nanopore sequencing offers a timelier and more sensitive alternative to DAS-ELISAs. Full article

Background/Objectives: Retinoblastoma represents the most common intraocular malignancy in childhood; however, the clinical applicability of mitomycin C (MMC) is restricted by dose-dependent ocular toxicity. Consequently, the development of pharmacological strategies that sensitize tumor cells to MMC while allowing dose reduction remains an unmet therapeutic objective. In this context, quercetin, a bioactive flavonoid with pleiotropic anticancer properties, has emerged as a potential chemosensitizing agent. Methods: Human retinoblastoma cell lines Y79 and WERI-Rb1 were exposed to MMC and quercetin, administered either individually or in fixed-ratio combinations. Cytotoxic responses were quantified through dose–response modeling and IC₅₀ determination following 24 and 48 h of treatment. Drug–drug interactions were quantitatively characterized using the Chou–Talalay combination index (CI) approach and isobologram analysis. Cell cycle distribution was assessed by propidium iodide (PI)-based flow cytometric analysis to evaluate treatment-associated alterations in cell cycle progression. Apoptotic cell death was assessed by Annexin V-FITC/PI flow cytometry, while transcriptional modulation of genes associated with apoptosis, cell cycle regulation, and oxidative stress (BAX, BCL-2, TP53, CASP3, CDKN1A, and HMOX1) was evaluated by qRT-PCR. Modulation of tumor-supportive signaling was examined by measuring VEGF and IL-6 secretion. Translational relevance was further investigated using a three-dimensional (3D) tumor spheroid model, and the functional contribution of reactive oxygen species (ROS) was interrogated through N-acetyl-L-cysteine (NAC) rescue experiments. Results: Quercetin significantly enhanced the cytotoxic activity of MMC in both retinoblastoma cell lines, with CI values below 1 across IC₅₀–IC₉₀ effect levels, indicating a synergistic pharmacological interaction. PI–FACS analysis revealed that combined MMC and quercetin treatment induced a pronounced accumulation of cells in the G2/M phase, consistent with cell cycle arrest, with a more marked effect observed in Y79 cells compared with WERI-Rb1 cells. Combination treatment resulted in a pronounced increase in apoptotic cell populations compared with single-agent exposure and triggered a coordinated pro-apoptotic transcriptional response, characterized by increased expression of BAX, TP53, CASP3, CDKN1A, and HMOX1, alongside suppression of BCL-2 and a marked shift in the BAX/BCL-2 ratio. Concurrently, VEGF and IL-6 secretion were significantly reduced, reflecting attenuation of pro-angiogenic and pro-inflammatory signaling. Notably, synergistic cytotoxicity was maintained in 3D tumor spheroids, where combined treatment induced spheroid shrinkage, architectural disruption, and reduced viability. NAC pretreatment diminished ROS accumulation and partially restored cell viability, indicating that oxidative stress contributes to, but does not solely account for, the observed synergistic cytotoxic effect. Conclusions: Collectively, these findings indicate that quercetin appears to function as an effective chemosensitizing adjuvant to MMC in retinoblastoma models, through transcriptional changes consistent with p53-associated apoptotic signaling at the transcriptional level, G2/M cell cycle arrest, and partial involvement of ROS-related cellular stress responses, along with suppression of tumor-supportive signaling pathways. The preservation of synergistic activity in 3D tumor spheroids supports the potential preclinical relevance of this combination. However, these findings are based on transcriptional and phenotypic analyses and should be interpreted as hypothesis-generating, requiring further validation through protein-level and in vivo studies before translational application. Full article

Annexin A1 (ANXA1) is a proresolving protein regulated by glucocorticoids, the standard care for severe and critical COVID-19 patients. As part of a larger project including hospitalized COVID-19 patients, this study aimed at evaluating ANXA1 and its FPR2 receptor in these patients, focusing on longitudinal profiles and comparison across disease severities and outcomes, and exploring their correlations with inflammation, endotheliitis and other proresolving mediators. Blood was collected in “severe” (n = 27), “critical” (n = 17) and “critical on veno-venous extracorporeal membrane oxygenation” (n = 17) COVID-19 patients at admission, days 3–4, 5–8, and weekly thereafter, and in controls (n = 23) at a single time point. We quantified ANXA1, resolvin D1, resolvin E1 (RvE1) and endocan by ELISA, cytokines and other endothelial markers by multiplex immunoassays, and FPR2 and Chemerin₁ receptors by RT-qPCR. Most patients underwent a 10-day dexamethasone regimen. Admission ANXA1 and FPR2 were significantly higher in all patient groups. Throughout hospitalization, ANXA1 increased mainly in “severe” patients and survivors, becoming higher at weeks 3 and 4 in survivors versus non-survivors. Variable cumulative dexamethasone doses did not differentially affect ANXA1 or FPR2. ANXA1 was associated with higher RvE1 during the dexamethasone effect period. Exploratory analyses showed that ANXA1 inversely correlated with RvE1 receptor and endotheliitis, whereas both ANXA1 and FPR2 positively correlated with inflammation. In conclusion, ANXA1 may be involved in COVID-19 recovery processes, and its interplay with RvE1 may ameliorate hyperinflammation. Full article

The global prevalence of osteoporosis is rising, particularly among the elderly and post-menopausal population. Although natural flavonoids can inhibit osteoclast overactivation, their low abundance and extraction challenges limit clinical translation. In this study, we synthesized a flavonoid derivative, SZQ-4, and evaluated its therapeutic potential for post-menopausal osteoporosis (PMO). Using an RANKL-induced osteoclastogenesis model in vitro, we demonstrated through TRAP staining, RT-qPCR, and bone resorption assays that SZQ-4 significantly suppresses osteoclast formation and activity. Mechanistically, RNA-seq, Western blot, siRNA knockdown, and plasmid-based overexpression experiments revealed that SZQ-4 reduces RANKL-induced reactive oxygen species (ROS) production, regulates SIRT3 expression, and improves mitochondrial function, thereby attenuating osteoclast differentiation. In an ovariectomy-induced bone loss mouse model, SZQ-4 treatment markedly alleviated femoral bone loss, decreased osteoclast numbers, and lowered ROS levels in the bone marrow microenvironment. Collectively, our findings indicate that SZQ-4 inhibits osteoclast-driven bone resorption by modulating the ROS-SIRT3–mitochondrial function axis, highlighting its potential as a candidate for preventing pathological bone loss. Full article

Carcass growth and development are crucial evaluation indicators influencing the economic efficiency of goats (Capra hircus). This study aimed to screen the nucleotide variation sites (SNPs) of the APOE gene in Guizhou white goats and explore the correlation between APOE gene variations and body size traits, as APOE had been identified as a key candidate gene regulating growth and development in this breed through transcriptome sequencing screening. A total of 324 Guizhou white goats were used in this study for SNP detection, population genetic analysis, real-time fluorescence quantitative PCR (RT-qPCR) and association analysis. The results showed that one nucleotide mutation site (g.353 A > G) was detected in the APOE gene, which yielded two alleles (A and G) and three genotypes (AA, AG and GG). The site exhibited moderate polymorphism and conformed to Hardy–Weinberg equilibrium. The mRNA expression level of APOE in longissimus dorsi muscle was significantly higher in males than in females. Association analysis revealed a sex-specific effect of this locus on body size traits. The A allele and AA genotype were significantly associated with increased body weight and heart girth in females, whereas no significant effect was detected in males. Therefore, the identified APOE gene mutation site can serve as a candidate molecular marker for the early selection of growth traits in Guizhou white goats. Full article

NIN-like protein (NLP) transcription factors are key regulators of plant nitrate signaling and stress responses. Although extensively studied in Arabidopsis thaliana and various crops, it has rarely been reported in woody plants, particularly in drought-tolerant tree species. In this study, 10 PeNLP genes were identified in the drought-tolerant tree Populus euphratica Oliv. through comparative genomics. These genes were unevenly distributed across seven chromosomes, and the gene-family expansion was mainly driven by whole-genome duplication (WGD). Analysis of conserved domains showed that PeNLPs contained 4–10 characteristic motifs, and most members possessed the typical RWP-RK and PB1-related domains. Collinearity analysis identified 18 NLP orthologous gene pairs between P. euphratica and its relatives (Populus pruinosa and Salix sinopurpurea), which exceeded the 15 pairs detected between P. euphratica and A. thaliana, indicating that the NLP family is more conserved within the Salicaceae. Phylogenetic analysis divided PeNLPs into three subfamilies, and their promoter regions harbored diverse cis-acting elements associated with hormone signaling, environmental stress, growth, and light response. Transcriptome and qRT-PCR analyses further demonstrated that PeNLPs were generally downregulated under drought stress. Overall, this study systematically characterized the evolution, structure, and drought responsiveness of the PeNLPs, providing a theoretical basis and genetic resources for improving nitrogen use efficiency and drought resistance in trees. Full article

Herbal melanin (HM), previously reported for its antiproliferative and pro-apoptotic properties, has garnered interest as a promising anti-colorectal cancer drug. However, HM’s biological effects and underlying molecular mechanisms and the related signaling pathways in colorectal cancer (CRC) cell motility are poorly investigated. To evaluate the impact of various concentrations (50, 100, and 200 μg/mL) of HM on cell migration, invasion, and tumorigenicity on human HT29 and SW620 CRC cell lines, a real-time cell analyzer instrument and colony formation assays were employed, respectively. An angiogenesis-related protein array was also used, and the levels of protein expression contributing to colony formation and extracellular proteolysis-driven cell migration and invasion, such as E-cadherin, N-cadherin and urokinase-type plasminogen activator receptor (uPAR), were monitored using Western blotting and RT-qPCR technologies. HM significantly decreased CRC cell motility, invasiveness, and formation of colonies, associated with E-cadherin upregulation and N-cadherin downregulation. In addition, HM specifically inhibited uPAR expression levels, which were also decreased by the pharmacological mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor UO126 and Jun N-terminal kinase (JNK) inhibitor SP600125, in both CRC cell lines, including metastatic CRC (mCRC) SW620 cell line. Addition of HM to cells pretreated with JNK and MEK inhibitors attenuated the blockade of JNK and ERK phosphorylation and alleviated HM-downregulated uPAR expression and HM-inhibited mCRC cell migration. In conclusion, our in vitro studies demonstrate that HM exhibits an inhibitory effect on CRC migration and invasiveness, associated with uPAR downregulation through JNK and ERK pathways. Full article

Endometriosis affects a large number of women of reproductive age, and its pathogenesis is still unclear. It causes severe chronic pelvic pain, which is often misdiagnosed as irritable bowel syndrome, or other disorders. Metabolomics and transcriptomic approaches enable the study of changes in various physiological or pathological pathways to identify new potential biomarkers. We employed gas chromatography–mass spectrometry (GC–MS) to investigate metabolic alterations, and quantitative real-time polymers-chain reaction (RT-qPCR) to assess changes in miR-451a and miR-125b in saliva in endometriosis. Serum and saliva samples of patients with symptomatic endometriosis and volunteers without it were collected and subjected to GC–MS and qPCR-RT analysis, respectively. Multivariate and univariate statistical analyses were performed. Orthogonal partial least squares discriminant analysis has shown the differences between the two groups. Eicosadienoic acid, arachidonic acid, and miR-451a increased significantly in endometriosis patients. Machine learning methods were used to build the predictive model, which can be used in early low-invasive diagnostics of endometriosis. Receiver operating characteristics analysis has tested the diagnostic power of metabolites. The combination of metabolic and microRNA profiling may improve our knowledge of the pathophysiological and signaling mechanisms in endometriosis and the discovery of new efficient biomarkers of endometriosis. Full article